MOLECULAR CHARACTERIZATION OF Entamoeba invadens

CHITINASES: AN ENCYSTATION SPECIFIC PROTEIN

Tuli Dey
MOLECULAR CHARACTERIZATION OF Entamoeba invadens
CHITINASES: AN ENCYSTATION SPECIFIC PROTEIN

Thesis submitted to the

Indian Institute of Technology, Kharagpur

For award of the degree

of

Doctor of Philosophy
by

Tuli Dey

Under the guidance of

Dr. Sudip K. Ghosh

DEPARTMENT OF BIOTECHNOLOGY

INDIAN INSTITUTE OF TECHNOLOGY KHARAGPUR

JULY 2009
© 2009 Tuli Dey. All rights reserved.
 Declaration

I certify that,

a. The work contained in this thesis is original and has been done by me under the
guidance of my supervisor, Dr. Sudip K. Ghosh.

b. The work has not been submitted to any other Institute for any degree or diploma.

c. I have followed the guidelines provided by the Institute in preparing the thesis.

d. I have conformed to the norms and guidelines given in the Ethical Code of Conduct
of the Institute.

e. Whenever I have used materials (data, theoretical analysis, figures, and text) from
other sources, I have given due credit to them by citing them in the text of the thesis
and giving their details in the references. Further, I have taken permission from the
copyright owners of the sources, whenever necessary.

(Tuli Dey)

ii
 Acknowledgement

I would like to take this opportunity to express my deep sense of gratefulness to my

supervisor Dr. Sudip K. Ghosh (Associate Professor, Department of Biotechnology,
IIT Kharagpur) for his constant encouragement, thoughtful and constructive criticisms
during the course of investigation and preparation of this manuscript.
I am thankful to Prof. Ananta K. Ghosh, Head Department of Biotechnology for
providing facilities and necessary help during the course of this work.
I wish to thank all members of my Doctoral Scrutiny Committee Prof. Amit K. Das,
Dr. Tapas K. Maiti, Dr. Anindya Sundar Ghosh and Dr. Joykrishna Dey for their
counsel and guidance in all aspects of my PhD work.
I am extremely indebted to all faculty members of Department of Biotechnology for
their advice and cooperation during the course of this study. I wish to thank all the
staff members of Department of Biotechnology. I would like to thank to Dr. Koel
Chaudhury, School of Medical Science and Technology for her generous help. I am
grateful to Prof. John Samuelson, Boston University for providing the antibodies. I
would like to thank Prof. T. Pathak and Prof. P. Pramanik from chemistry department
for collaborative study. I would also like to acknowledge Central Research facility,
IIT Kharagpur for providing instrumentation and Council of Scientific and Industrial
Research India funding research fellowship during my PhD tenure.
I would specially like to express my gratitude to my lab mate Dr. Dibyarupa Pal,
Arindam Chakraborty and Sintu Samanta for their constant support and help for four
years. Thanks to Raunak for helping me in bioinformatics study.
I wish to thank my senior colleagues Dr. B. Mahendran, Dr. Rupesh Dash, Dr.
Chitrangada Acharya, and Dr. Sampurna Sattar for their help. Among my friends
Sujit, Sanjaya, Ratna, Biman, Sangeeta, Shobhon, Subhankar, Mrinmoy Da,
Palashpriya, Rashmi, Soumen, Banani, Saikat and Swatilekha need special mention.
Special thanks are due to Moumita, Sanghamitra, Sadhana, Shawsati and at last
Poulomi for being there always as wonderful friend and much more. Thanks to Dr. S.
Samaddar, Dr. A. Chakraborty, Dr. Saikat Roy and Dr. Sayak Roy for being my long
distance friend in the time of need. Thanks to Soma di and Jhana da for being there
with me.
Last but not the least I want to express my gratitude towards my late father who
dreamed of something like this, and to my mother and brother who believed and have
faith on me.

IIT Kharagpur
July 2009 (Tuli Dey)

iv
 List of Symbols

˚C Degree Centigrade
4MU 4-methylumbelliferone
ABS Adult bovine serum
Amp Ampicilline
APS Ammonium per sulphate
AU Arbitrary Unit
BLAST Basic Local Alignment Search Tool
bp Base pair
BSA Bovine serum albumin
cDNA Complemenatary DNA
CFW Calcoflour white
Da Dalton
DAB Diaminobenzidine
DAPI 4′,6-Diamidino-2-phenylindole dihydrochloride
DMSO Dimethyl sulphoxide
DNA Deoxyribonucleic acid
DNase Deoxyribonuclease
DTT Dithiothreitol
E. coli Escherichia coli
EDTA Ethylenediaminetetraacetic acid
EGTA Ethylene glycol tetraacetic acid
Eh Entamoeba histolytica
Ei Entamoeba invadens
ELISA Enzyme-linked immunosorbent assay
EtBr Ethidium bromide
gm Gram
GalNac N-Acetylgalactosamine
GlcNac N-Acetylglucosamine
HPLC High-performance liquid chromatography
hr Hour
HRP Horseradish peroxide
Ig Immunoglobulin
IPTG Isopropyl β-D-1-thiogalactopyranoside
kana Kanamycin
KCl Potassium chloride
kDa KiloDalton
LB Luria broth
min Minute
ml Milliliter
mM Milimolar
mRNA Messenger ribonucleic acid
Mtz Metronidazole
N.D Not detected
NaCl Sodium chloride
ng Nanogram
nm Nanometer
nM Nanomolar
O.D Optical density
v
PAGE Polyacrylamide gel electrophoresis
PBS Phosphate buffered saline
PCR Polymerase Chain Reaction
pM Picomolar
PMSF Phenylmethylsulphonyl fluoride
rDNA Ribosomal DNA
RNA Ribonucleic acid
RNase Ribonuclease
rpm Rotation per minute
rRNA Ribosomal RNA
RT-PCR Reverse transcriptase Polymerase Chain Reaction
s Second
SDS Sodium dodecyl sulfate
SLS Sodium lauryl sulfate
Taq Thermus aquaticus
TEMED N,N,N',N'-Tetramethylethylenediamine
Tetra Tetracycline
TIGR The Institute for Genomic Research
TRITC Tetramethyl Rhodamine Isothiocyanate
UNICEF United Nations Children's Fund
UV Ultra violet
WHO World Health Organization
X-gal 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside
YT Yeast Extract Tryptone
β-ME β-mercaptoethanol
µg Microgram
µl Microliter
µm Micrometer
µM Micromolar

vi
 Abstract

Amoebiasis remains as a morbid disease throughout the tropical counties, due to the
unhygienic living conditions. Entamoeba histolytica (Eh), the causative agent is an
anaerobic protozoa completes its life cycle between the cyst and trophozoite stage.
Current post genomic knowledge of Eh was used to identify new drug targets within
different molecules, including encystation and excystation specific ones. Chitinase of
other parasites were used successfully as a drug target or for the antigen mediated
transmission blocking.
Detail molecular characterization of Entamoeba chitinases was necessary for
identifying exclusive and new drug target as several chitinases like proteins were
reported in human, with few conserved domains. Reptilian parasite Entamoeba
invadens (Ei) was used as a model organism for encystation study as Eh was not
amenable to encystation in axenic condition. Several encystation specific proteins
were categorized as potential drug targets, including chitinases, as its inhibitor was
able to restrain the encystation. Ei contains three chitinases, while Eh contains single
one.
In this study, three chitinases of Ei named as EiChit1, EiChit2 and EiChit3 were
characterized in silico, to identify the conserved aromatic residues, and substrate
binding mode. All Ei and Eh chitinases were cloned and expressed with N-terminal
His tag. Recombinant chitinases were found to have hydrolytic activity, and bound to
insoluble chitin, without any chitin binding domain present in EiChit2 and EiChit3.
Recombinant chitinases act as exochitinase; by cleaving dimers from the chitin
polymer. Several methylxanthine derivatives were found to inhibit the enzymes in
competitive or non-competitive way though in micro molar range.
Importance of ChBD of EiChit1 was determined by its replacement with lectin ChBD
and removal. Removal of ChBD was found to improve the enzymatic ability against
the soluble substrate. Fusion protein with lectin ChBD shows diminished activity.
Number and location of ChBD aromatic residues was found to influence the substrate
binding.
Initiation of encystation was instrumental in chitinase transcription, which was
profiled during encystation by RT-PCR. Localization of translated chitinases during
encystation was observed by fluorescence microscopy using polyclonal anti EiChit1
and anti EiChit2 antibody. EiChit2 antibody was able to identify Eh cysts due to the
sequence and structural similarity.

Key words: Amoebiasis, Entamoeba histolytica, Entamoeba invadens, Encystation,

Chitinase, Homology modelling, Chitin binding domain, Fusion protein, Chitinase
inhibitor.

vii
 CONTENTS

Title page i

Declaration ii

Certificate of Supervisor iii

Acknowledgement iv

List of symbols v

Abstract vii

Contents viii

Chapter 1 Introduction 1-16

1.1 Introduction 3
1.2 Amoebiasis and Amoebic parasite 3
1.3 Entamoeba histolytica and its life cycle 6
1.4 Metronidazole and Metronidazole resistivity 7
1.5 Encystation and Excystation specific molecules as
probable drug candidate 9
1.6 Entamoeba invadens as a model for Entamoeba
histolytica encystation 9
1.7 Encystation of Entamoeba 10
1.8 Chitin and Chitosan 12
1.9 Chitinases 13
1.10 Chitin Binding domain of Chitinases and Chitin lectin 15
1.11 Objectives 16

Chapter 2 Material and Method 17-60

2.1 Material 19
2.1.1 Entamoeba Strain 19
2.1.2 Bacterial Strain 19
2.1.3 Primer 20
2.1.4 Molecular Biology Material 21
2.1.5 Molecular weight marker 21
2.1.6 Immunodetection reagents 21
2.1.7 Chemicals 22
2.1.8 Instruments, Glass and Plastic wares 22
2.1.9 Recipes of medium, buffers and solutions 36
2.1.10 Kits 36
2.1.11 Vectors 36
2.2 Microbiological Methods 39
2.2.1 Growth of bacterial culture 39
2.2.2 Preparation of competent cell 39
2.2.3 Transformation 40
2.2.4 Selection of blue-white colony 40
2.2.5 Bacterial cell storage 40
viii
 2.3 Molecular Biology Methods 41
2.3.1 Genomic DNA isolation 41
2.3.2 Total RNA isolation 41
2.3.3 Plasmid DNA isolation 42
2.3.4 cDNA synthesis 43
2.3.5 RT-PCR 44
2.3.6 PCR amplification from genomic DNA 44
2.3.7 Restriction enzyme digestion 44
2.3.8 Agarose gel electrophoresis for DNA and RNA 45
2.3.9 Purification of the gel eluted sample 45
2.3.10 Spectrophometric estimation of DNA and RNA 46
2.3.11 Ligation 47
2.3.12 Automated dye terminator cycling sequencing
of DNA 47
2.4 Protein method 48
2.4.1 Expression of recombinant His-tagged protein
under native conditions 48
2.4.2 SDS-PAGE 49
2.4.3 Coomassie blue R-250 staining and destaining 49
2.4.4 Silver staining 49
2.4.5 Purification of recombinant His-tagged protein
under native conditions 50
2.4.6 Regeneration of used column 50
2.4.7 Quantification of protein 51
2.4.8 Western blotting 51
2.4.9 Electroelution 52
2.4.10 ELISA 52
2.4.11 Enhanced chemiluminescence 53
2.4.12 Raising polyclonal antibody in rabbit 54
2.5 Enzymatic methods 55
2.5.1 Enzyme assay 55
2.5.2 Inhibitor assay 55
2.5.3 Substrate binding assay 56
2.5.4 Substrate cleavage pattern 56
2.6. Entamoeba cell culture methods 57
2.6.1 Entamoeba cell culture 57
2.6.2 Encystation of Entamoeba invadens 57
2.6.3 Excystation of Entamoeba invadens 57
2.6.4 Entamoeba total protein isolation 58
2.6.5 Fluorescence Microscopy 58
2.6.6 Cryopreservation of Entamoeba cell 59
2.6.7 Revival of cryopreserved cell 59

Chapter 3 In silico characterization of Entamoeba chitinases 61-76

3.1 Abstract 63
3.2 Introduction 63
3.3 Results
3.3.1 Data mining and sequence identification 64

ix
 3.3.2 Preliminary information regarding Entamoeba
chitinases 65
3.3.3 Phylogenetic analysis of Entamoeba chitinase 66
3.3.4 Primer designing 68
3.3.5 Homology modelling 70
3.3.6 Molecular docking 72
3.4 Discussion 73
3.5 Conclusions 75

Chapter 4 Cloning, Expression and Purification of Entamoeba

chitinases 77-88

4.1 Abstract 79
4.2 Introduction 79
4.3 Results
4.3.1 PCR amplification of Entamoeba chitinases
from genomic DNA 80
4.3.2 Cloning of Entamoeba chitinases in pGEMT
vector 81
4.3.3 Cloning of Entamoeba chitinases in pQE30
vector 82
4.3.4 Expression of Entamoeba chitinases in bacterial
system 83
4.3.5Purification of Entamoeba chitinases using
Nickel NTA beads 84
4.4 Discussion 86
4.5 Conclusions 88

Chapter 5 Enzymatic characterization of Entamoeba

chitinases 89-102

5.1 Abstract 91
5.2 Introduction 91
5.3 Results
5.3.1 β (1-4) bond cleavage efficiency 93
5.3.2 Insoluble substrate binding efficiency 95
5.3.3 Insoluble substrate cleavage efficiency 97
5.3.4 Inhibitor assay using Methylxanthine compounds 98
5.4 Discussion 99
5.5 Conclusions 102

Chapter 6 Characterization of N-terminal Chitin binding

domain of EiChit1 103-120

6.1 Abstract 105

6.2 Introduction 105
6.3 Results
6.3.1 Entamoeba ChBD and fusion protein 108
6.3.2 Cloning of EiChit1CAT, EiChit1R-CAT, JacobChBD
and JessieChBD 109

x
 6.3.3 Construction of JacobChBD-EiChit1R-CAT and
JessieChBD-EiChit1R-CAT fragments 110
6.3.4 Cloning, Expression and purification of EiChit1CAT,
JacobChBD-EiChit1R-CAT and JessieChBD- EiChit1R-CAT
Fragments in pQE30 vector 112
6.3.5 β (1-4) bond cleavage efficiency of truncated and
fusion protein 113
6.3.6 Insoluble substrate cleavage efficiency of
JacobChBD-EiChit1R-CAT and
JessieChBD- EiChit1R-CAT 114
6.3.7 Substrate binding efficiency of
JacobChBD-EiChit1R-CAT and
JessieChBD- EiChit1R-CAT 116
6.4 Discussion 117
6.5 Conclusions 119

Chapter 7 Transcriptional profiling and localization of Ei chitinases

during encystation 121-134

7.1 Abstract 121

7.2 Introduction 121
7.3 Results
7.3.1 Standardization of axenic culture and Encystation
of Entamoeba invadens 124
7.3.2 Transcriptional profile of three Ei chitinase in
encysting cells 125
7.3.3 Raising polyclonal antibody in rabbit against
EiChi2 126
7.3.4 Localization of Chitinases in encysting
Entamoeba invadens 126
7.3.5 Identification of EhChit1 and Eh cyst with
anti EiChit2 antibody 129
7.4 Discussion 131
7.5 Conclusions 133

Chapter 8 Discussion 134-142

Chapter 9 Conclusions and future scopes 143-146

9.1. Conclusions 145

9.2. Future scopes 146

References 147-158

Appendices 159-164

Curriculum vitae 165

xi
 Chapter 1

Introduction
2
1.1 Introduction

Amid the numerous socio-economic problems of developing countries, diarrhoea act

as one of the major contributors to child-mortality causing an estimated death of 2.5
million per year (Kosek et al., 2003; Murray et al., 1997). One etiology of diarrheal
disease is amoebiasis which is endemic in third world country and emerging as a
potent threat in developed countries too, between the vulnerable group of immigrants
and travellers. Among the developing countries of tropical world like India, Mexico,
Bangladesh, and Pakistan poor sanitary condition, lowered living condition, with other
socio-economic problems seems to be the main reasons behind the occurrence of
amoebiasis (Clark et al., 1993; Guerrent, 1986). Total elimination of this morbid
disease is only possible through the remodelling of some very basic conditions such as
sanitary system, hygienic habits, and accessible safe drinking water. These
infrastructural problems can be answered with the change of socio-economic status
along with other long term goals. Another approach to address this disease using
molecular biology as a tool is to discover vaccine candidates and better drugs.
However the lack of proper community based observational studies is one of the major
causes behind the uncertain disease burden and natural history of amoebiasis, which
hinder the proper identification and evaluation of disease and its potential threat.
Entamoeba histolytica the causative agent of amoebiasis stands as the second most
important protozoa after plasmodium. It was included recently among the category B
priority bio-defense agent that heightened the interest in preventing the parasite.

1.2 Amoebiasis and Amoebic parasites

Amoebic colitis and amoebic liver abscess were known to the ancients, as Hippocrates
recognised that “Dysenteries, when they set in with fever, alvine (intestinal)
discharges of a mixed character or with inflammation of the liver … are bad” (Stanley
Jr., 2003). More than 200 years elapsed after amoebas were identified as a cause of
dysentery, by the St Petersburg physician Fedor Aleksandrovich Losch in 1875
(Losch, 1978). In early literature the genus names Endamoeba and Entamoeba were
used to describe Entamoeba histolytica. As per the ruling of The International
Commission on Zoological Nomenclature from 1954 onwards, the E. coli was
designated to be the type species and the term Entamoeba was used in place of

3
Endamoeba to describe E. histolytica (Bruckner, 1992). Several members of amoebic
populations were found to dwell the human gastrointestinal system such as Entamoeba
dispar, Entamoeba hartmoni, Entamoeba coli, Endomalix nana, though only the E.
histolytica, an anaerobic parasite, found to be the sole responsible for this disease, and
use human as the only reservoir. This parasite evolved early and survived till today,
without any major physiological changes (Clark, 2000). The Entamoeba lineage has
no close relatives among free living protozoan. The conclusion from ribosomal DNA
and other genomic sequence analyses shows that the genus Entamoeba diverged from
other eukaryotes much later than other “more” eukaryotic cell (Fig1.1). This
speculation probably indicates the secondary loss of several eukaryotic structures in
response to the obligatory parasitic life cycle of this organism (Clark and Roger, 1995).

Fig. 1.1: The fragment comigration data derived from riboprint patterns were converted into
estimated genetic distances by the method of Nei and Li and the tree constructed by the Fitch-
Margoliash method (Clark and Diamond 1997)

A very significant percent of tropical world population (almost 90%), is being

reported to harbor this parasite within their small intestine. But most human infections
are asymptomatic, as the parasite acting as a harmless commensal organism (Haque et
al., 1998). Many asymptomatic hosts never become symptomatic and usually act as an

4
infectious host for a short time only. Among the 48 million cases of probable infection
throughout the world, only 10% have found to show clinical symptoms with intestinal
and /or extra intestinal pathology (Walsh, 1986). Recently the division of E.
histolytica into pathogenic E. histolytica (invasive) and non-pathogenic E. dispar
(non-invasive) has been done depending on isoenzyme typing as well as gene
sequencing study.

Transmission occurs by ingestion of amoebic cysts with fecally contaminated food or

water or sexually by oral-anal contact. Depending on the affected organ, the clinical
manifestations of amebiasis are termed as intestinal or extra-intestinal. Intestinal
amoebiasis is frequently asymptomatic and varies from fulminant dysentery with
fever, chills, and bloody or mucoid diarrhoea to mild abdominal discomfort with
bloody diarrhoea alternating with periods of constipation or remission. There are four
clinical forms of invasive intestinal amebiasis, all of which are generally acute:
dysentery or bloody diarrhoea, fulminating colitis, amoebic appendicitis, and
ameboma of the colon. Dysenteric and diarrheic syndromes account for 90% of cases
of invasive intestinal amebiasis. Patients with dysentery have an average of three to
five mucosanguineous evacuations per day, with moderate colic pain preceding
discharge, and rectal tenesmus. In patients with bloody diarrhoea, evacuations are few
but the stools are composed of liquid faecal material stained with blood. Fever and
systemic manifestations are generally absent.

These syndromes constitute the classic ambulatory dysentery and can easily be
distinguished from that of bacterial origin. Although E. histolytica can infect almost
every organ of the body, the most frequent form of extraintestinal amebiasis is the
amebic liver abscess. This condition, which results from the migration of trophozoites
from the colon to the liver through the portal circulation, is 10 times more common in
adults than in children and 3 times more frequent in males than in females (Sepulveda
et al., 1986). Dissemination of amoebic infection to extra-intestinal sites most
commonly involves the liver, lungs, pericardium, brain and skin through the
circulating system. Infections with E. histolytica may or may not be symptomic,
though in 20% of infected cases parasite invades beyond the gastrointestinal
periphery, which if left untreated, results in death.

5
1.3 Entamoeba histolytica and its life cycle

The life cycle of E. histolytica is simple and consists of two stages, the motile
invading stage known as trophozoites, and non-motile infecting stage termed as cyst.
Mature cysts ingested with contaminated food or water, pass through the acidic
environment of stomach and become active after entering the neutral or slightly
alkaline condition of small intestine. Four trophozoites (small metacystic trophozoites)
emerged from single cyst and developed into the normal trophozoites which become
established within the mucous layer of intestine.

Fig. 1.2: Faecal oral route of transmission of E. histolytica.

Trophozoites- Living trophozoites vary in size from 12-60-µm in diameter.

Difference of size is noted between the collected trophozoites from symptomic and
asymptomic hosts. The cytoplasm appears finely granular with “ground-glass”
appearance. A clear outer ectoplasm and granular inner endoplasm are clearly
differentiated with several small vacuoles present within the later one. One well
developed and transparent nucleus is being found to contain peripheral chromatin and

6
karyosome (in stained sample). Mitochondria like structure or crypton or mitosome
and Golgi like structure are reported in some recent publications (Mai et al., 1999;
Ghosh et al., 2000). Developments of endoplasmic reticulum like structure are being
observed under electron microscopy (Teixeira et al., 2008).

Depending on the localization of the trophozoites, the symptoms vary, such as

mucosal trophozoites may give rise to the intestinal symptoms and after entering the
circulatory system they may generate the extra-intestinal symptoms in liver or brain or
lungs. After entering the large intestinal tract they condensed into a round mass in
presence of few unidentified stimulants.

Cyst- Immature cyst may contain a glycogen mass and a highly refractile
chromatoidal bodies with smooth rounded edges. In spherical mature cyst (metacyst)
the diameter varies from 10-20 µm (usually 12-15 µm), and the glycogen mass
disappears completely. Four well distinct nucleuses along with several chromatoidal
bodies are being found throughout the cytoplasm which is covered with a 20 nm thick
wall constituted with protein and polysaccharide mesh. The excystation occur in the
terminal ileum giving rise to four trophozoites from each cyst (Espinosa-Cantellano et
al., 2000).

1.4 Metronidazole and metronidazole resistivity

Two classes of drugs are generally used depending on the localization of this parasite.
Paramomycin, Diloxanide fluroate, and Idoquinol act on the intestinal parasite and
classified as Luminal ameobicides, while metronidazole, chloroquine and
Dehydroemetine affect the tissue-invading amoebas, and named as tissue ameobicides.
Metronidazole (Mtz) (1-[2hydroxyethyl]-2-methyl-5-nitroimidazole) was first
introduced as trichomonicide in 1959 (Cosar et al., 1959). At present this drug is used
as a single treatment against several anaerobic bacterial and protozoan infections,
along with other derivatives of nitroimidazole [2-(2-methyl-5-nitro-1H-imidazol-1-yl)
ethanol] (Gillin, 1981). This prodrug is marketed under the trade name “Flagyl” and
gets activated selectively after the reduction of nitro group by several electron carriers
and reducing enzymes, within the anaerobic parasites (Fig. 1.3) (Upcroft et al., 2001,
Pal et al., 2009).

7
Fig. 1.3: Metronidazole structure. Black arrow indicates the position of reduction by which the
drug gets activated.

This unique feature of anaerobic activation made it a wonder drug against several
anaerobic bacteria and protozoa’s, as it remains inactive within highly aerobic
environment of eukaryotic cells. The reduced products are responsible for disrupting
the DNA structure and produce adduct structure with proteins, inhibiting the cell cycle
(Leitsch et al., 2007).

Extensive and indiscriminate use due to easy availability of Mtz and its derivatives is
lead it’s resistivity among different group of prokaryotes. Metronidazole resistance
has been reported in pathogenic bacteria like Helicobacter pylori (Goodwin et al.,
1998; Koivisto et al., 2004), Bacteroides spp. (Narikawa et al., 1991; Diniz et al.,
2004) and Clostridium difficile (Pelaez et al., 2008). Among protozoan parasites,
Trichomonas vaginalis, Trichomonas foetus (Johnson, 1993) and Giardia intestinalis
(Ellis et al., 1993) have shown resistance towards Mtz. Reports of cross-resistance of
Bacteroides spp against nitroimidazoles are well documented now, and becoming very
common (Haggoud et al., 2001). Current literature shows the growing trend of cross-
resistance against Mtz or 5-nitroimidazole family of drugs (Dunne et al., 2004), which
can lead to the failure of current drug therapy. Absence of proper species level
identification of Entamoeba in pathogenic or non-pathogenic carrier increase the risk
of unwanted drug exposure towards the non-pathogenic strains also. Sufficient
evidence is available to classify this drug as an animal carcinogen, as it shows against
rodent (in vivo and in vitro) (Trzos et al., 1978; A-Kareem et al., 1984) though its
genotoxicity has not been proved yet in human (Bendesky et al., 2002). In spite of
ambiguous data regarding human study, chronic and indiscriminated use of Mtz
renders its role as a possible carcinogen among individuals with Crohn’s disease
(Krause et al., 1985). This situation demands a new array of putative drug candidates
as it is frightening to imagine the consequences if large-scale resistance is to emerge.

8
1.5 Encystation and Excystation specific molecules as probable drug candidate

Cyst, the infectious form of E. histolytica, is a chitin walled dormant stage, which
propagate the infection. Excystation or the emergence of four rapidly diving
trophozoites from a cyst occurs in the alkaline environment of small intestine
(Eichinger, 1997). Encystation or the cyst formation occurs in the large intestine as a
result of unknown cues, which may range from low nutrients, high population or host
conditioning (Arroyo-Begovich et al., 1980). Removal of nutrient factors such as
serum and glucose from the culture medium results in the formation of multinucleated
cyst-like structures in E. histolytica, however complete encystation of E. histolytica
not yet been observed (Barron-Gonzalez et al., 2008).

This imposed trigger suggests that when the environment is not suitable for
trophozoite survival this protozoan enters the encystation procedure and probably this
process is irreversible. Thus disrupting the encystation process can lead to eventual
cell death due to nutrient depletion and high osmotic pressure. A chemotherapeutic
agent can be designed to block the encystation and can produces a fatal outcome for
the protozoan, which could at the very least serve to reduce transmission (Jarroll et al.,
2003).

Inhibition of cyst formation may curtail the infection of Entamoeba, and may help to
identify novel chemotherapeutic targets. With the completion of the E. histolytica
genome project (Loftus et al., 2005) chances of identification of potential therapeutic
or vaccine targets increased rapidly (Laughlin et al., 2005).

1.6 E. invadens as a model for E. histolytica encystation

Till now no axenic synchronous encystation protocols have been reported for E.
histolytica, therefore, studies in this area have concentrated on a related reptilian
parasite Entamoeba invadens which have similar basic morphology and life cycle. E.
invadens exists within reptiles such as turtle and snakes as a commensal, but act as
pathogen among a subset of hosts and create a similar type of colon pathology like E.
histolytica (Donaldson et al., 1975).

9
Genome wide survey of E. invadens shows higher GC content than that of E.
histolytica. E. invadens gene sequences shows 62% sequence identity at the genome
level and 74% sequence identity at coding regions with E. histolytica genes, with a
high amount of laterally transferred genes of metabolic pathways as similar to E.
histolytica. Several highly repetitive sequences such as rRNAs, tRNAs, CXXC-rich
proteins, and Leu-rich repeat proteins are detected in E. histolytica genome and find to
be repetitive in E. invadens genome also. E. histolytica proteins involved in amoebic
virulence, drug resistance, cell cycle, vesicular trafficking, signal transduction,
en/excystation and cell growth have homologous components in E. invadens genome
confirming its candidature as a potential model of E. histolytica study (Wang et al.,
2003).

Another reason behind using E. invadens as a model of encystation is because of its

amenability for encystation in vitro. Only the removal of glucose and sodium chloride
along with low serum level in the culture media mediate some intra and intercellular
changes causing the amoebae to round-up, and form multicellular aggregates which
followed by the formation of chitin-coated cysts within 72 hrs (Vazquezdelara-
Cisneros et al., 1984; Avron, et al., 1986). Among the upregulated genes of Gal
deprived E. invadens, ubiquitin and chitinase were recognized after 24 hrs (Coppi and
Eichinger, 1999), which can be reversed depending on the addition of free Gal to the
encystation medium, implying the importance of Gal-binding lectins in the encystation
specific gene expression.

1.7 Encystation of Entamoeba

Encystation of several protozoan parasites was studied before in vitro and existence of
a similar pathway was observed which creates same morphological changes (Chavez-
Munguia et al., 2007).

Actual reasons behind such transformation of Entamoeba sp. within the host gastro-
intestinal tract is still unknown, though several factors such as high population,
scarcity of nutrients expected to play the key role. To mimic such condition, in vitro
encystation of E. invadens is optimized by using low nutrient and hypertonic medium
termed as LG (Low glucose) medium, (Sanchez et al., 2002) containing approximately

10
47% of the constituents of the normal culture medium (TYIS-33) (Diamond et al.,
1978).

In the course of encystation several morphological changes were observed, like the
clumping of cells, and appearance of cord like structures (Carranza-Rosales et al.,
2000). Mature cyst formation occur within the time period of 60-72 hr, with the
formation of four nuclei along with the fabrication of cyst wall with microfibrillar
appearances surrounding the plasma membrane. The cyst wall was found to be of
different thickness depending on the maturation stage, ranging from 30-50 nm and
120-150 nm. In another study, chitinase filled vesicles are reported in encysting E.
invadens (Ghosh et al., 1999). An ultra structural study of E. invadens has shown the
presence of several vesicles within the cytoplasm, containing electron dense material
similar to the cyst wall composition (Chavez-Munguia et al., 2003). The phenomenon
of multiple vesicle deposition within the cleft between cyst wall and membrane was
also observed. During excystation creation of the operculum may be caused by the
bursting of such vesicles signalled by the environmental stimuli.

Highly insoluble nature of cyst wall material along with tightly packed meshwork is
essential for the survival of the cyst outside the host, and surpasses the water
disinfection system (Erlandsen et al., 1989). Major component of many of the
protozoan cyst wall is expected to be chitin while in a series of studies on Giradia
cyst, filamentous cyst wall is being reported to contain β(1-3) linked N-acetyl
galactosamine polymer, not the chitin (Jarroll et al., 2002). In previous studies
presence of chitin is being documented in the E. invadens cyst (Arroyo-Begovich et
al., 1980 and1982), though a latest study reports the presence of chitosan (deacetylated
chitin) also (Das et al., 2006).

Cyst-specific proteins were supposed to bind the polysaccharides, through

hydrophobic and ionic interaction, to balance the positive charge of chitosan in
Entamoeba cyst wall. Among the cyst wall proteins Jacob, a lectin with multiple
chitin-binding domains was reported to present in maximum amount (Frisardi et al.,
2000). Other carbohydrate modifying enzymes such as chitinases (de la Vega et al.,
1997) and chitin synthases (Das et al., 1991; Campos-Gongora et al., 2004) have been
identified in E. histolytica, E. dispar and E. invadens, though very little is known
about the synthesis of chitin during Entamoeba encystation. Interestingly, chitin

11
degrading enzyme Chitinase was recognized to be important during the encystation
process, as one of its inhibitors was found to inhibit the encystation (Villagomez
Castro et el., 1992) along with the chitin synthase inhibitors (Avron, et al., 1982)

1.8 Chitin and chitosan

As the 2nd most abundant polysaccharide on earth, chitin was first investigated in 1811
within the cell walls of mushrooms. In 1830 it was also detected in insects and named
as chitin. Chitin is a (1-4)-linked 2-acetamido-2-deoxy-β-D-glucan (Fig. 1.4), and
occur in two different polymorphic forms. These two forms α-Chitin and β-Chitin
differentiate in their packing and polarities of adjacent chains in successive sheets. All
the chains were found to be aligned in parallel manner in β types, while in α type it
occur in anti-parallel manner. In most hydrozoans, nematodes, arthropods, molluscs α-
chitin occur as a major type, while β-chitin occurs in brachiopods, cuttlefish.

Chitin is highly hydrophobic in nature and completely insoluble in water and most of
the organic solvents but solubilises in hexafluoro-isoprapanol, or hexafluoro-acetone.
Solubility of the chitin is remarkably poor due to the high crystallinity supported by
hydrogen bonds between the acetamido groups and depends on the degree of N-
acetylation i.e. the ratio of 2-acetamido-2-deoxy-D-glucopyranose to 2-amino-2-
deoxy-D-glucopyranose (Muzzarelli and Jolles, 1999).

With the advent of several chemical modifications, more soluble polymers are created
from chitin, making it useful for hundreds of application (Muzarelli et al., 1986). Little
more soluble Chitosan was first produced in 1859 belongs to the family of
deacetylated chitins (Fig. 1.4).

Fig. 1.4: Structure of chitin and chitosan

12
Commercially chitosan can be produced by removing the acetyl groups by NaOH
treatment. Despite of its deacetylated structure, chitosan still possesses the inherent
crystal structure within, and is sparsely soluble in dilute acids such as acetic or formic
acid. Though most of the natural polysaccharides are neutral or acidic in nature, the
chitin and chitosan form a highly basic group, which is being found to be instrumental
in preparing films, chelate metal ions and polyoxysalt formation.

Along with the soluble counter parts, several other enzymatic products of chitin such
as dimer, trimers were found to exhibit antioxidant activity (Chen et al., 2003), and
other immuno-modulatory effects (Wu et al., 2007) as only di and trimers were able to
pass through the intestinal barrier (Chen et al., 2005).

Biological degradation of chitin into the oligomers, is solely depends on the hydrolytic
enzyme chitinase, which is available in most bacteria, fungal and plant in order to help
in their different endeavours ranging from food digestion to host invasion.

1.9 Chitinases

Chitinase (EC 3.2.1.14) (Nomenclature Committee of International Union of

Biochemistry and Molecular Biology, 1992) is established as the single group of
hydrolytic enzymes other than lysozyme (EC 3.2.1.17), which degrade chitin into
different oligomers by cleaving the β (1-4) bonds. Chitinase is found within a huge
range of organisms, from bacteria to human, retaining its conserved sequences and
three dimensional structures throughout the evolutionary pathway. The reasons behind
the synthesis of chitinases are varied from defence to cell proliferation.

From the structural point chitinases are classified into two groups, family 18 and
family 19 of glycosyl hydrolase. Family 18 chitinases, contain (β/α)8 catalytic domain,
mainly comprise of bacterial, fungal and mammalian chitinases. This family consists
of more than 200 enzymes, which are further classified by their enzymatic properties.
Most of the family 18 chitinases are well characterized and comprise of single
catalytic domain, along with one or more extra domains at N- or C-terminal end. The
catalytic domain contains several aromatic residues along within the cleft for substrate
binding, with a Glutamine as a conserved amino acid.

13
More than 150 members of family 19 are mostly of plant origin, and not properly
characterized. Difference between these two classes continued from their origin,
sequence, and configuration of hydrolytic cleavage, concluding them as evolutionary
distinct from each other. Other than these two important classes, few minor classes are
also formed, containing chitosanses (family 46).

Chitinases are also characterized depending on their substrate cleavage pattern and
product formation. Chitinase producing fixed length oligomers from any end of the
chitin polymer, classified as exochitinase, while randomly cleaving chitinases are
termed as endochitinase (Deane et al., 1998). Further classification is done to
differentiate the enzymes producing monomers, dimers or oligomers. Evident of
multiple chitinase with different substrate cleavage pattern are observed, and
concluded as instrumental for increased processivity (Horn et al., 2006).

All Entamoeba chitinases belongs to family 18 group and few of them contain the
extra substrate binding domain also. Other than Entamoeba, a large number of
important parasites are reported to produce stage specific chitinase like proteins,
suggesting their importance in those stages (Brydon et al., 1987; Schlein et al., 1991).
Inhibition of such parasitic chitinases either by inhibitor or transmission blocking
vaccine shows promising results in inhibiting parasitic transmission (Shahabuddin et
al., 1993; Dissanayake et al., 1995; Villagomez Castro and Lopez-Romero, 1996).

Chitinase inhibitors may have a potential to act as a pesticide, as the chitinase is being
found throughout the fungal and bacterial kingdom. Allosamidine a
pseudotrisaccharide, obtained from Streptomyces sp. (Sakuda et al., 1986) is found to
be the most potent and efficient inhibitor of both family 18 and 19 of chitinases.
Several other novel chitinase inhibitors such as cyclic peptides (Izumida et al., 1996),
argifin (Shiomi et al., 2000) and argadin (Arai et al., 2000) are being designed and
characterized. Identification of other potential inhibitor by screening drug library may
found to be useful in future (Rao et al., 2005).

One intriguing problem, regarding the usage of parasitic chitinase as a drug target or
vaccine, emerged due to the recent discovery of human chitinases and chitinase like
proteins (Boot et al., 1995 and 1998). Human chitinase and other chitinase like protein
belong to family 18 of glycosyl hydrolase and share an evolutionary common
structure. Though the structural similarity of human and other parasitic chitinases are

14
not that extreme, cross reactivity may occur as an unavoidable result, which can be
circumvented by the studies of structurally unique regions of parasitic chitinases.

1.10 Chitin binding domain of chitinase and chitin lectins

Majority of the well known plant and insect chitinases contains single or multiple
domains at either N- or C- terminal end which termed as chitin binding domain
(ChBD) other than the conserved catalytic domain

These domains remain connected to the catalytic domain directly or by means of some
linker peptide. The plant ChBD or the hevein domain is mostly composed of 30-43
residues including eight cysteines, three aromatic residues and glycines (Iwanaga et
al., 1998). Invertebrate chitin-binding domain is being assumed to consist of 65
residues with conserved six cysteine and aromatic residues (Kawabata et al., 1996).
While the invertebrate and plant chitin-binding proteins have similar domain structure
and function, no significant similarity was observed at amino acid level. Different
patterns of disulfide bond formation conclude the absence of any common ancestor for
plant and invertebrate ChBDs. The purpose of the conserved residues among the
insect and plant ChBDs are being evaluated, and it is assumed that all cysteine,
proline, and glycine residues play the structural role, while the conserved polar and
hydrophobic residues are responsible for chitin-binding (Suetake et al., 2000).

Till date, numerous ChBDs are identified and classified as ChBD type I, II and III
(SMART Database) depending on the number of cysteine residues, polar residue and
domain length. The substrate binding completely depends on the aromatic residues,
which are seems to be exposed (Boraston et al., 2004). The reason behind the origin of
these domains is not clear, though in one study it is assumed that, the addition of
ChBD in chitinase occurred only after the separation of chitinases from their
ancestors, as no such domains are observed among lysozymes the near relatives of
chitinases. The reason of this addition is predicted in two possible ways, such as the
gradual evolution from a non-chitin-binding sequence along with the insertion of CBD
from elsewhere in the genome (Shen et al., 1999). The acquisition of a CBD by a
chitinase could increase its activity for insoluble chitin. Number and hydrophobicity
of exposed aromatic residues dictates the substrate binding capacity (Kikkawa et al.,
2008). As well as addition of excess ChBD to any chitinases or subtraction of existing

15
domain drastically changes the enzymatic profile indicating its importance in
insoluble substrate binding (Carmen et al., 2006).

1.11 Objectives of this study

To recognize the potential drug target against E. histolytica, we select the encystation
and excystation specific protein chitinase as a plausible candidate. E. invadens
chitinases are used in this study as a model protein along with E. histolytica chitinase
to identify their role during the encystation. in vitro and in vivo characterization of
these proteins are presumed to be an important task and we focused on the following
objectives in the present study.

a) In silico characterization of E. invadens chitinases.

b) Cloning and expression of Entamoeba chitinases in bacterial system.

c) Enzymatic characterization of recombinant Entamoeba chitinases.

d) Characterization of chitin binding domain of EiChit1.

e) Transcriptional profiling and localization of chitinases in encysting E.

invadens.

16
 Chapter 2

Materials and Methods

18
 2.1 Materials

2.1.1 Entamoeba stains

For all experimental studies, Entamoeba histolytica (NIH:200 strain) and Entamoeba
invadens (IP1 strain) were used.

2.1.2 Bacterial strains

Table 2.1: E. coli strains used in this study and their genotypic details

Strain Genotype Use

F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR
DH5α For clone
nupGΦ80dlacZ∆M15∆(lacZYA-argF)U169, hsdR17
(Invitrogen) maintenance
(rK- mK+), λ–

F-mcrA∆(mrr–hsdRMS -mcrBC) φ80lacZ∆M15

Top 10 For clone
∆lacX74 nupG recA1 araD139 ∆(ara-leu)7697 galE15
(Invitrogen) maintenance
galK16 rpsL(StrR) endA1 λ-

JM109 (New endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB+

For clone
England ∆(lac-proAB) e14- [F' traD36 proAB+ lacIq
maintenance
Biolabs) lacZ∆M15] hsdR17(rK-mK+)

XL1-Blue endA1 gyrA96(nalR) thi-1 recA1 relA1 lac glnV44 For expression
(Stratagene) F'[ Tn10 proAB+ lacIq ∆(lacZ)M15] hsdR17(rK- mK+) vector maintenance

M15 with For protein

lacI Kanr on pREP4, F- recA+ uvr+ lon+ lac
pREP4 (Qiagen) expression

[F- = Does not carry the F plasmid, F+ = Carries the F plasmid, F'[ ] = Carries an F
plasmid that has host chromosomal genes on it, Chromosomal genes carried in the F
plasmid are listed in brackets, rB/K+/- = The (B/K) defines the strain lineage, the +/-
indicates whether the strain has or hasn't got the restriction system, mB/K+/- = The
(B/K) defines the strain lineage. The +/- indicates whether the strain has or hasn't got
19
the modification (methylation), hsdS = Both restriction and methylation of certain
sequences are deleted from the strain, hsdR = For efficient transformation of cloned
un-methylated DNA from PCR amplifications, INV = Chromosomal inversion
between locations indicated, ara-14 = Cannot metabolize arabinose, araD = mutation
in L-ribulose-phosphate 4-epimerase blocks arabinose metabolism, ∆( ) =
Chromosomal deletion of genes between the listed genes, deoR and nupG =
Regulatory gene that allows constitutive expression of deoxyribose synthesis genes,
endA1 = For cleaner preparations of DNA due to the elimination of non-specific
digestion by Endonuclease I, (e14) = Excisable prophage like element containing
mcrA gene, galE = Mutations are associated with high competence, increased
resistance to phage P1 infection, and 2-deoxygalactose resistance, glnV = Suppression
of amber (UAG) stop codons by insertion of glutamine; required for some phage
growth, gyrA96 = Mutation in DNA gyrase; conveys nalidixic acid resistance,
lacZ∆M15 = Partial deletion of the lacZ gene that allows α complementation of the β-
galactosidase gene, mcrA = Mutation eliminating restriction of DNA methylated at the
sequence CmCGG (possibly mCG), mcrB = Mutation eliminating restriction of DNA
methylated at the sequence RmC, (φ80) = Cell carries the lambdoid prophage φ80. A
defective version of this phage carrying lacZM15 deletion (as well as wild-type lacI,
lacYA, and flanking sequences) is present in some strains, recA1 and recA+ = For
reduced occurrence of unwanted recombination in cloned DNA; cells UV sensitive,
deficient in DNA repair, relA = Relaxed phenotype; permits RNA synthesis in absence
of protein synthesis, Tn10 = Transposon normally carrying Tetracycline resistance,
Tn5 = Transposon normally carrying Kanamycin resistance]

2.1.3 Primers

For cloning and RT-PCR purpose primers were designed using Oligo Perfect primer
designing software (Invitrogen). During primer designing Tm was determined
depending on the primer length and GC content. Restriction enzyme sites and ATG
sequences were added depending on the requirement. All the primers were obtained in
de-salted condition.

20
2.1.4 Molecular Biology Materials

All restriction enzymes, T4 DNA Ligase, Taq polymerase, Hi-fidelity polymerase,

RT-PCR enzymes, were procured from Roche (Germany) and New England Biolabs
(USA). Pre-stained and Un-stained Protein ladders were availed from Bio-Rad and
Fermentas. Protein-A HRP was brought from Bangalore Genei (India). Gene specific
primers for PCR amplification, RNase, were obtained from Sigma (USA), IDT
(Bangalore) and MWG (Bangalore). PVDF blotting membrane and 0.2 µM filter
membrane were brought from Millipore.

2.1.5 Molecular weight Markers

DNA markers were obtained from NEB and Invitrogen.

100 bp marker from NEB contains fragments of 1517, 1200, 1000, 900, 800, 700, 600,
500, 400, 300, 200, 100 bp.

1 kb marker from NEB contains fragments of 10,000, 8000, 6000, 5000, 4000, 3000,
2000, 1500, 1000, 500 bp.

1 kb plus marker from Invitrogen, contains bands ranging from 500 bp to 12 kb.

Protein markers procured from Bangalore Genei, Bio-Rad and Fermentas.

The PMWM (Medium) range from Bangalore Genei contain these following bands of
97.4, 66.0, 43.0, 29.0, 20.0, 14.3 kDa. The Fermentas unstained marker range contains
proteins of 116, 66.2, 45.0, 35.0, 25.0, 18.4, 14.4 kDa. The pre-stained marker from
Fermentas contains band of 129.0, 85.0, 50, 36, 28 and 21 kDa. Kaleidoscope pre-
stained marker obtained from Bio-Rad contains bands of 250, 150, 100, 75, 50, 37, 25,
20, 15 and 10 kDa.

2.1.6 Immunodetection reagents

For Immunodetection studies, Anti Rabbit IgG conjugated with TRITC, Phalloidain-
FITC, DAPI, Sytox Green, FITC were obtained from Sigma and Molecular Probes.

21
Anti rabbit IgG conjugated with HRP and Protein A-HRP for western blot were
obtained from Sigma and Bangalore Genei respectively.

2.1.7 Chemicals

Antibiotics such as ampicillin, kanamycin sulphate, tetracycline, and Peniciline-

Streptomycine solutions were brought from Sigma and Hi-Media. Luria broth, YT
broth and agar agar were procured from Hi-Media. Molecular biology grade Agarose
were obtained from Seakem Pvt Ltd. Protein work reagents like Bradford reagent,
DAB, and TMB were obtained from Bio-Rad and Bangalore Genei. BSA fraction V
was brought from Sigma. Ni-NTA agarose beads and DEAE sepharose beads were
obtained from Qiagen and GE-Amersham respectively. HPLC grade acetonitrile,
analytical grade methanol, isopropanol and chloroform were brought from Merck.
Adult Bovine Serum was obtained from HyClone. All other chemicals and reagents
used for microbiological or cell culture work were of molecular biology grade and cell
culture grade respectively if not stated otherwise and purchased from Sigma, Merck,
and Hi-Media. All the mediums, buffers and solutions were prepared either in Milli Q
water or Elix water (Millipore).

2.1.8 Instruments, Glass and plastic wares

All Polymerase Chain Reactions were done in Perkin Elmer 2400 series or Applied
Biosystems Thermocycler. Ligation was done using Eppendorf thermostat ligation
bath. Centrifugations were done using Eppendorf refrigerated and non-refrigerated
centrifugation system with fixed angle rotor and swing bucket rotor as per the
requirement. All microbiological work and cell culture work were done in BioKleanzd
Biosafety Laminar hood. For sonication purpose pulse sonicator of Mysonix was used.
Agarose and polyacrylamide Gel electrophoresis, Western blotting, electro-elution
were performed with Tarson, GE-Amersham, and Bio-Rad apparatus.

Enzymatic assays were done in SPEX Fluromax-3 of Horiba Zobin Vyon and Varion
Spectroflurometer. HPLC analysis was done in Agilent 1100 series. DNA
visualization in agarose gel was done by UV Transilluminator from Bioview,
Germany. DNA and Protein concentration were checked in Nanodrop

22
spectrophotometer, UV-Visible Spectrophotometer of Beckman Coulter and Varian.
For pipetting, Pipetman from Tarson, Aqupippete from Gilson and Steripipette from
Erba BioHit were used. Sterilized and filter tips were obtained from Tarson and
Axygen. Microcentrifuge tubes of 0.2, 0.5, 1.5 and 2 ml were brought from Tarson.
All the glass wares were obtained from Borosil and plastic wares brought from Tarson.
Bacterial culture and Entamoeba cultures were incubated in New Brunswick Shaker-
incubator and Beckman Coulter incubator respectively. All the Entamoeba cell culture
was maintained in 50 ml Nunc cell culture flask and 15 ml glass culture tube of
Borosil. Bright field Microscopy of Entamoeba cells was done with Leica S4E and
Olympus CKX31. For fluorescence microscopy Leica MPS 60 and Olympus IX51
were used. For confocal microscopy Olympus Flow view FV1000 was used, with
argon and HeNe laser.

2.1.9 Recipes of Mediums, Buffers and Solutions

Bacterial Medium preparation

Preparation of LB medium (1 liter, pH 7.4)

1. Powdered LB of 20 gm was dissolved in 900 ml of water. pH of the solution was

adjusted to 7.4 with NaOH and then volume made up to 1 liter. The medium was
autoclaved for 15 minutes at 15 psi and 122°C in an autoclave.

2. Tryptone of 10 gm, 5 gm of Yeast extract and 5gm of sodium chloride were

dissolved in 900 ml of water. pH of the solution was adjusted to 7.0 with NaOH and
then the volume was made up to 1 liter. The medium was autoclaved for 15 minutes at
15 psi and 122°C in an autoclave.

Preparation of YT Medium (1liter, pH 7.0)

1. Powder YT of 15.5 gm dissolved in 900 ml deionized water. NaOH was used to

adjust the pH of the solution to 7.0. The medium was autoclaved for 15 minutes at 15
psi and 122°C in an autoclave. Medium was stored at 4oC for future use.

2. Tryptone of 16 gm, 10 gm of Yeast extract and 5gm of sodium chloride were

dissolved in 900 ml of water. pH of the solution was adjusted to 7.0 with NaOH and
23
then volume made upto 1 liter. The medium was autoclaved for 15 minutes at 15 psi
and 122°C in an autoclave.

Preparation of LB agar plates

LB medium was prepared as mentioned above and 1.5% agar was added before
autoclaving. After autoclaving medium was cooled to 55oC and antibiotics were added
to the medium. Solidified plates were stored at 4oC for future use.

Antibiotic solution

Ampicillin (100 mg/ml)

Ampicillin of 1 g was dissolved in 10 ml of sterile deionized water. The solution was

filter sterilized through 0.22 µm filter (Millex) and aliquots of 1 ml were stored in -
20oC. Working concentration of ampicillin was 100 µg/ml.

Kanamycin (50 mg/ml)

Kanamycin monosulfate of 500 mg (m.w. = 582.60) was dissolved in 10 ml of sterile

deionized water and filter sterilized through 0.22 µm filter. Aliquots of 1 ml were
stored in –20oC for future use. Working concentration of Kanamycin was 50 µg/ml.

Tetracycline (12.5 mg/ml)

Free base form of tetracycline of 25 mg was dissolved in 10 ml of absolute ethanol and

aliquoted and stored in aluminum foil wrapped microcentrifuge tubes at -20oC for
future use. Working concentration of tetracycline was 12.5 µg/ml.

DNA buffers

Preparation of 1 M Tris (pH 8.0)

Tris base of 12.11 gm was dissolved in 60 ml of water. pH of the solution was

adjusted to 8.0. Then the solution was allowed to cool before making up the volume to
100 ml and autoclaved.

24
Preparation of 0.5 M EDTA (pH 8.0)

EDTA of 18.61 gm was added to 70 ml of sterile water. The solution was stirred
vigorously in a magnetic stirrer and NaOH pellets were added to adjust the pH of the
solution to 8.0. Approximately 8 gm of NaOH pellet required to dissolve the EDTA
completely. The volume was adjusted to 100 ml, and the solution was autoclaved.

Preparation of 10% SDS (pH 7.2)

To make 1 liter of 10% SDS (pH 7.2), 100 gm of electrophoresis grade SDS was
dissolved in 900 ml of water. The solution was heated at 68oC to dissolve the SDS
completely. The pH of the solution was adjusted to 7.2 by adding a few drops of
concentrated HCl and then volume was adjusted to 1 liter. The solution was then
dispensed into aliquots.

3 M Sodium acetate (pH 5.2)

Anhydrous Sodium acetate of 24.6 gm was dissolved in 80 ml of sterile water and pH

was adjusted to 5.2 with glacial acetic acid. The volume was made upto 100 ml and
sterilized by autoclaving.

6X DNA gel loading buffer

Bromophenol blue (0.25%) and glycerol (30%) was dissolved in 10 ml of water and
stored at 4°C.

50X Tris–acetate buffer

Stock of 50X TAE (Tris-Acetate-EDTA) buffer was prepared by dissolving 242 gs of

Tris base, 57.1 ml glacial acetic acid and 100 ml of 0.5 M EDTA (pH 8.0) in water.
The volume of water was adjusted and sterilized by autoclaving.

100 mM CaCl2 solution

To prepare 100 mM CaCl2 solution, 0.73 g of CaCl2 salt was dissolved in 40 ml of

water. The volume was made up to 50 ml, and sterilized by autoclaving. The solution
was stored in 4°C.

25
Tris saturated Phenol

Phenol was melted at 68oC in water bath. To 50 ml of melted phenol, 8-

hydroxyquinoline was added to final concentration of 0.1%. This compound imparts
yellow colour to organic phase and act as antioxidant, partial RNase inhibitor and
weak chelator of metal ions.

The liquid phenol was then extracted several times by vigorous shaking at room
temperature with equal volumes of 1.0 M Tris-HCl (pH 8.0) followed by 0.1 M Tris-
HCl (pH 8.0) at room temperature. The aqueous layer was removed carefully and the
extraction procedure continued until the pH of the aqueous phase was >7.6. Extracted
phenol can be stored at 4°C at 0.1 M Tris-HCl or equilibration buffer for months.

X-gal

Amorphous X- gal dissolved in Dimethyl formamide to make 40 mg/ml of stock and

stored in 4°C for long time.

RNase A (10 mg/ml)

Pancreatic RNase A of 10 mg was dissolved in 1 ml of 10 mM sodium acetate (pH

5.0) and kept in boiling water bath for 15 minutes to inactivate any contaminating
DNase.The pH of the solution was adjusted to 7.5 with 1 M Tris-HCl. Aliqouts are
stored at -20°C for future use.

Ethidium Bromide (10 mg/ml)

Amorphous form of EtBr of 1 g was dissolved in 100 ml of sterile water and stored in
room temperature.

Alkaline Lysis buffers for mini preparation of Plasmid DNA

Preparation of solution I
To make solution I, 25 mM Tris-base (pH 8.0) was mixed with 10 mM EDTA (pH 8).

26
The volume was made up to 100 ml with sterile water. The solution was autoclaved
for 15 minutes at 10 psi and stored at 4oC.

Preparation of solution II

Solution II was prepared fresh every time prior to plasmid DNA isolation. To make
fresh solution II, 0.2 N NaOH (freshly diluted from 10 N stocks) was added to 1%
SDS (from 20% SDS stock). The volume made with distilled water and mixed
thoroughly several times.

Preparation of solution III

Potassium acetate solution of 5 M was prepared dissolving 49.07 g of potassium

acetate in 70 ml of distilled water. The volume was made up to 100 ml, after the salt
dissolved completely. The stock solution was sterilized by autoclaving. 11.5 ml of
Glacial acetic acid added to 60 ml of 5 M potassium acetate solution. pH of the
mixture was checked to be of 4.8, and the final volume was made up to 100 ml by
adding distilled water. The solution was stored in 4°C for future use. Over saturated
solution of Ammonium acetate in water also used as solution III sometimes.

Protein buffers

30% Acrylamide solution (Sol A)

SDS-PAGE protein gel was prepared using 30% acrylamide/bisacrylamide stock

solution. Acrylamide saliter of 29.2 g and 0.8 g of N, N- methylenebisacrylamide was
dissolved in sterile water and the volume was adjusted to 100 ml. The solution was
stored in dark bottle at 4°C.

4X Resolving gel buffer (Sol B)

Tris-HCl solution of 1.5 M (pH 8.8) and 0.4% of SDS was dissolved and volume was
adjusted with water. The solution was stored at 4°C.

4X stacking gel buffer (Sol C)

Tris-HCl (pH 6.8) of 1 M and 0.4% of SDS was dissolved and volume adjusted with
water. The solution was stored at 4°C.

27
10% ammonium persulfate

Ammonium persulfate of 0.5 gm was dissolved in 5 ml of water. The solution was

stored at 4°C.

TEMED

TEMED was obtained commercially from Sigma as liquid and stored at 4°C.

1X Tris-glycine gel running buffer

Tris base of 3 gm (25 mM), 14.4 gm of glycine (192 mM) and 1gm SDS (0.1%)
dissolved in water and volume made up to 1 liter. pH was found to be 8.3 without any
adjustment.

5X Sample buffer

Tris-HCl (pH 6.8) solution of 60 mM final concentration, 25% Glycerol, 2% SDS,

0.1% Bromophenol blue, and 14.4 mM of 2-mercaptoethanol were mixed. The
solution was stored at 4°C.

Coomassie brilliant blue Staining solution

Coomassie Brilliant Blue R250 of 1 gm was dissolved in 450 ml of methanol, 450 ml

water and 100 ml of glacial acetic acid. The solution was filtered through filter paper
and stored at room temperature.

Destain solution for Coomassie brilliant blue stained gel

To prepare 1 liter destain solution, 100 ml methanol dissolved in 100 ml glacial acetic
acid and 800 ml water and stored in dark bottle at room temperature.

Electrodialysis buffer

Ammonium hydrogen carbonate and SDS was dissolved in 1liter of distilled water to
the final concentration of 15 mM and 0.025% (w/v) respectively.

28
Silver staining buffer

Fixing solution

Methanol and formalin of 40% and 13.5% of final volume was mixed and final
volume was made 250 ml by adding distilled water.

Sodium thiosulphate solution

Sodium thiosulphate of 0.05 gm was dissolved in 250 ml distilled water to make the
final stock of 0.02%.

Silver nitrate solution

Silver nitrate of 0.25 gm was dissolved in 250 ml water to make the final stock 0.1%.

Developing solution

To prepare 250 ml of fresh developing solution, 3% Sodium carbonate, 0.05%

formalin and 0.000016% sodium thiosulphate solution was mixed.

Stopping solution

Stopping solution was prepared by adding 6 gm of Citric acid in 12.5 ml of water to

make 2.3 M final stock.

Phosphate Buffered Saline (1X PBS) (pH 7.2, 1 liter)

To prepare 1X PBS solution, 8 gm of NaCl (137 mM), 0.2 gm of KCl (2.7 mM), 1.44
gm of Na2HPO4 (dibasic anhydrous) (10 mM), and 0.24 gm of KH2PO4 (monobasic
anhydrous) (2 mM) were mixed in 900 ml of water. Final pH was adjusted to 7.2 with
HCl and then final volume of water was adjusted to 1 liter. The solution was
autoclaved and stored at room temperature.

Western blotting buffer

All the buffers were prepared fresh and used in chilled condition.

29
Equilibration buffer

Tris base of 3gm and 14.4 gm of Glycine were dissolved in distilled water and the
final volume was made up to 1000 ml.

Transfer buffer

To 1000 ml of equilibration buffer 200 ml methanol was added and the buffer was
kept in 4°C till further use.

Blocking buffer

To prepare 3% BSA (w/v) solution 0.3 g of BSA was dissolved in 10 ml of PBS.

Washing buffer (PBS-T)

Tween-20 (0.01%) was dissolved in 1000 ml of 1X PBS (pH-7.2).

Primary antibody solution

Polyclonal or monoclonal Primary antibody was raised in rabbit or brought from the
supplier respectively and diluted in 1X PBS at 1:1000 ratio.

Secondary antibody solution

Protein A-HRP or Anti Rabbit IgG conjugated with HRP was obtained from the
supplier and diluted in 1X PBS at 1:1500 ratio.

Detection solution

Supplied DAB of 1 mg was dissolved in 10 ml of supplied dilution buffer and

detection was done by hydrogen peroxide.

Immunostains

4% PFA

Double distilled water of 80 ml heated to 55-65°C in beaker with a stirrer.

Paraformaldehyde powder of 4 gm was mixed and the solution was stirred until all the
paraformaldehyde gets dissolved. Few drops of 10N NaOH were added to clear the
milky solution. The solution was cooled and 10 ml of 10X PBS was added to make up
30
the final volume 100 ml. Aliquots of 10 ml was prepared in 15 ml centrifuge tubes and
stored in -20°C freezer.

DAPI

To make a 5 mg/mL stock of DAPI, contents of one vial (10 mg) was dissolved in 2
ml of deionized water or dimethylformamide. Aliquots of 200 µl was prepared and
stored in -20°C

Phalloidin-FITC and Phalloidin TRITC

Stock solutions of Phalloidin conjugates have been made in methanol or DMSO at 0.1
to 5 mg/ml. Aliquots was prepared and kept in -20°C. Final dilutions made in aqueous
physiological buffers.

ConA-Texas Red and ConA-Alexa fluor 488

Stock solutions was made 1–5 mg/ml in 0.1 M sodium bicarbonate (approximate pH
8.3) and stored in -20°C in aliquots.

Polyclonal Anti Ei Chitinase 2 Antibody

Polyclonal anti EiChit2 antibody was raised in a rabbit following the protocol of 3
boosters and 3 collections. After final booster, 15 ml blood was collected from the
heart of the rabbit and the serum was stored in -20°C.

Anti Rabbit IgG conjugated with TRITC

Anti rabbit IgG solution was kept in 2-8°C and diluted at 1:500 times in 1X PBS.

1 M Sodium di-hydrogen Phosphate and 1M Disodium hydrogen phosphate

Buffer

Stock solutions of 1 M of NaH2PO4 (monobasic) and 1 M of Na2HPO4 (dibasic) were

prepared by dissolving 138 gm of NaH2PO4.H2O (monobasic, 138 g/mole) and 142 gm
of Na2HPO4 (dibasic, anhydrous, 142 g/mole) in sufficient water to make a final
volume of 1 liter. The stocks were stored at room temperature.

31
200 mM Sodium phosphate buffer (pH 6-8)

Sodium phosphate buffers of 200 mM of different pH were prepared using required

amount of 1M sodium monobasic and 1M sodium dibasic solution after calculating the
pKa.

200 mM sodium acetate (pH 3-5)

Sodium acetate trihydrate and glacial acetic acid were dissolved in water to obtain
different pH after pKa calculation.

Buffers for Ni-NTA chromatography

Lysis buffer for protein purification under native condition (pH 8.0)

Lysis buffer or sonication buffer was prepared by adding 6.9 gm of NaH2PO4.H2O (50
mM), 17.54 gm NaCl (300 mM), and 0.68 gm imidazole (10 mM) in double distilled
water. The solution pH was adjusted to 8.0 with 10N NaOH.

Wash buffer (pH 8.0)

To prepare 1 liter of washing buffer, 6.9 gm of NaH2PO4.H2O (50 mM), 17.54 gm of

NaCl (300 mM), and 1.36 gm of imidazole (20 mM) was dissolved in water. The pH
was adjusted to 8.0 with 10N NaOH.

Elution buffer (pH 8.0)

To prepare 1 liter of elution buffer, 6.9 gm of NaH2PO4.H2O (50 mM), 17.54 gm of

NaCl (300 mM), and 17 gm of imidazole (20 mM) was dissolved in water. The pH
was adjusted to 8.0 with 10N NaOH.

Regeneration Buffer F for Ni-NTA column

The solution was consisting of 6 M Guanidium hydrochloride and 0.2 M acetic acid
dissolved in required amount of sterile water. The solution was stored at room
temperature.

32
Sodium azide (0.02%)

Sodium azide of 20 mg was dissolved in 100 ml of sterile water. The solution was
used for storing columns.

1 M Dithiothreitol (DTT)

DTT (DL-DTT, anhydrous, m.w. = 154.25) of 1.5 gm was dissolved in 8 ml of

deionized water. The volume is adjusted to 10 ml and aliquots of 1 ml were dispensed
in microcentrifuge tube. The tubes were wrapped in aluminum foil and stored in -20°C.

Bovine Serum Albumin (1mg/ml)

BSA (fraction V, Sigma) of 1 mg was dissolved in 1 ml of sterile water. The solution

was stored at -20°C for future use.

100 mM IPTG

Amorphous saliter of IPTG (mol. wt. 238.3 g/mole) of 238 mg was dissolved in 10 ml
of sterile water and sterilized by 0.22 µm disposable filter. Aliquots of 1 ml are made
and stored at -20°C. Final concentration for induction was used as 1 mM or lower than
that.

Colloidal chitin

Chitin powder was obtained from the suppliers, and incubated with concentrated HCL
at 4°C for overnight with continuous stirring. The supernatant was mixed drop-wise
with 50% chilled ethanol. The supernatant was washed vigorously with distilled water
until the pH became neutral. The colloidal material was freeze dried and stored.

33
Entamoeba Cell culture medium

Complete TYIS-33 medium

To prepare 1000 ml of complete medium, 100 ml of Adult Bovine Serum (10%), 30

ml of premixed Vitamin mixture (3%), 10 ml of antibiotic solution (1%) were added to
870 ml of TYI broth. The total mixture was filtered through 0.2 µM filters.

Adult Bovine serum

Triple filtered adult bovine serum was obtained from the suppliers. Heat inactivation
was done by incubating the serum in 65°C for 30 min. The serum was then cooled and
kept in 4°C in aliquots. To prepare 1000 ml of TYI broth, Bacto peptone of 30 gm,
Glucose of 10 gm, NaCl of 2gm, L-Cysteine Hydrochloride of 1gm, Di-Potassium
hydrogen phosphate of 1gm, Potassium Di-hydrogen phosphate of 600 mg, Ascorbic
acid of 200 mg and 1 ml of Ferric ammonium citrate (22.8 mg/ml) were dissolved in
500 ml of double distilled water and pH was adjusted to 6.8 using 10N NaOH. Final
volume was made up to 870 ml and sterilized by autoclaving for 15 min. The
incomplete medium was used for preparing complete medium, and stored in 4°C.

Complete Vitamin Tween-80 Mixture

To prepare 1200 ml of complete vitamin, 12 ml of Vitamin B12 (40 mg/ml), 4 ml of

Thioctic acid (dissolved in ethanol to prepare 1mg/ml solution) and 4ml of Tween-80
were added to 1000 ml of incomplete Vitamin Mixture (NCTC 107). The final mixture
was filtered through 0.2µm filter. The filtered solution was kept in -20°C.

Incomplete Vitamin mixture (NCTC 107)

Different compositions named as Sol I, Sol II, Sol III, Sol IV and Sol V were prepared.

Sol I- 25ml of part I, 25 ml of part II and 10 ml of part III was dissolved in 40 ml of

water.

Part 1 was prepared by adding 25 mg of p-aminobenzoic acid and 12.5 mg of Niacin

in boiling distilled water and the final volume was made to 25ml.

34
Part II was prepared by mixing 12.5 mg of Niacinamide, 12.5 mg of Pyridoxine-HCl,
12.5 mg of Pyridoxal-HCl, 5 mg of Thiamine-HCl, 5 mg of Ca-pantothenate, 25 mg of
Myo-inositol, and 250 mg of Choline chloride to double distilled water and the final
volume made up to 25 ml.

Part III was prepared by adding 10 mg of Riboflavin was mixed in 10 ml of distilled

water. Drop wise 0.1N NaOH was added to dissolve the substrate and the final volume
was made up to 20 ml.

Sol II- 10 mg of D-Biotin and 1ml of 1N HCl was added to double distilled water and
the final volume was made to 100 ml.

Sol III- 10 mg of Folic acid was added to 100 ml of distilled water (80°C).

Sol IV- 10 mg of Calciferol (Vit D), 10 mg of Vitamin A, 2 mg of Menadione (Vit K)

were added to 50 ml of distilled water and mixed with 10 ml of 5% (v/v) aqueous
solution of Tween-80. The final volume was made up to 100 ml.

Sol V- 10 mg of Tocopherol acetate (Vit E) was added to 100 ml of distilled water.

Stock solutions of sol I, sol II, sol III, sol IV, and sol V were prepared at first and
added in following amount to prepare the NCTC mixture, Sol I: Sol II: Sol III: Sol IV:
Sol V:: 2: 1: 1: 10: 1 part.

Encystation medium

To prepare 1000 ml of complete encystation medium, 50 ml of Adult Bovine Serum

(5%), 30 ml of Premixed Vitamin mixture (3%), 10 ml of antibiotic solution (1%)
were added to 910 ml of LG broth. The total mixture was filtered through 0.2 µM
filter. To prepare 1oo ml of LG medium Bacto peptone of 14.1 gm, L-Cysteine
Hydrochloride of 470 mg, Di-Potassium hydrogen phosphate of 470 mg, Potassium
Di-hydrogen phosphate of 282 mg, Ascorbic acid of 94 mg and 470 µl of Ferric
ammonium citrate (22.8 mg/ml) were dissolved in double distilled water and pH was
adjusted to 6.8 with 10 N NaOH. Final volume was made up to 910 ml and sterilized
by autoclaving for 15 min, and stored in 4°C.

35
Cryopreservation solution

Axenic culture medium base (TYI) and serum (heat inactivated bovine serum) were
mixed in same volume (1:1) to prepare the Solution I. Cell culture grade DMSO of
0.75 ml was mixed to 5 ml of this solution I, to reach the final concentration of 15%
and the mixture was termed as solution II.

2.1.10 Kits

Genomic DNA isolation kit was obtained from Promega (USA) and used for
Entamoeba genomic DNA isolation. Total RNA isolation kit or TRI Reagent solution
was obtained from Ambion (USA). Plasmid DNA miniprep kit was obtained from
Promega. QIA quick Gel extraction kit was brought from Qiagen (Germany).
Retroscript or the cDNA synthesis kit was purchased from Ambion (USA).

2.1.11 Vectors

TA vector was used to facilitate one step cloning after PCR amplification. Blue-white
selection was used for preliminary screening of recombinant clones.

Vectors from two different sources were used in this study and their genetic structure
and specificity were described below.

pGEM-T TA cloning vector (Promega)

pGEM-T system is very convenient for one step PCR product cloning. Originated
from pGEM-5Zf (Promega, USA), this vector contains one 3’ terminal thymidine to
both ends (Fig. 2.1).

pGEM-T vector contains T7 and SP6 RNA polymerase promoters flanking MCS,
within the α peptide-coding region of the enzyme β-galactosidase, and the origin of
replication of the filamentous phage f1. Insertional inactivation of α-peptide allows
colour screening of recombinant clones. The pGEM-T vector contains multiple
restriction sites within the MCS for easy release of cloned fragment. The vector is of 3
kb molecular weight, and ampicilline resistant gene was incorporated as antibiotic
marker.
36
 Fig. 2.1: Schematic diagram and sequence map of pGEM-T vector (Promega manual).

pTZ57R/T TA cloning vector (Fermentas, USA)

This vector represents the same basic mechanism and facility for one step PCR
product cloning (Fig. 2.2).

Fig 2.2: Schematic diagram and sequence map of pTZ57R/T vector (Fermentas manual)

37
Commercially obtained from Fermentas, this plasmid is informally known as pFERM.
The vector is of 2.8 kb, and contains f1 replication origin. The MCS is flanked by α-
peptide coding region and T7 promoter. β-Lactamase gene (bla) confers the
ampicilline resistance. The linear vector contains two 3’ thymidine at the open ends
for insert binding. Multiple restriction sites are available within MCS for insert
release. Blue-white screening was used for preliminary clone identification.

pQE30 vector (Qiagen, Germany)

Over expression of heterologous protein within E. coli system generally done by pQE
vector system commercially obtained from Qiagen (Fig.2.3). 6xHis-tagged protein was
over expressed using T5 promoter transcription-translational system.

Fig.2.3: Schematic diagram and sequence map of pQE30 vector (Qiagen Handbook)

Primarily originated from pDS family of plasmids, this group of vector emerges after
several derivations and weights 3.4 kb. Phage T5 promoter and two lac operator
ensure efficient repression of expression in absence of induction, and resulted in
strongly controlled expression. 6xHis-tag coding sequence attached to N terminal of
cloned sequence. MCS and translational stop codons were available in all reading
frames, flanked by several restriction sites. Origin of replication belongs to ColE1, and
β-lactamase gene confers the ampicillin resistance. Strong ribosomal binding site

38
ensure high translation rate, and dual transcriptional terminator ensure the expression
stability.

2.2 Microbiological methods

All microbiological methods were done by following Sambrook and Russell (2001) if
not stated otherwise.

2.2.1 Growth of bacterial culture

The bacterial culture with/without plasmid was grown overnight in L.B or in YT

medium supplemented with required antibiotics at 37°C at 250 r.p.m.

2.2.2 Preparation of competent cell

For the transformation purpose competent cells were prepared by using calcium
chloride method. Overnight grown bacterial culture of 200 µl was used as innoculum
to 20 ml of native LB and incubated for 3-4 hr at 37°C to reach the logarithmic phase.
After 3-4 hr, the O.D of the culture media reached 0.5-0.6, which indicates the log
phase of bacterial growth. The bacterial solution was kept in ice for 5 min and then
centrifuged at 5000 r.p.m. for 5 min at 4oC. After the centrifugation, the supernatant
was removed and the bacterial pellet was suspended in 3 ml of ice cold 100 mM
calcium chloride and kept in ice for 45 min. Exposure to calcium ions enables the cells
to uptake DNA or to become “competent”. Thereafter centrifugation was done again at
5000 r.p.m. for 5 min at 4°C. Supernatant was discarded and bacterial pellet re-
suspended in 1 ml of 100 mM calcium chloride solution. Dissolution of the pellet
slowly gives rise to competent cell.

For long time storage, the cells were dissolved in 1 ml of 100 mM cold calcium
chloride: glycerol (85:15) solution. Suspensions of 50 µl were stored in sterile
microcentrifuge tubes, flash frozen in liquid nitrogen and stored at -70oC for future
use. Such frozen cells retain their competency for months.

39
2.2.3 Transformation

The process of uptake of exogenous DNA by competent cell is termed as

transformation and is achieved by heat shock method in this study. Competent cells of
50 µl were mixed with required amount of ligation mix. The cells are then kept in ice
for 45 min to chill them without disturbance. Heat shock was given at 42oC in a water
bath for 2 min and the cells were again kept in ice for 10 min. Then 950 µl of fresh
L.B was added to the cells in the microcentrifuge tube and incubate in shaker-
incubator for 1 hr at 37°C. This stage helps the cells to grow in non-selective media
for synthesis of antibiotic resistant genes. After 1 hr the microcentrifuge tubes were
taken out, centrifuged briefly and 60 µl of supernatant was plated on L.B-agar plates
with required antibiotic. Cells were spread with help of spreader and the plates were
incubated at 37oC overnight for the colonies to grow.

2.2.4 Selection of blue-white colonies

The rationale behind the blue white screening is the disruption of the lacZ gene
responsible for the β-galactosidase enzyme and unavailability of the enzyme for X-gal
degradation. With the cloning of insert in the MCS site, the gene gets disrupted and
the absence of β-galactosidase enzyme resulted in the white colonies on the X-gal
coated plate, where the non-recombinant clones produce the enzyme and degrade the
substrate forming blue colored colonies. The white colonies were picked up and grown
in 3 ml LB medium supplemented with 100 µg/ml of ampicillin or required
antibiotics.

2.2.5 Bacterial Cell storage

Once the right clone was detected, glycerol stock was made for long term storage.
Single colony was isolated and grown for overnight in 1 ml of LB medium containing
required antibiotic at 37°C with vigorous shaking at 250 rpm. 800 µl of such overnight
grown culture was mixed with 200 µl of sterile glycerol (20% v/v) and vortexed
briefly to mix the contents well. The cryovials were flash frozen in liquid nitrogen and
stored at -70oC for future use.

40
2.3 Molecular Biology methods

All molecular biology methods were done by following Sambrook and Russell (2001)
if not stated otherwise.

2.3.1 Genomic DNA isolation

50 ml culture flasks with log phase Entamoeba cells were chilled with ice and cells
were harvested and transferred in to 1.5 ml microcentrifuge tube. Cells were pelleted
down by centrifugation at 1500 r.p.m. for 3 min. The genomic DNA was isolated
following the manufacturer’s protocol. Briefly the supernatant were removed and 200
µl PBS was added to wash the cells. The cells were re-suspended in 1xPBS followed
by the addition of 600 µl Nuclei Lysis solution to lyses the cells. 3 µl of RNase
Solution was added to the mixture and the sample was mixed by inverting the tube 2-3
times. The mixture was incubated for 15-30 min at 37°C. The sample was cooled to
room temperature for 5 min before proceeding further. To the mixture, 200 µl of
Protein precipitation solution was added and vortex vigorously at high speed for 20 s.
The sample was chilled on ice for 5 min. The mixture was centrifuged for 4 min at
13,000×g. The precipitated protein forms a tight white pellet. The supernatant
containing the DNA was removed carefully and transfer it to a clean 1.5 ml
microcentrifuge tube. 600 µl of isopropanol was added to the solution and gently
mixed by inversion until the white thread-like strands of DNA form a visible mass.
The tube was centrifuged for 1 min at 13,000×g at room temperature. The DNA was
precipitated as small white pellet. The supernatant was decanted carefully and washed
with 600 µl of 70% ethanol. The tube was centrifuged for 1 min at 13,000×g at room
temperature. The ethanol was carefully aspirated and the DNA pellet was air-dried for
10-15min. 100 µl of DNA rehydration Solution was added to rehydrate the DNA by
incubating at 65°C for 1 hr. The DNA was stored at 2-8°C.

2.3.2 Total RNA isolation

The culture flasks containing Entamoeba cells were chilled by incubating them in ice,
and the cells were pelleted down, by centrifugation at 1500 r.p.m. for 3 min. The total
RNA was isolated following the manufacturer’s protocol. Briefly 1 ml of TRI Reagent
41
solution per 5-10 x 106 cells was added and incubated for 5 min at room temperature.
The mixture was centrifuged at 12,000x g for 10 min at 4°C and the supernatant was
transferred to a fresh microcentrifuge tube. 200 µl of chloroform per 1 ml of TRI
Reagent solution was added, mixed well, and incubated at room temp for 5-15 min.
The tube was centrifuged at 12,000 x g for 10-15 min at 4°C, and the aqueous phase
was transferred to a fresh tube. 500 µl of isopropanol per 1 ml of TRI Reagent solution
was added to the aqueous solution and mixed well for 5-10 s, followed by incubation
at room temp for 5-10 min. The tube was centrifuged at 12,000 x g for 8 min at 4-
25°C, and the supernatant was discarded. 1 ml of 75% ethanol per 1 ml of TRI
Reagent solution was used to wash the pellet. The ethanol was washed away by
centrifugation at 7,500 x g for 5 min. The RNA pellet was briefly air dried and
dissolved in nuclease free water. Isolated RNA was stored in -20°C.

2.3.3 Plasmid isolation

Small scale and large scale plasmid isolation was done by alkaline lysis method using
the standard protocol. For small scale isolation of plasmid DNA, 3 ml of full grown
bacterial culture was used. The cells were grown overnight for 16 hrs at 37oC in L.B
medium supplemented with appropriate antibiotics at required concentrations at 250
r.p.m. The cells were pelleted down by centrifugation at 5000 r.p.m. for 5 min.
Supernatant was removed completely and the bacterial pellets were re-suspended
completely in 200 µl of cold solution I. The cells were then vortex well to loosen the
cell pellet so that each and every cell gets exposed to lysis solution. 400 µl of freshly
prepared solution II was added. SDS from sol II solubilize the phospholipid and
protein components of cell membrane leading to cell lysis and release cell components
and denatures the chromosomal and plasmid DNA. The solution was mixed properly
by inverting the microcentrifuge tube slowly (to avoid shearing of chromosomal
DNA) and kept in ice for exactly 5 min which allows maximal release of plasmid
DNA from the cell. The cell lysate was then neutralized by the addition of 300 µl of
solution III and the tubes were inverted slowly several times and kept in ice for 10
min. Here the high salt content precipitates the denatured protein, chromosomal DNA
and cell debris forming insoluble salt-detergent complex. Plasmid DNA being circular
and covalently closed renatures quickly and remains in solution.

42
To separate out the plasmid DNA, the tubes were centrifuged at high speed of 12,000
r.p.m. for 10 min. Supernatant was saved in a new sterile microcentrifuge tube and
equal volume of isopropanol was added. The microcentrifuge tube was allowed to
stand in room temperature for 20 min followed by centrifugation at 10,000 r.p.m. for
10 min. Supernatant was discarded and 200 µl of 70% cold ethanol was added to wash
the pelleted plasmid DNA. Ethanol precipitates out intact nucleic acid molecules. The
residual ethanol was removed by brief centrifugation, pellet was air dried and then
dissolved in required volume of sterile water. RNaseA (5mg/ml) was added to the
solution to a final concentration of 20 µg/ml and the solution was incubated at 37°C in
a water bath for 30 min.

Equal volumes of Tris saturated phenol: chloroform (1:1 v/v) was added to the RNase
treated solution, thoroughly mixed by vortexing and centrifuged at 10,000 r.p.m. for
10 min. Phenol being denser than water remains below where the nucleic acids remain
in the upper aqueous layer. After centrifugation the upper aqueous layer was slowly
pipetted out into a sterile microcentrifuge tube, without disturbing the double layer of
phenol and aqueous material. The same procedure was repeated again with equal
volume of chloroform and the upper aqueous layer was collected. The aqueous layer
containing the DNA was precipitated out with 1/10 volume of 3 M Sodium-acetate
and 2.5 times of dehydrated alcohol. The solution was kept at -20°C and then
centrifuged at 10,000 r.p.m. for 10 min to pellet plasmid DNA. Supernatant was
discarded and 200 µl of 70% cold ethanol was used to wash pelleted DNA. The
residual ethanol was removed by brief centrifugation.

The DNA pellet was air dried completely to make it absolutely ethanol free. Then
plasmid was dissolved in required amount of autoclaved distilled water. For large
scale isolation of plasmid DNA, cells were harvested from 250 ml LB/YT medium
supplemented with required antibiotics at 37°C following the same procedure with
scaled up solution.

2.3.4 cDNA synthesis

From the isolated total RNA 1-2 µg was used for cDNA synthesis by following the
manufacturer’s protocol. Briefly in a typical reaction of 20 µl, sequentially 2 µl Oligo

43
(dT), 2 µl 10X RT Buffer, 4 µl dNTP mix, 1 µl Placental RNase Inhibitor, 1 µl
Reverse Transcriptase were added. The final volume was made up to 20 µl by adding
Nuclease-free Water. The mixture was mixed gently centrifuged briefly and incubated
at 44°C for 1 hr followed by 92°C for 10 min to inactivate the Reverse Transcriptase.
The reaction mixture was stored at -20°C before proceeding to the PCR.

2.3.5 RT-PCR

RT-PCR was carried out by using gene specific primers designed to amplify a small
region (~450bp) of the gene of interest. A typical RT-PCR mixture of 50 µl was
prepared containing 1-5 µl RT reaction mixture, 5 µl 10X PCR buffer, 2.5 µl dNTP
mix, 2.5 µl PCR primers, 1 unit Taq DNA Polymerase and nuclease free water. The
reaction was carried out for a cycle of 94°C for 2- 4 min, 30 cycles at 94°C for 30 s,
55°C for 30 s, 72°C for 40 s and then by a final extension at 72°C for 5 min. Positive
and negative controls were done by using Actin template and cDNA mixture
respectively.

2.3.6 PCR amplification from genomic DNA

PCR amplification of gene of interest was completed by using gene specific primers.
For cloning purpose Hi-fidelity Expand Taq polymerase was used, while Taq
polymerase was used otherwise.

In a typical reaction of 50 µl, 20 ng of template DNA, 0.5 unit of Taq polymerase, 0.5
µl of dNTP mixture (100X stock solution; 10 mM of each), 20 pmole of sense and
antisense primers, 5 µl of Hi fidelity Expand buffer or PCR buffer (10X stock
solution) were used, and the rest of the volume was made up to by adding autoclaved
PCR water. The whole PCR reaction was carried out for a cycle of 95°C for 5 min, 35
cycles at 95°C for 30 s, 55°C for 30 s, 68°C for 60 s (72°C for Taq) (60 s for every
1000 bp long fragment) and then by a final extension at 72°C for 5min.

2.3.7 Restriction enzyme digestion

All restriction enzyme digestion was done by following supplier’s instructions.

44
In a standard reaction mixture of 100 µl containing 10 µg of plasmid DNA, 1 µl of
required enzymes were added (1unit/100 µl). Required buffer (10X stock) of 10 µl and
BSA (100X stock) of 1 µl (if suggested) were added. The final volume was made up
by autoclaved water. Both double and sequential digestions were carried out by
incubating the reaction mixture for 3 hr at 37°C water bath. In the situation of double
digestion, the buffer and enzymes of previous digestion were removed by alcohol
precipitation. After the completion of first digestion 1/10 times ice cold 3 M sodium
acetate and 2.5 times of dehydrated alcohol was added to the reaction mixture. After
20 min incubation at -20°C, the reaction mixture was centrifuged at 10,000 r.p.m. for
10 min. Further washing was done by adding 200 µl ice cold ethanol and
centrifugation for 10 min at 10,000 r.p.m. The pelleted DNA was air dried and
dissolved in distilled water and treated further with the next restriction digestion
enzyme, with high salt containing buffer.

2.3.8 Agarose gel electrophoresis of DNA and RNA

To visualize the isolated DNA or RNA, agarose gel electrophoresis was done which
helps to separate the oligonucleotide fragments. To cast 0.8% agarose gel, 0.4 g of
molecular grade agarose was dissolved in 50 ml water containing 0.01%
electrophoresis buffer. The agarose was then cooled down to 60°C, and Ethidium
bromide was added to a final concentration of 0.5 µg/ml. The melted agarose solution
was poured in the pre-assembled electrophoresis tray with the comb in proper position
and the gel was allowed to solidify. After solidification, the comb was removed
carefully and the gel was mounted in an electrophoresis tank containing enough 1X
TAE buffer to cover the gel completely. The DNA/RNA samples were mixed well
with the 6X loading dye and then slowly loaded in to the slots of the submerged gel
using micropipettes. The gel was run at 70 volt until the bromophenol blue dye
migrated to about 1 cm from the bottom of the gel. The ethidium bromide stained gel
was then observed using an UV Transilluminator.

2.3.9 Purification of the gel eluted samples

To elute DNA from agarose gel QIA quick Gel Extraction kit (QIAGEN) was used.

45
The kit also used to cleans up DNA from enzymatic reactions. After the gel
electrophoresis, DNA fragment was excised from the agarose gel with a sterile sharp
blade. The gel piece containing the DNA was weighed in clean microcentrifuge tube
and thrice volume of QG buffer was added per amount of gel weight (100 mg of
gel~300 µl). The solution was incubated at 50°C for 10 min and vortex vigorously to
dissolve the gel completely.

The QIA quick spin column was placed in a 2 ml collection tube and centrifuged at
10,000 r.p.m. for 1 min, which allows binding of the DNA to the membrane. Flow
through was discarded and 750 µl of ethanol mixed PE buffer was added to QIA quick
column and centrifuged at 10,000 r.p.m. for 1 min. This PE buffer washes the
membrane. Flow through was discarded and again centrifuged for an additional 1 min
at 10,000 r.p.m. to remove residual PE buffer.

The QIA quick column was placed in a fresh 1.5 ml microcentrifuge tube. To elute the
DNA 30 µl of autoclaved distilled water was added to the QIA quick column and then
centrifuged at 10,000 r.p.m. for 1 min. The eluted DNA was collected in the
microcentrifuge tube and it was checked by agarose gel electrophoresis.

2.3.10 Spectrophotometric estimation of DNA and RNA

DNA and RNA concentration was estimated spectrophotometrically at 260 nm by

comparing serial dilutions. The equation used is Nucleic acid concentration (µg/µl) =
Absorbance260nm x extinction coefficient x dilution value (Extinction coefficient is
0.055, 0.040, 0.033 for ds DNA, RNA and ss DNA, respectively).

Purity of nucleotide can be evaluated by ratio of A260/A280.

DNA concentration was estimated assuming that an O.D260nm of 1 corresponds to 50

µg/µL of double stranded DNA. Thus the formula stands: O.D260nm x 200 [dilution
factor] x 50 µg/ml = DNA µg/ µl.

For protein and RNase free DNA the ratio of A260/A280 should be close to 2. RNase
contamination will increase the ratio.

46
For estimating RNA, concentration was estimated assuming that an O.D260nm of 1
corresponds to 40 µg/µL of RNA. Thus the formula stands: O.D260nm x 200 [dilution
factor] x 40 µg/ml = RNA µg/ µl.

2.3.11 Ligation

For ligation with pTZ57R/T or pGEM-T, the ligation mixtures were prepared by
following the manufacture’s protocol. For ligation of the pQE30 vector and insert
molecule, the 1:5 molar ratio of vector to insert was used. With that respect the
concentration of vector and insert was taken in the ligation mix. A typical ligation
mixture contains 1 unit T4 DNA Ligase enzyme (1unit/1µl), 1µl Ligase buffer, vector
and insert to make up the final volume up to 10 µl. The reaction mixture was
incubated at 16°C for 16 hr.

2.3.12 Automated dye terminator cycle sequencing of DNA

To check the fidelity of the sequences, sequencing reaction was done using Applied
Biosystems automated DNA Sequencer. Sequencing reaction was consist of 300 ng of
purified plasmid DNA, 2.0 µl of 2.0 pico mole primers, 2.0 µl of Big DyeTM
Terminator Cycle Sequencing Ready Reaction Mix (Applied Biosystems, USA) and
sterile double distilled water to a final volume of 10 µl prepared in sterile 0.2 ml tubes.
All the additions were carried out in ice. Thermal cycling was carried out following
the ABI recommended cycling proge of 96°C for 30s, 50°C for 15 min and 60°C for 4
min. The sequence products were purified using ABI recommended ethanol
Tm
precipitation for Big Dye Terminators by adding 60 µl of 100 % ethanol to the 10
µl sequencing reaction volume. The solution remained at room temperature for a
minimum of 15 min before centrifuging at 3000 r.p.m. for 45 min using swing bucket
rotor system of refrigerated centrifuge. The supernatant was discarded by inverting the
plates followed by short centrifugation at 1000 r.p.m. for 1 min. The DNA pellet was
washed with 70% cold ethanol, briefly centrifuged and the remaining supernatant was
removed by pipette tip. The sequencing tubes were stored in dark at 4°C prior to use.
The purified DNA samples were re-susupended in 12 µl of Hi-Di-Formamide and

47
were denatured at 96°C for 5 min. The samples were then loaded to the Sequencer for
reading.

2.4 Protein methods

Both the growth of expression culture and purification of the protein were done
following the Ni-NTA Spin Handbook (Qiagen) and Protein methods (Bolleg et al.,
1996). The principle is based on IMAC principle (Immobilized Metal Affinity
Chromatography) where the poly-Histidine tag acts as the metal chelating group. The
pQE-30 expression plasmid consists of a full-length gene cloned between the BamHI
and SalI cloning sites of the expression plasmid. The gene is fused in-frame with N-
terminal peptide containing six tandem Histidine residues, and expression of the His-
tag protein is driven by a T7 RNA polymerase promoter.

2.4.1 Expression of recombinant His-tagged protein under native conditions

Recombinant pQE30 plasmid was transformed into E. coli M15 and plated on
Amp/Kana LB agar plates. Colonies were picked and inoculated in 3 ml culture for
overnight incubation at 37°C and shaking at 250 r.p.m. 25 ml of LB media was
inoculated further for protein expression study and shake vigorously at 37°C until the
O.D600nm reaches between 0.5-0.7. The recombinant protein expression was induced by
adding 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to the culture and the
incubation continued at 37°C for 5 hr. After the incubation period, cells were harvested
by centrifugation at 5000 r.p.m. for 6 min and the supernatant was discarded. Bacterial
pellet were then washed and re-suspended in 2.5 ml of 1X PBS (pH 7.2) containing 1
mM PMFS and 1 mM DTT. Cells were sonicated by using ultrasonic cell disrupter, for
15 min with 30s pulse on ice. The clear solution was centrifuged at 13,000 r.p.m. for
45 min at 4°C to precipitate out the cell debris and impurities. The supernatant was
stored separately and the pellet was washed thoroughly with 1X PBS. The pellet was
dissolved in same amount of 1X PBS and vortex briefly. To detect the solubility of the
recombinant expressed protein, samples were aliquoted from supernatant and the
pellet fraction, treated for SDS-PAGE analysis.

48
2.4.2 SDS-PAGE

For SDS-PAGE, the method of Laemmeli was followed. Bio-Rad gel apparatus was
used for gel casting. The glass plates of mini gel apparatus were cleaned and
assembled properly on a gel caster.

The ingredients Solution A (4 ml), Solution B (2.5 ml), Water (3.5 ml), 10% APS (50
µl) and TEMED (5 µl) were mixed to cast 10 ml of 12% resolving gel.

After 15 min, 5% stacking gel of 5 ml was casted using the following components
such as Solution A (0.67 ml), Solution C (1 ml), Water (2.3 ml), 10% APS (30 µl) and
TEMED (5 µl) above the resolving gel.

The comb was inserted in proper position. After 30 min of polymerization, the comb
was removed carefully and the wells were washed properly with 1X running buffer to
remove un-polymerized acrylamide. Gel was fitted into the electrophoresis tank and
1X Tris glycine electrophoresis buffer was poured into the tank to cover the vertical
glass plates. 20 µl of protein samples were boiled with 4 µl of 5X sample buffer for 3
min at 100°C, and loaded into the wells. The gel was run at 70 Volt till the protein
sample entered the resolving gel after crossing the stacking gel. Then the gel was run
at 100 Volt till the dye font is 1 cm away from the end. After completion of the run gel
apparatus was dismantled carefully and the gel was taken out. Detection of migrated
bands was done either by Coomassie blue staining or by silver staining.

2.4.3 Coomassie blue R-250 staining and destaining

Coomassie brilliant blue R-250 binds nonspecifically to almost all proteins, which
allows detection of protein bands in polyacrylamide gels. Gels are soaked in 1X
staining solution with gentle shaking in rocker for 1 hr or for longer time at room
temperature. After that gels are washed with 1X Destain solution with several changes
till the bands were visualized in naked eye.

2.4.4 Silver staining

Protein bands in SDS-PAGE gel containing less than microgram of protein were not
visible by Coomassie blue staining. Silver staining technique was done to detect nano
49
gram level of protein. The gel was soaked for 10 min in fixing solution. Washing of
the gel was done twice by water for 5 min each. Impregnation of the gel was done by
soaking it in 0.02% Na2S2O3 for 1 min. Further washing was done twice by water for
20 s. Silver staining was done by immersing the gel in 0.1% AgNO3 for 10 min. The
stained gel was rinsed with water and again with a small volume of developing
solution. For development of the stain, the gel was soaked in fresh developing solution
until band intensities are adequate (~1-3 min). 12.5 ml of 2.3 M citric acid was added
to stop the development and incubated for 10 min, followed by the washing for 10 min
in water. The developed gel was then soaked in water for 30 min or longer before
drying.

2.4.5 Purification of recombinant His-tagged protein under native conditions

After the expression profile was analyzed, protein purification was done utilizing the 6
Histidine tag at the N-terminal end of the protein sequence. The cytosolic protein was
purified following the suppliers protocol. Ni-NTA agarose beads were obtained from
Qiagen. Slurry (5%) of 1 ml volume was loaded on a glass column and washed with
water followed by the equilibration with water or 1X PBS. After the beads were
settled, the supernatant was applied on the column and the flow through was collected.
To minimize the non-specific bindings, the column was washed several times with the
wash buffer (pH-8). Washing was continued till O.D280nm reaches less than 0.1. Bound
proteins were eluted stepwise with elution buffer containing gradient of 50, 100, 200
and 500 mM imidazole. The elution was monitored by watching the O.D280nm. Initially
all the fractions were collected and stored at 4°C for further analysis by SDS-PAGE.

2.4.6 Regeneration of used column

Single column was used for at least 3-4 times for a single protein. To regenerate the
leached Ni ions, following steps were done by following the manufacturer’s protocol.
Used columns were washed with 2 column volumes of Regeneration Buffer, followed
by the wash with 5 volumes of water. 2% SDS solution of 3 volumes were used to
solubilise the excess protein, which was further gradually washed by sequential
addition of 1 volume of 25%, 50% and 75% ethanol, followed by 5 volumes of 100%
Ethanol. Further rehydration of column was done by washing with 1 volume of 75%,
50
50%, and 25% Ethanol, followed by 1 volume of water. Finally the column was
washed with 5 volumes of 100 mM EDTA (pH 8.0). Enough water was used to wash
away the residual alcohol and debris. The column was then replenished with 2
volumes of 100 mM NiSO4, and excess NiSO4 was washed with 2 volumes of water.
Finally the column was washed 2 volumes of Regeneration Buffer, and equilibrate
with 2 volumes of 1X PBS.

2.4.7 Quantification of protein

Protein in the samples was estimated by Bradford method using BSA as standard. A
standard curve of serially diluted BSA was prepared. Protein samples (2, 4, 6, 8 and 10
µl) were taken in clean test tubes. The volume was made 0.8 ml with water in all test
tubes. 0.8 ml of water in one test tube served as blank. 0.2 ml of Bradford reagent was
added to each test tube mixed well and incubated in dark for 5 minutes. The
absorbance was recorded at 595 nm. Total amount of protein in each sample was
calculated from the standard curve of BSA.

The NanoDrop® ND-1000 spectrophotometer A280 modules are used to determine

the concentration of purified protein samples. Standard graphs are not required in this
module. The Beer-Lambert equation (A = E * b * c) is used for all protein calculations
to correlate absorbance with concentration [A is the absorbance value (A), E is the
wavelength-dependent molar absorptivity coefficient (or extinction coefficient) with
units of liter/mol-cm, b is the path length in centimeters, c is the analyte concentration
in moles/liter or molarity (M)]. For this experiment a general reference setting was
used based on a extinction coefficient value of 0.1% (1 mg/ml) protein solution
producing an Absorbance of 1.0 at 280 nm (where the path length is 10 mm or 1 cm).

2.4.8 Western blotting

Western blotting was done to confirm the nature of over expressed proteins by using
monoclonal or polyclonal antibody against the over expressed protein. After running
the SDS-PAGE with pre-stained protein marker, the gel was transferred in the chilled
equilibration buffer. The PVDF membrane was cut and activated by immersing it in
chilled methanol for 10 min. In western blotting cassette, all the components were

51
placed in the following order from the cathode to anode end; sponge, whatmann filter
paper, gel, activated membrane, whatmann filter paper and sponge.

In wet transfer method the cassette was filled with transfer buffer and the transfer was
done for 20 hr at 20 m.v. After 20 hr the membrane was transferred to 3% BSA
solution and incubated overnight at 4°C or for 1 hr at room temperature with constant
rocking. Blocked membrane was further washed for three times for 10 min each with
PBS-T solution and incubated with diluted primary antibody (1:1000) for 1 hr at room
temperature with constant rocking. Washing was followed by three times for 10 min
each with 1X PBS-T. Washed membrane was incubated with diluted secondary
antibody conjugated with HRP (1:1500) for 1 hr at room temperature with shaking.
Detection was done by adding the diluted DAB substrate and hydrogen peroxide.

2.4.9 Electroelution

Electroelution of protein from the SDS-PAGE gel for further use was done by
Electroelution apparatus obtained from Bio-Rad. The SDS-PAGE gel was stained with
Coomassie blue staining for 30 min and destained for 1 hr. The purified band of
protein of interest was sliced from the gel and cut into small pieces. The gel pieces
were placed within the poly carbonate tubes and the apparatus was assembled. After
the final set up, all the air bubbles were removed from the tubes and the tubes were
placed within the gel tank. 1liter of electrodialysis buffer was poured in to the tank,
and electroelution was done at 20 volt, for 8-10 hr. Afterwards the destained gel pieces
were removed from the tube, and the eluted protein solution of blue colour was
recovered from the lower trap of the tube. For further use the eluted protein was
lyophilized.

2.4.10 ELISA

ELISA or Enzyme Linked Immuno Sorbent Assay was done to detect the level of
antibody titre raised in rabbit. Antigen or the purified protein was diluted to 0.1 µg/25
µl (4.0 µg/ml) in coating buffer (0.1 M NaHCO3, pH 8.6), and each well was coated
with 25 µl (0.1 µg). The plate was covered with plastic film and incubated at 4°C
overnight. On next day the coating solution was washed out of wells and the wells was
52
washed twice vigorously with distilled water. The wells was blocked by the addition
of 50 µl 3% BSA/PBS solution and followed by the incubation for 1 hr at room
temperature, wrapped in plastic or in a moist sealed container. The blocking solution
was shaked off (without washing or drying) and washed with 1X PBS for three times.
The primary antibody dilution in 1X PBS was done in 1:10, 1:100, 1:1000 1:10,000
ratio. Serially diluted primary antibody solution of 25 µl was added in each well, in
triplicate manner. The plate was wrapped in plastic wrapper and incubated at room
temperature for 1 hour. The primary antibody solution was shaked off and the wells
were washed with 1XPBS. 25 µl of 1:1000 times diluted secondary antibody
conjugated with HRP was added to each well and the plate was wrapped in plastic
wrapper. 1 hr incubation was done at room temperature, followed by three times
washing with 1X PBS. TMB substrate was diluted and added to each well, followed
by the addition of hydrogen peroxide and detection of signal at 495 nm.

2.4.11 Enhanced chemiluminescence (ECL)

For Enhanced chemiluminescence detection, all the preliminary steps such as

electrophoresis of SDS-PAGE gel, blocking, followed by primary, and secondary
antibody binding was done in the same fashion as done for western blotting. After the
final washing of the blot it was kept in 1X PBS solution. Equal volumes of luminol
and enhancer solutions were mixed together, followed by the preparation of peroxide
solution in enough volume to cover the total membrane. The detection mix was
incubated for at least five min to equilibrate. Excess buffer from the washed blot was
drained, and the detection mix was directly added to the blot (protein side up) and
incubated for 1-3 minutes at room temperature. Excess detection mix was drained off
and the membrane was wrapped well in saran wrap. Remaining air pockets were
gently removed. The blot was placed in the film cassette placing the protein side up.
The cassette was then handled in the dark and a sheet of film was placed on the blot.
The cassette was closed and the film was exposed for 30-60 seconds. The exposed
film was replaced with a new one, and developed in the mean time with developer and
fixer solution. The second film was exposed for a longer time. The developed and
fixed film was then analyzed.

53
2.4.12 Raising polyclonal antibody in rabbit.

Polyclonal antibody was raised in rabbit, following all standard animal ethics and
maintaining the rabbit in a hygenic condition. For raising antibody against one of the
expressed protein, a male white rabbit of 500 gm weight was obtained from the local
suppliers. Before raising the antibody, 1 ml of blood was collected from the central ear
artery of that rabbit to obtain the pre immune serum. The antigen/protein was purified
by affinity chromatography and eluted from the gel, followed by 5 hr lypholization.
Concentration of the the lypholized protein was checked. 500 µg of lypholized protein
was diluted to 1 ml with 1X PBS and mixed with 1ml of complete Freund’s adjuvant
(CFA). The mixture was vortex vigorously to obtain an emulsified solution, which was
injected intramuscular way to the rabbit. All the subsequent injections were done by
using Incomplete Freund’s Adjuvant (IFA). The following schedule (Table 2.2) was
maintained to obtain maximum antibody titer.

All the blood except the final bleed was collected from the central ear artery with a 19
gauge needle. The final bleed was drawn from the heart, and allowed to clot and
retract at 37°C overnight. The clotted blood is then refrigerated for 24 hr and the
serum clarified by centrifugation at 2500 r.p.m for 20 min. Straw colored serum was
decanted and aliquted to store in -20°C.

Table 2.2: Schedules of injection and blood collection

Week 0 Week 1 Week 3 Week 6 Week 7 Week 9 Week 10 Week 11

Bleed 5 Immunize Immunize Immunize Bleed Bleed Immunize Bleed 15

ml with with with 5ml 5ml with ml
(1 ml antigen in antigen in antigen in (1 ml (1 ml antigen in (10 ml
pre- CFA IFA IFA serum) serum) IFA serum)
immune
serum)

54
2.5 Enzymatic methods

2.5.1 Enzyme assay

The chitinase activity against β-1-4 glycosidase bond was determined with soluble
artificial substrate 4-methylumbelliferyl-tri-N-acetylchitotrioside, which release
fluorosence product. The substrate was dissolved in pure DMSO and 1 mM stock was
prepared. In a typical reaction volume of 300 µl, 200 mM of sodium phosphate buffer
(pH 7.2) varying amount of substrate (1-10 µM) was added with required amount of
purified enzyme. The reaction was continued and the mixture was incubated for 15-30
min at 25°C. 100 µl of 200 mM sodium carbonate solution was used to stop the
reaction. DMSO was used as negative control.

Different pH were maintained by different buffer, such as 200 mM sodium phosphate

buffer were used for 5-8 pH range, while 200 mM sodium acetate buffer were used for
pH 3-5 range. Same experiments were repeated in different range of temperature
between 5°C-45°C. Product quantity per time was performed by measuring
fluorescence emission (excitation at 360 nm and emission at 450 nm) using Simple
reading mode of Varian spectroflurometer, model Cary-Eclipse equipped with a
Xenon lamp. Standard graph was prepared using 4Mu to quantify the product. One
unit of chitinase activity was defined as the amount of enzyme required to release
1 nmol of 4-methylumbelliferone (4MU) per second at 25°C. Km and Vmax values were
calculated by applying reaction rate to the Michaelis–Menten equation [v = Vmax
[S]/(Km + [S])] where v is the reaction rate at a specific substrate concentration, Vmax is
the maximum reaction rate and [S] is substrate concentration. The reaction rate was
plotted as a function of the substrate concentration. Kinetic module of Sigma Plot
software was used to calculate the Km and Vmax values by using Lineweaver-Burk
plots.

2.5.2 Inhibitor assay

Inhibitor studies were done against the chitinase enzymes by using three previously
reported methylxanthine/methylxanthine derivative compounds namely Pentoxifylline,
Theophylline and Caffeine. 10 mM Stocks were prepared by dissolving all the

55
inhibitors in double distilled water. 300 µl of reaction mixture was prepared with 1
mM 4-methylumbelliferyl-tri-N-acetylchitotrioside (1-5 µM), 5 µg of purified enzyme
and 200 mM sodium phosphate buffer (pH-7.2) containing different amount of
inhibitors (50-150 µM). Reactions were stopped after 15-30 min of incubation at 25°C
by adding 100 µl of 200 mM sodium carbonate solution. Product formation was
determined by measuring fluorescence emission (excitation at 360 nm and emission at
450 nm) using Simple reading mode of Varian spectrofluorometer. All the results were
analyzed using single inhibitor-single substrate mode of inhibitor analysis programme
of Enzyme Kinetics module of Sigma Plot software. Ki values of the used inhibitors
and mode of inhibition was calculated using Lineweaver-Burk plots.

2.5.3 Substrate binding assay

Substrate binding specificity and affinity of the enzymes were determined by

incubating the purified enzyme, with the following materials such as chitin beads
(New England Biolabs), colloidal chitin, and Shell crab chitin dust (Sigma). Briefly
the substrates were washed with 1X PBS and incubated with purified enzyme for 2 hr
with occasional shaking, at room temperature. After incubation, the supernatant was
removed by centrifugation for 5 min at 10,000 r.p.m., and the pellets were washed
twice with wash buffer. Both the supernatant and pellet were boiled with 5X sample
dye and the binding ability was qualitatively analyzed in 12% SDS-PAGE. BSA
(fraction five) was used as a control under same condition to nullify the adsorption
probability. Quantification of binding ability was done by measuring the protein
content in unbound/supernatant fraction at 280 nm.

2.5.4 Substrate cleavage pattern

Colloidal chitin was prepared as described in materials. Colloidal chitin and chitin dust
was used as insoluble natural substrate to understand the substrate cleavage pattern of
chitinases. 100 µl of colloidal chitin or 1 mg of chitin dust was washed with and
resuspended in 200 mM sodium phosphate buffer (pH-7.2). Required amount of
purified enzymes were added to the mixture and the reaction mixture was incubated
for different time span (1 hr - 24 hr) at room temperature with constant agitation. 200
mM sodium carbonate solution was added to stop the reaction. The mixture was
56
centrifuged at 10,000 r.p.m. for 10 min and the supernatant was analyzed by reverse
phase HPLC. As mobile phase water-acetonitrile was used, along with C18 column as
the stationary phase, and the elution was done by distilled water with a flow rate of 1
ml/min. Product detection was done at 210 nm at room temperature. Standard
oligosaccharides were used to standardize the elution peak profile.

2.6 Entamoeba cell culture methods

2.6.1 Entamoeba cell culture

Entamoeba histolytica (NIH strain) and Entamoeba invadens (IP1 strain) were
maintained axenically in TYIS-33 medium at 37°C, and 25°C respectively.
Entamoeba histolytica cells were routinely sub cultured in every 48 hr, depending on
the cell confluency. Entamoeba invadens cells were sub cultured twice in a week.

2.6.2 Encystation of Entamoeba invadens

For the initiation of encystation log phase Entamoeba invadens trophozoites were
harvested and centrifuged at 1500 r.p.m. for 3 min to pellet down. Single washing with
TYIS-33 medium was followed by three washes with encystation medium. Cells were
counted by hemocytometer, and diluted to reach the final concentration of 107 cells per
ml. Cells were resuspended in Nunc flask filled with 40 ml of encystation medium and
incubated at 25°C. Clumping of encysting cells was observed after 12 hr, and mature
cyst was obtained within 60-72 hr. Maturity of the cyst was determined by CFW
staining. For different time dependent studies, encysting cell of different hr was
collected and treated as per the requirement.

2.6.3 Excystation of Entamoeba invadens

Entamoeba invadens mature cysts were obtained from 72 hr encystation culture, and
washed with encystation medium. This population contains encysting trophozoites as
well as mature cyst. Washing with chilled 1X PBS was followed by 5 min incubation
with 0.05% SLS solution. Only the mature cysts will remain in the solution, while the
trophozoites or encysting cells were get lysed by SLS. Viability of the mature cyst was
57
verified by Trypan Blue exclusion. The mature cysts were washed several times with
1X PBS to wash away the SLS. Finally the pellet was washed twice with TYIS-33
medium and resuspended in it. Within 12 hr of incubation emerging trophozoites were
observed under phase contrast microscope.

2.6.4 Entamoeba total protein isolation

Whole protein of encysting Entamoeba invadens was collected for immunoblot.

Encysting cells of different time point was chilled and pelleted down by centrifugation
at 1500 r.p.m. for 3 min. The pellet was washed twice and resuspended in 1 ml of
chilled 1X PBS, followed by the addition of 1 µl of Protease inhibitor cocktail. The
cell suspension was sonicated for 15 min with 5 sec pulse, and centrifuged for 10 min
at 10,000 r.p.m. The supernatant was collected and stored in -20°C for further study.

2.6.5 Fluorescence Microscopy

Entamoeba invadens trophozoites, encysting cells and cysts were subjected to

immuno-staining microscopic analysis. The cells were chilled and pulled down by
centrifugation at 1500 r.p.m. for 3 min. The pellet were washed twice with chilled 1X
PBS and resuspended in 1 ml of 1X PBS. All the washing and incubation was done in
4°C. For fixing 1 ml of 4% PFA was added to the cell suspension to make the final
concentration 2%. The cells were incubated in ice for 10 min, followed by three times
wash with 1X PBS. For permeabilization, cell pellet were treated with 0.01% Trition
X 100 for 5 min at room temperature. The permeabilized cells were washed thrice
with chilled 1X PBS and kept in 4°C. For primary staining, anti EiChit1 and EiChit2
antibodies were diluted to 1:200 times in 1X PBS. Permeabilized cells were incubated
for 1 hr with primary antibody solution at 4°C. Cells were washed thrice with chilled
1X PBS, and incubated for 1 hr with 1:500 times diluted TRITC conjugated anti rabbit
IgG for fluorescence staining. Stained cells were washed thrice with chilled 1X PBS,
followed by the slide preparation with 10 µl of cell suspension. Phalloidin conjugated
with FITC used for F-Actin counter staining, and nucleus was stained by DAPI or
Sytox green. 100 mM Calcofluor (CFW) was used to stain mature cyst chitin wall.

58
Olympus Flowview FV1000 confocal Laser scanning microscope with argon and
HeNe lasers was used for sample analysis, using respective laser and fluorescence
channels.

2.6.6 Cryopreservation of Entamoeba cell

A cell freezer was pre-chilled with isopropanol at -80°C for 24 hrs before starting the
cryopreservation procedure. Fresh log phase axenic culture of Entamoeba was chilled
on ice for 5 min. Chilled cells were centrifuged at 1500 r.p.m. for 3 min, and the
medium was decanted. The cells were resuspended in 2 ml of solution I in a 2 ml tube.
From there cell suspension of 0.5 ml was transferred in 1 ml cryotubes. Prepared
solution II (15% Cell culture grade DMSO solution) of 0.5 ml was then added to that
cryotubes to make up the final volume to 1 ml. Filled tubes were incubated in 37°C
incubator for 15 min, followed by rapid cooling. The cryotubes were directly
transformed into the cell freezer, kept at -80°C. The cell freezer (with the cryotubes)
was kept in -80°C for 48 hr, followed by a liquid N2 storage.

2.6.7 Revival of cryo-preserved cell

Cryo-preserved tubes were collected from the liquid N2 tank, and immersed in 37°C
water bath immediately to thaw the medium. The tubes were incubated for 10 min in
37°C, and used to inoculate 15 ml culture tube filled with pre-warmed TYIS-33
medium. The culture tubes were kept in 37°C incubator in slanted position for 48 hr
without any disturbance. After 48 hr, fresh medium was added and again the tubes
were incubated for another 24 hr more, after which subculture was started.

59
60
 Chapter 3

In silico Characterization of Entamoeba chitinases

62
3.1 Abstract

Computational characterization of three Entamoeba invadens chitinases are reported in

this chapter. BLASTN search reveals the presence of three chitinases belongs to family
18 of glycosyl hydrolyase in E. invadens genome. Entamoeba chitinase sequences show
no significant similarity with other prokaryotic chitinase validating the idea of convergent
evolution. Protein models of Ei chitinases are prepared by homology modelling, using
human chitotriosidase as template. The predicted models demonstrate very high similarity
and conserved catalytic domain structure within themselves. Several aromatic residues
within the catalytic cleft were identified by docking of penta NAG molecule, may play
important role in substrate binding and β(1-4) bond cleavage activity.

3.2 Introduction

Entamoeba histolytica has a haploid genome, though its ploidy was not maintained well
throughout the life cycle. In a previous observation E. histolytica DNA is being found to
resolved in 6 to 9 bands between 300 and 2000 kilobases, distributed in 16-22 large and
up to 31 small chromosomes (Valdes et al., 1990). The total genome of E. histolytica
HM1: ISS strain is find to be more than 20 Mb containing 14 chromosomes while most of
its extra-chromosomal DNA content was made up of rDNA. Amoebic genes are
reportedly have limited numbers of introns with short and intergenic sequences. The
completed E. histolytica genome project was a collaborative effort involving both TIGR
and the Sanger Institute Pathogen Sequencing Unit. The random shotgun strategy was
used for E. histolytica genome sequencing. Randomly 2.0 kb average insert size plasmid
was prepared to make 6-8 kb pHOS2 libraries of the E. histolytica genome as well as a
larger-insert BAC library. Sufficiently large numbers of randomly selected clones
obtained from sequencing libraries and sequenced from both ends to generate a high
quality draft (Loftus et al., 2005).

Several phylogenomic analyses support the proposal of lateral gene transfer among the
Entamoeba genome, specifically among the metabolic repertoire. The genome encodes a
large number of novel receptor kinases and contains expansions of a variety of gene
families, including those associated with virulence. E. invadens a reptilian parasite
reportedly mimic the symptoms of human enteric pathogen E. histolytica, and used

63
widely as a model of encystation study because E. histolytica is not amenable to encyst in
axenic culture. In pulse field gradient electrophoresis of E. invadens, 4 to 5 bands
between 300 and over 2000 kb were resolved and discriminated in 4 large and 6 small
chromosomes (Valdes et al., 1990). At the nucleotide level E. invadens genome has on
average 60% sequence identity with that of Entamoeba histolytica with a 70% genic
identity and 50% intergenic identity. Highly repetitive sequences such as of rRNAs,
tRNAs, CXXC-rich proteins, and Leu-rich repeat proteins are found to be in the E.
invadens genome as they are in E. histolytica genome. E. invadens proteins involved in
amoebic virulence, vesicular trafficking, signal transduction, and drug resistance are
found to have homologs in E. histolytica genome (Wang et al., 2003).

Presence of a single chitinase gene in E. histolytica genome has already been reported,
followed by its sequence analysis (de la Vega et al., 1992). Presence of multiple
chitinases within E. invadens genome was also reported (Wang et al., 2003) and was
partially characterized (Villagomez Castro and Lopez-Romero, 1996). Occurrence of
multiple chitinases in E. invadens genome is not an isolated incident, as gene multiplicity
is common among kinetoplastid protozoan’s (Landfear et al., 1983) expressing same
protein (Seebeck et al., 1983). Multiplicity of other chitin-binding domain may occur due
to domain shuffling as it happens in other fungal and bacterial chitinase and carbohydrate
binding proteins (Shen and Jacobs-Lorena, 1998).

3.3 Results

3.3.1 Data mining and sequence identification

Chitinase (E.C 3.2.1.14) is a well-documented enzyme and close to lysozyme group, that
breaks down the β (1-4) glycosidic bonds of polysaccharide chitin. The enzyme belongs
to glycosyl hydrolase group, and was classified into two well distinct families depending
on its three dimensional structure. Family 18 includes the chitinases and homologous
chitin binding proteins termed as chito-lectins from viruses, bacteria, fungi and animals as
well as classes III and V from plants. Family 18 enzymes were known to adopt the TIM
(triosephosphate isomerase) barrel structure consisting of strongly conserved (α/β)8 motifs
containing separate chitin-binding domains in the carboxyl terminal region. Chitinases
belonging to this family shows a well-conserved catalytic domain sequence containing
several key aromatic amino acids needed for substrate binding. Family 19 chitinases are

64
identified mostly from plants (classes I, II and IV), nematodes, and some bacteria
(Funkhouser et al., 2007).

Eh chitinase sequence (Accession no. XP652205) obtained from the already published
genome, and used to perform BLAST within the E. invadens genome. Three nucleotide
sequences, named as EiChit1 (Accession no. AAB52724), EiChit2 (Accession no.
ABC59330) and EiChit3 (Accession no. ABC59331) were obtained from E. invadens
genome. ORF finder was used to detect the longest reading frames, and respective protein
sequences were selected for further studies. Protein sequences of other chitin binding
proteins of E. invadens such as Jacob1 (Accession no. AF175527) and Jessie3b
(Accession no.ABC59329) were obtained from the NCBI database. All accession no. are
enlisted in appendix A.

3.3.2 Preliminary information regarding Entamoeba chitinases

From the obtained nucleotide and protein sequences, we gathered several preliminary
informations using ExPASy proteomics server and its primary structure analysis tool.
Theoritical mol wt, pI, and other primary sequence related measurements of each protein
are presented in Table 3.1. Secondary structures containing α helices, β sheet and random
coils were obtained from Predictprotein server, and their probable percentages are
tabulated in Table 3.1.

Table 3.1: Theoretical characterization of EiChit1, 2, 3 and EhChit1

Protein Mol. Wt pI Hydrophobicity Signal Secondary structure

identifier (Da) sequence
Alpha Extended Random
helix strand coil
EiChit1 55797.1 4.72 -0.647 Present 19.11% 27.16% 53.72%

EiChit2 41862.1 5.30 -0.445 Present 19.95% 28.96% 51.09%

EiChit3 41613.4 5.01 -0.549 Present 32.97% 20.05% 46.98%

EhChit1 55758.3 5.51 -0.646 Present 18.29% 29.47% 52.24%

65
Hydropathy plot or hydrophobicity of the proteins were calculated using Kyte-Doolittle
scale (Kyte and Doolittle 1982). SignalP, TMHMM, TMpred, nnPredict servers were
used to understand the localization and vesicular transportation of these chitinases due to
posttranslational modifications. All the Entamoeba chitinases found to belong to the
family 18 glycosyl hydrolase, by Conserved domain search programme.

3.3.3 Phylogenetic analysis of Entamoeba chitinase

Family 18 of glycosyl hydrolase have many members from bacteria, insect, and higher
animals including humans, and bear some signature domain and conserved motif within
the catalytic domain. Protein sequence of Ei Chitinase 1 was used as query sequence in
blastp programme to search similar sequences within the non-redundant database. Twenty
sequences were selected depending on the highest score and lowest e value. Accession
number of retrieved sequences are listed in appendix B and Fragments of aligned
sequences are represented in Fig.3.1.

Protein sequences of EiChit1 (gi1685364), EhChit1 (gi67472835), EdChit1

(gi167380579), EiChit2 (gi91983531), EiChit3 (gi84620156), Human Chit1
(gi73909055), Pan Chit (gi114571914), Paralichthys Chit (gi46240808), Branchiostoma
Chit (gi219437504), Danio Chit (gi213625929), Equus Chit (gi219689080), Drosophila
Chit (gi195374614) Helicoverpa Chit (gi33325189), Pediculus Chit (gi212509328),
Rattus Chit (gi119120779), Aedes Chit (gi157107967), Anopheles Chit (gi158286667),
Aspergillus Chit (gi115400105), Bacillus Chit (gi75759179), and Listeria Chit
(gi47092241), were aligned using ClustalW 2.0 software of EMBL-EBI server.

66
 Fig. 3.1: Mutiple sequence alignment of 20 chitinases using ClustalW. Fragments of catalytic
domain sequences presented here and four- consensus motif I, II, III and IV indicated within box.

All default gap value and penalties (such as Open gap is -12, extend gap is -2, end gap -1,
and gap distance is 4) were used for alignment along with the default Gonnet matrix. The
aligned sequences show four consensus motif containing consensus aromatic residues
such as VGGW (domain I), DWEYP (domain II), MTYDF (domain III), and VGM
(domain IV).

Twenty chitinase sequences used to prepare the phylogenetic tree by minimum evolution
method with bootstrap values using MEGA software (Kumar et al., 2007; Rzhetsky and
Nei 1992). The bootstrap consensus tree inferred from 1000 replicates is taken to
represent the evolutionary history of the taxa analyzed (Fig 3.2) (Felsenstein, 1985).

The maximum probable was searched using the Close-Neighbor-Interchange (CNI)

algorithm (Nei and Kumar, 2000) at a search level of 1. The Neighbor-Joining Algorithm
(Saitou and Nei, 1987) used to generate the initial tree. Branches corresponding to
partitions reproduced in less than 50% bootstrap replicates were collapsed, and bootstrap
percentage was shown next to the branches.

The tree was drawn to scale, with branch lengths as the evolutionary distances computed
using the Poisson correction method (Zuckerkandl and Pauling, 1965), and is in the units
of the number of amino acid substitutions per site. All positions containing gaps and
missing data were eliminated from the dataset (Complete deletion option). Phylogenetic
study in relation with other well-known chitinases clearly predict the difference between

67
the sequence of bacteria (IV), protozoan (III), insect (II) and higher animals (I), clustering
them in different clades.

Fig. 3.2: The Phylogenetic tree presented constructed using Mega (version 4) software. Minimum
evolution method was used with Poisson correction for multiple amino acid substitution and with
1000 random bootstrap replicates. The Fig. shows the minimum evolution tree. Alignment of
amino acid sequences of 20 different chitinase was done using ClustalW. The chitinases comprise
four clusters denoted as I, II, III and IV (discussed in the text). The scale at the bottom left is in
units of amino acid substitutions per site. Bootstrap values ≥ 50% are shown.

3.3.4 Primer designing

For RT-PCR, different primers were designed by using Oligo Perfect server (Invitrogen).
All the nucleotide sequences were uploaded, and primers were designed to amplify ~400
bp long fragment of the C terminal of the protein of the Ei Chitinase 1, 2 and 3 (Table
3.2). For cloning and expression in bacterial system, primers were designed using Oligo
perfect software (Invitrogen) excluding the predicted N-terminal signal sequences for
both Eh and Ei Chitinases (Table 3.3). For expression in bacteria, Methionine codon
(ATG) was added to the 5’ of the every forward primer.

68
 Table 3.2: Primer sequence (all 5’Æ3’) used for RT-PCR.

Gene Primer Sequence

E.invadens Actin Sense CTC CCA CAC GCC ATC CTC AG

Antisense GGA CGA TGG CTG GGC CAG AC

E. invadens Chi1 Sense CCA GCT GCA AAT ATC CAG

Antisense TTA GCA CCG ATC AAG CTC

E.invadens Chi2 Sense GAC GAC ACG TCG AAG ACT C

Antisense TCA ATC AGA TGC TAA AAC

Sense GAT GGC AAA CAT CGA AAT CG

E. invadens Chi3
Antisense TTA GTT TTT CAA CTC ATC G

Table 3.3: Primer sequences (all 5’Æ3’) used for expression study. Incorporated restriction sites
were underlined and italicized.

Gene Name Primer Sequence

E.invadens Chi1CBD-R-Cat Sense GGATCC ATG TGT GAA GGA CTC GAC AAC GG

Antisense CTCGAG TTA GCA ACC GAT CAA GCT CTT TC

E.invadens Chi1Cat Sense GGA TCC ATG AAG GTT GTC TCG TAT TAC ACC

Antisense CTC GAG TTA GCA ACC GAT CAA GCT CTT TC

E.invadens Chi2Cat Sense GGA TCC ATG AAG GTG GTT GGG TAC TAT AC

Antisense CTC GAG TCA ATC AGA TGC TAA AAC AC

E.invadens Chi3Cat Sense GGA TCC ATG AAA GTG ATT GGA TAC

Antisense CTC GAG TTA GTT TTT CAA CTC ATC GAT C

E.histolytica Chi1CBD-R-Cat Sense GGA TCC ATG CAC AAC TGT GAA GGT C

Antisense GCG CTC GAG TTA ACA TTT CTC AAT TAG

E.histolytica Chi1Cat Sense GGA TCC ATG AAA ACA GTT GCT TAT TAT AC

Antisense GCG CTC GAG TTA ACA TTT CTC AAT TAG

E. invadens JacobCBD Sense GGA TCC ATG CAA ACA TGT ACA ATT GAT GG

Antisense AGA TCT GTA GTT TCT TCT TTG TAA CCA CAT G

E. invadens JessieCBD Sense GGA TCC A TGC AAA CAT GTA ACG GGC T

Antisense AGA TCT GTC CAC AAA ATA GTC TCC TGG TT

E.invadens Chi1R-Cat Sense AGA TCT AAG TCG TCT GAG GAA CCA CTC

Antisense GTC GAC TTA GCA CTG ATC AAG CTC TTT TTC

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3.3.5 Homology modelling

To understand the three-dimensional structure of all E. invadens chitinase, in silico

modelling was done using homology modelling method. The chitinase sequences were
aligned against existing protein sequences at RCSB Protein Data Bank. Human
chitotriosidase and allosamidine structure (PDB identifier-1HKK) shows maximum score
and used as template for modelling (Rao et al., 2004). Models were built using
Modeler9v1 (Sali et al., 1993), and verified using the Procheck (Laskowski et al., 1993),
Modeler DOPE function, PROSA server (Sippl et al.,1993), Anolea server (Melo et al.,
1997), and Castp server (Binkowski et al., 2003). Predicted models of E. invadens
chitinases were selected depending on several aspects such as active site pocket similarity,
normality values (G-score), amino acid conformation (Ramachandran plot), Non local
atomic interaction energy, and sequence structure compatibility values (DOPE score and
Z score). The selected models have more than 85% residues in most favored region of
Ramachandran Plot, G-factor of 0.02, very similar ProsaZ score with template, and
94.39%, 95.25% similarity in the substrate binding pocket area and volume respectively.

EiChitinase1 and EiChitinase3 models were superimposed on EiChitinase2 model, to

elucidate their similarity using the visualization software Chimera (Pettersen et al., 2004).
The superimposed models of EiChitinase1, 2 and 3 show high similarity within catalytic
domain except in some loop regions (Fig 3.3)

A B

Fig. 3.3: Predicted models of EiChit1 (yellow) and EChit2 (green) [A], and EiChit2 (green) and
EiChit3 (yellow) [B] were presented in ribbon style, superimposed using Chimera. Top views of
the TIM barrel structure of the superimposed models are shown. It is evident that the predicted
models of all three chitinases are very similar except few loop regions.

70
Due to the conserved three-dimension structure, it was assumed that all E. invadens
contain similar primary sequences throughout the catalytic domain. To identify the
extension of sequence similarity within different species of Entamoeba, sequence
alignment was done (Fig 3.5).

Fig. 3.5: Aligned catalytic domains of Entamoeba chitinases. Entamoeba histolytica (XP652205),
Entamoeba invadens (Chi 1 -AAB52724, Chi2- ABC59330, and Chi3- ABC59331) and
Entamoeba dispar (XP001735375) chitinases were aligned using ClustalW server of EMBL.
Conserved amino acid residues of active site of the enzyme were indicated within box. Identical,
similar and somewhat similar amino acids are indicated with asterics (*), colon (:) and dot (.).

3.3.6 Molecular docking

Penta-N-Acetyl Glucosamine (NAG5) structure was used as substrate and superimposed

on the model of E. invadens Chitinase 2 protein to analyze the substrate binding
properties, following the superimposition technique (Tews et al., 1997). In the
superimposed NAG5 model on chitinase 2, the -1 and +1 subsite binding NAG molecules
were taken from S. marcescens chitobiase-chitobiose complex, the -2 subsite binding
NAG molecule was taken from chitotriosidase-chitobiose complex, the -3 subsite binding

71
NAG molecule were taken from hevamine-chitrotetrose complex and the +2 subsite
binding NAG molecule was taken from S. marcescens chitinase A-chitotriose complex.

Docked molecule was visualised after the energy minimization with Chimera (Pettersen
et al., 2004) (Fig.3.6). Interaction of the sugar residues with the conserved aromatic
amino acid residues was observed. The bound complexes were scored using SCORE
(Wang et al., 1998) and they gave pKd values of 5.38, 4.58, 5.27. Hence it could be
clearly seen that the protein prefers occupancy at the +1, +2 subsite rather than at the -1
subsite alone, resulting in the cleavage of dimers from the substrate end.

Fig. 3.6: Molecular docking of NAG5 within the catalytic cleft of predicted model of E. invadens
Chitinase 2. Penta N-acetyl Glucosamine (NAG5) bound to the modeled catalytic cleft of
E.invadens chitinase2. The thin line structure in cyan represents the oligo chitin (NAG5) with the
number beside each sugar ring representing the subsite. Amino acid residues of the active site
W84, N85, D128, E130, Y131, Y203, D204, R253 and E282 interacting with the sugar molecule
are indicated. The surface is shown to highlight the catalytic cleft. The image is generated using
PyMOL (DeLano Scientific). Sugars are numbered -3 through +2 from the non-reducing end as is
the current convention.

3.4 Discussion

Total genome sequencing of E. histolytica reveal the presence of single chitinase, while
three chitinases were identified in its reptilian counterpart E. invadens. To understand the
enzymatic profile of these enzymes, molecular characterization is a necessity, which is
often proceeds with the in silico characterization. In silico characterization includes the

72
understanding of phylogenetic relation, reorganization of structural aspect, and its
behaviour with the probable substrate.

Different Bioinformatics software were used to identify the preliminary aspects of these
proteins, such as the theoretical mol. wt. and pI calculation. Different range of pI signify
the overall surface charge and may involved with substrate binding efficiency, as lower pI
value increase the chitin binding capacity.

All four chitinases show the presence of N-terminal signal sequences with a chance of
post-translational cleavage of approx 15 amino acids, which signify the chance of protein
transport and occurrence of specific destination. Maximum chance of cleavage was
calculated mostly between polar-polar and nonpolar-nonpolar residues such as Serine-
Glutamine (EiChit1), Glycine-Leucine (EiChit2), Glutamine-Lysine (EiChit3) and
Alanine-Histidine (EhChit1). nnPredict server predicted the secondary structures which
shows more or less similar distribution of helices, sheets and random coils within
EiChit1, 2 and EhChit1, which is different than EiChit3. This difference may change the
three dimensional orientation, resulting in different substrate binding efficiency.

Phylogenetic analyses have shown that all known family 18 chitinases contains four
conserved regions within the catalytic domain (de la Vega et al., 1998). Conserved region
I is KXXXXXGGW, where X is a non-specific amino acid. Conserved region II is
comprised of FDGXDLDWEYP, which is believed to be located in the catalytic region of
the enzyme and the residue E is a putative proton donor in the substrate hydrolysis.
Conserved regions III and IV are MXYDXXG and GXXXWXXDXD, respectively.
Aligned sequences shows consensus motif with polar and aromatic residues such as
VGGW (domain I), DDWEYP (domain II), MTYDF (domain III) and VGM (domain
IV). All Entamoeba chitinases along with the representatives of other group confirms the
presence of such conserved motifs, which further strengthen their position in family 18 of
glycosyl hydrolase.

Phylogenetic investigation was done using the MEGA software and minimum evolution
method. Bootstrap consensus tree shows four different clusters named as I, II, III and IV
containing vertebrate, insect, protozoa and bacteria chitinase respectively. All Entamoeba
chitinases clustered among a distinct clade separated from other group ruling out the
possibility of lateral gene transfer, which happened to be the reason behind the origin of
most Entamoeba gene (Samuelson, 2002). The distribution of Entamoeba chitinases

73
clearly show that they are not evolutionary related to any bacterial, fungal, or insect
chitinases and have no bacterial ancestors. Among the different clades, Entamoeba
chitinases show comparatively high resemblance with the chitinases of evolutionary
advanced organism such as with insects and vertebrates. This similarity explained by
convergent evolution of different proteins under same evolutionary selective pressure. All
three E. invadens chitinases shows different time point of origin and remain separated
from each other. E. histolytica and E. dispar probably originated at in same evolutionary
period. Among three E. invadens chitinases, Ei Chitinase 3 probably evolved primarily,
followed by Ei Chitinase 2 and Ei Chitinase 1. Addition of extra domain may occur in
evolutionary pathway, to serve some other purpose such as substrate binding, rather than
only substrate hydrolysis. The origin of multiple chitinase in E. invadens probably occur
long ago to serve different purposes during encystation and excystation, while in
advanced E. histolytica and E. dispar, a single chitinase probably found to be enough to
serve all requirements, explain the absence of other two chitinases.

Homology modelling of E. invadens chitinase was done to identify the active residues
responsible for substrate hydrolysis. Human chitotriosidase used as template for
modelling study due to the high sequence similarity and score value. All three Ei
chitinase models were superimposed with each other without any significant structural
distortion. In spite of different secondary structure distribution, their tertiary conformation
of the catalytic domain remains same except of some loop regions. Multiple alignment of
only the catalytic domain of Entamoeba chitinases detects identical aromatic residues,
which may play important role in binding with negatively charged substrates.

Molecular docking with oligomers molecular was done to characterize the role of those
aromatic residues. Docking was done by superimposition using penta and hexa NAG
molecule. Docked protein shows preference for +2 and +1 subsites along with -1 subsite.
Tyr131 and Glu130 along with Tyr203 and Asp204 may play some important role in
substrate binding.

The hydrolysis reaction mechanism may follow the common anchimeric stabilization
hydrolysis mechanism of family 18 chitinases where the substrate is distorted to a boat
conformation by forming the oxazolinium intermediate. Chitinases exhibiting this mode
of substrate hydrolysis, can be inhibited by allosamidine, the pseudosaccharide molecule

74
which mimic the oxazolinium group. Other inhibitor molecules mimicking the basic
structure of allosamidine also found to inhibit such enzymes to some extent.

3.5 Conclusions

• Nucleotide sequences of all the Entamoeba chitinases were obtained from the
complete or ongoing genome project.
• All the Entamoeba chitinases belongs to the family 18 chitinases and contain
TIM barrel structured catalytic domain.
• All Ei and Eh chitinases contain signal sequences and destined to be in the
outside of the cell membrane or in vesicle.
• Primers for cloning and RT-PCR were designed using bioinformatics tool.
• Phylogenetic studies shown high similarity between all Entamoeba chitinases,
while they do not show enough resemblance with other bacterial, fungal or
plant chitinases eliminating the chance of lateral gene transfer. Similarity with
insect or vertebrate chitinase may occur due to convergent evolution.
• Homology modelling of all the Ei chitinases were done, which show highly
similar catalytic cleft structure, with few exception at loop regions.
• Molecular docking of penta chitosaccharide (NAG5) within Ei Chitinase 2
model shows the substrate binding sites and possible substrate cleavage
pattern.

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76
 Chapter 4

Cloning, Expression and Purification of Entamoeba chitinases

78
4.1 Abstract

Molecular cloning and expression of all three Ei and single Eh chitinases are done in
bacterial system and discussed in this chapter. All four genes were fished out by PCR
amplification and subjected to further cloning. For single step cloning, pGEM-T
vector was used followed by the sub cloning in pQE30 vector for bacterial expression.
Recombinant proteins were expressed with N-terminal His tag, after induction with
IPTG. Over-expressed Protein of 56 kDa (EiChit1 and EhChit1) and 45 kDa (EiChit2
and EiChit3) were confirmed with monoclonal anti His antibody and purified to 80-
90% homogeneity with Ni-NTA agarose beads

4.2 Introduction

Entamoeba histolytica, the amoebic parasite encyst and excyst to survive through the
harsh environment and propagate within the host with ease. This transformation
requires the synchronized initiation of several pathways, which finally completed the
chromosomal rearrangement, compartmentalization of several stage specific proteins,
creation of the chitinious cyst wall. Detail understanding of these procedures is still in
its infancy. In encysting Entamoeba invadens, several proteins were found to be
controlled transcriptionally (Coppi and Echinager, 1999; Ehrenkaufer et al., 2007) and
thus predicted to play an important role. Among these transcriptionally regulated
proteins, very few were characterized in details. Molecular characterization of these
proteins may enlighten their specific roles and may initiate the search of new drug
molecules against them (Laughlin et al., 2005). Protozoan chitinase which was found
to play important role in parasite transmission or host invasion, used as a potential
drug target, and its inhibition in these organism were found to produce less virulence
or low transmission ability within host (Shahabuddin et al., 1993; Welburn et al., 1993
and Rogers et al., 2008).

Chitinase belongs to such transcriptionally regulated protein group (de la Vega et al.,
1997) and required during encystation, as the well known chitinase inhibitor
“Allosamidine” was found to inhibit the E. invadens encystation (Villagomez Castro et
al., 1992). Though its role during excystation was not clear, degradation of chitinious
cyst wall was proposed to be the aim of these enzymes. Genome wide study of E.
histolytica reveals the presence of single chitinase, (Loftus et al., 2005) while E.
79
invadens was reported to have a complete chitinolytic system consisting of three
chitinases (Wang et al., 2003). Cloning and expression of three Ei chitinase and one
Eh chitinase was done in bacterial system to characterize these enzymes.

4.3 Results

4.3.1 PCR amplification of Entamoeba chitinases from genomic DNA

Axenic culture of E. invadens and E. histolytica was maintained in TYIS-33 medium

at 25°C and 37°C respectively (Diamond 1977) and used for genomic DNA isolation.
Gene specific primers containing BamHI and XhoI restriction enzyme sites were
designed (Table 2.3) to amplify the following fragments of all four Entamoeba
chitinases excluding the signal sequences (Fig.4.1). EiChit1 and EhChit1 contain
chitin binding domain (ChBD), heptapeptide repeat and the catalytic domain, while
EiChit2 and EiChit3 consist of only the catalytic domain. The protein sequences are
enlisted in appendix D.

Fig. 4.1: Schematic diagrams of E. histolytica (EhChit1) and E. invadens (EiChit1, EiChit2
and EiChit3) chitinases used for cloning and expression. CBD represents N-terminal chitin
binding domain of approximately 60 amino acids. R represents heptapeptide serine rich linker
connecting CBD and catalytic domain. Catalytic domain displays the longest C-terminal
domain responsible for substrate hydrolysis.

PCR amplification was done using the Expand High Fidelity PCR system. Ei chitinase
1, 2, 3, and Eh chitinase 1 were amplified by using the following cycle, of 95°C for 5
min, 35 cycles at 92°C for 30 s, 55°C for 30s, 68°C for 1.5min (1min for 1000bp ) and
then by a final extension at 72°C for 5min. All the amplified products of
approximately 1500 bp (EiChit1 and EhChit1) and 1200 bp (EiChit2 and EiChit3)
were checked in 0.8% agarose gel with 1kb DNA ladder (Fig. 4.2)
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 Fig. 4.2: Amplified EiChit1, EiChit2, EiChit3 and EhChit1 and 1kb DNA
marker. Amplicons are shown with black arrows

4.3.2 Cloning of Entamoeba chitinases in pGEMT vector

The amplified products were purified further by gel elution and used to determine the
product concentration at 260nm. Approx 75 ng of PCR product (approx length 1.2-
1.5kb) was ligated to 50 ng of supplied vector mixture in 3:1ratio using the following
equation; where vector size is 3kb, and insert:vector ratio is 3:1.

{(ng of vector × kb size of insert)/ kb size of vector}× insert:vector molar ratio = ng

of insert

The ligated products were transformed into competent E. coli, DH5α cells. Retardation
of plasmid DNA from white colonies in comparison to blue colonies in agarose gel
electrophoresis was used as a preliminary screening for recombinants, followed by
restriction digestion with BamHI and XhoI.

Digested plasmids were run in 0.8% gel in comparison to undigested plasmids, and
inserts of expected length were detected (Fig.4.3). Sequencing was done for
confirmation.

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 Fig. 4.3: Agarose Gel electrophoresis of restriction enzyme digested TA clones of EiChit1,
EiChit2, EiChit3 and EhChit1. Lane1- TA-EiChit1, Lane 2- Digested TA--EiChit1, Lane 3-
TA-EiChit2, Lane 4- Digested TA-EiChit2, Lane 5-TA--EiChit3, Lane 6- Digested TA-
EiChit3, Lane 7- TA-EhChit1, Lane 8- Digested TA-EhChit1. Lane M represents 1kb DNA
Marker. Inserts are shown with black arrows.

4.3.3 Cloning of Entamoeba chitinases in pQE30 vector

Sub-cloning of all the chitinases was done in bacterial expression vector pQE30
(Qiagen, Germany) for expression study. BamHI/XhoI digested chitinase fragments
were cloned in the BamHI and SalI site of the pQE30, as SalI and XhoI are compatible
with each other. Digested vector and insert were gel purified and ligated in 3:1 ratio.

Chemically competent XL1 Blue cells were transformed with ligation mixture.
Recombinant colonies and confirmed by the restriction digestion with BamHI and
HindIII, as the SalI and XhoI sites get destroyed followed by the ligation. Digested
plasmids were run in 0.8% agarose gel, and cleaved bands of expected lengths were
identified by comparing to 1kb DNA ladder (NEB) (Fig. 4.4)

Fig. 4.4: Agarose gel electrophoresis of pQE30 clones of EiChit1, EiChit2, EiChit3 and
EhChit1, digested with BamHI and HindIII. Lane M represents 1kb DNA marker. Inserts are
shown with black arrows.

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4.3.4 Expression of Entamoeba chitinases in bacterial system.

Recombinant proteins were expressed in M15 (pREP4) strain of E. coli containing the
lac repressor expressing plasmids (pREP4). The pREP4 plasmid is derived from
pACYC and multiple copies of pREP4 were present in the host cells ensuring the high
levels of the lac repressor protein and tight regulation of recombinant protein
expression

Confirmed pQE30 clones were used for transforming chemically competent M15
cells, and plated on Amp-Kan LB plate. Single colonies were selected and used for
expression study. Freshly grown log phase culture was induced with 1 mM IPTG
solution, followed by 5 hr post induction incubation at 37°C. The total bacterial
proteins were electrophoresed in 12% SDS-PAGE gel in comparison with un-induced
total protein, to detect the induced band. The induced lanes showed the bands of
expressed proteins, at expected region. Induced bands of expressed proteins were
observed at different molecular weight which is slightly higher than their expected
length (Fig. 4.5) (EiChit1- 65 kDa, EiChit2- 45 kDa, EiChit3- 45 kDa and EhChit1- 65
kDa).

Fig. 4.5: SDS-PAGE analysis of total protein of IPTG induced M15-EntChitinaes-pQE30

clones. Over expressed protein bands are marked with black arrows. Lane M15 represents
uninduced bacterial protein and Lane M represents Protein marker.

The over expressed protein contains 6 Histidine residue at their N-terminal. Further
confirmations of expressed proteins were done by western blot using monoclonal Anti
His antibody as primary antibody (Fig. 4.6). HRP conjugated Protein A was used as
secondary reagent, and Di-amino-benzoic acid (DAB) detection system was used for

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band development. Single band of His tagged proteins were observed in total protein
fraction of induced M15-Entamoeba chitinase clones at their respective region.

Fig. 4.6: Western blots of expressed, EiChit1, EiChit2, EiChit3 and EhChit1. Total bacterial
proteins from induced clones were used, and over expressed bands were detected with
monoclonal anti His antibody and marked with black arrows.

4.3.5 Purification of Entamoeba chitinases using Nickel NTA beads

The N terminal 6xHis affinity tag of pQE30 system was used for purification, with Ni-
NTA agarose beads by affinity chromatography. Low immunogenicity and small
length of 6xHis tag do not interfere with protein compartmentalization, folding and
activity. Presently Nitrilotriacetic acid (NTA) (Qiagen) a tetradentate chelating
adsorbent, used solely for IMAC allowing the purification of very low amount of
protein, which results in more than 90% homogeneity in single step purification.

Expression profiling was done to identify the nature of expressed proteins. Induction
was done with 1 mM IPTG, followed by 5 hr post induction incubation at 37°C. The
induced bacterial pellets were sonicated and centrifuged to separate the soluble or
membrane bound proteins, and analyzed in 12% SDS-PAGE. Induced proteins were
found mostly in membrane bound fractions in every case.

To obtain maximum protein in soluble fractions, several combination of IPTG

concentration, post induction incubation period and post induction temperature were
checked. As for preliminary optimization, 0.5 mM, 0.2 mM, 0.1 mM and 0.05 mM
IPTG were used along with 16hr post induction incubation period at 15°C.

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Level of expression in soluble fraction was further confirmed by the western blotting
of the recombinant proteins, using monoclonal anti His Antibody. The optimized post
induction incubation period and temperature was tabulated in Table 4.1.

Purification was done using Ni-NTA agarose beads, as mentioned in “Methods and
Materials”. In every case 200-250 mM was found to elute more than 90% of the bound
protein. Purification quality was determined by 12% SDS-PAGE, followed by staining
and destaining (indicated by black arrow in Fig. 4.7). Purified proteins were further
concentrated using centricon with 30 kDa cut off membrane (Pall Sciences) and
quantified with Bradford reagent using BSA fraction five (1mg/ml) as standard. Total
protein yield were tabulated in table 4.1.

Fig. 4.7: Single step purification profiles of EiChit1, EiChit2, EiChit3 and EhChit1.
Purified bands are marked with black arrows. Lane M- Protein marker

Table 4.1: Optimum IPTG Conc., post induction incubation temperature for maximum protein
yield and obtained protein.

Protein IPTG Conc. Post induction Yield

(mM) Temperature mg/Lt
EiChit1 0.20 15°C 2.00
EiChit2 0.20 15°C 5.00
EiChit3 0.05 15°C 0.64
EhChit1 0.20 15°C 1.30

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4.4 Discussion

Chitin, the polymer of GlcNAc molecule ranked as the 2nd most abundant one, after
cellulose and involved in different structural aspects of lower and higher group of
organisms. In many fungi and bacteria, occurrence of multiple chitinase is a very
common phenomenon.

Several human parasites were reported to contain single or multiple chitinases for
transmission through vectors, or to continue their life cycle. So these chitinases were
evaluated as a possible drug target either as a target molecule or a source for
transmission blocking antigen (Langer and Vinetz, 2001). Presence of any chitin or
any similar polysaccharide was not discovered in human system, multiple human
chitinase or chitinase like protein was discovered in last few years (Renkema et al.,
1995). Though their exact role was not characterized yet, their involvement in
different disease and genetic deficiencies made them an interesting molecule.

Other than Plasmodium (Sahabuddin et al., 1995) and Leshmania chitinase (Rogers et
al., 2008), Entamoeba chitinases were treated as a potential drug target to combat with
the amoebiasis. E. histolytica contain a single chitinase as Entamoeba dispar and the
exact role of this parasitic enzyme in its life cycle is still unknown. Interestingly the
genome sequence of “in vitro encystation model” E. invadens, a reptilian parasite,
comprises of three chitinases. Partial characterization of those enzymes was followed
by their isolation and purification from encysting cells (Villagomez Castro and Lopez
Romero, 1996). All Entamoeba chitinases contain conserved sequence throughout the
catalytic domain and belongs to family 18 of chitinase.

Molecular characterization of these enzymes was not possible by using the

endogenous molecules, as these molecules are stage specific and comprise a very
small amount of total cell protein. So, to achieve that goal, molecular cloning and
expression of recombinant protein in a bacterial system was proposed and carried out.

From the genome sequence it was observed that Eh chitinase contains a N-terminal
substrate binding domain, a C-terminal catalytic domain and a heptapeptide linker in
between. In a different note the Ei chitinase complex contains three enzymes having
different composition. Ei chitinase 1, the longest protein of them, has similar structure

86
as Eh chitinase, though the other two enzymes show a different structure by having
only the catalytic domain.

For PCR amplification, gene specific primers were designed and four chitinases were
fished out from the genomic DNA. Amplicons of EiChit1 and EhChit1 were found to
be of 1500 bp, while EiChit2 and EiChit3 produce 1200 bp amplicons. During PCR, a
mixture of amplicons were produced either containing “A” in their ends, or not. TA
vectors were used for cloning purpose by using those “A” overhangs as a linker. The
TA-chitinase clones were confirmed by restriction enzyme digestion and sequencing.

For expression purpose bacterial system was used for high protein yield and easy
maintenance. pQE30 expression system was used which contain N-terminal 6xHis tag
for one step purification. This 6 Histidine tag was rarely found to disturb the protein
folding and modulate the three dimensional structure, so removal of this tag from
expressed protein is not a necessity. All cloned fragments were cleaved by using
restriction enzymes and ligated to linearized pQE30 vector.

Confirmed pQE30 clones were used for protein expression in M15 strain of E. coli.
M15 strain contains pREP4 plasmid which produces the lac repressor to help tight
transcription regulation. IPTG was used as inducer. Over-expressed proteins were
observed only in IPTG induced bacteria. All four expressed proteins were analysed in
SDS-PAGE, and they appear to be a little bit heavier than their calculated weight due
to different SDS binding and different level of denaturation. Addition of 6xHis tag and
restriction sites also put some extra amino acid. Confirmation of expression was done
by western blot with monoclonal anti-His antibody.

All expressed proteins were identified primarily in membrane bound fraction, which
obstruct the purification and further characterization. Appearance of over-expressed
proteins in membrane bound fraction may occur due to high translation rate and
improper folding. So, IPTG concentration was lowered to reduce the expression level.
Lower post induction incubation temperature was introduced to inhibit the improper
protein folding. Finally all the proteins were obtained in soluble fraction after using
0.2-0.5 mM IPTG and 15°C as post induction incubation temperature.

Purification of soluble protein was done in single step using Ni-NTA agarose beads.
Nitrilotriacetic acid (NTA), the chelating adsorbent, occupies four of the six ligand

87
binding sites in Ni+2 ion. Two free sites in the coordination sphere of nickel ion
interact with the Histidine tag. The histidine residues become protonated below pH 4-
5, and no longer remain in bound condition with the nickel ions. But lowering the pH
of elution buffer may create improper protein folding, so high concentration of
imidazole was used instead, as 6xHis tagged protein couldn’t compete with them for
binding sites on Ni-NTA beads. Maximum amount of protein was eluted with 250 mM
imidazole solution.

4.5 Conclusions

• All Ei and Eh chitinases were amplified from genomic DNA using gene
specific primers.
• Amplified fragments were cloned in pGEMT vector using T/A overhangs.
• Recombinant clones were selected and confirmed by sequencing.
• Sub-cloning was done in pQE30 vector and confirmed.
• Recombinant protein expression was confirmed with 12% SDS-PAGE and
western blotting.
• Induction conditions were standardized to obtain maximum soluble protein.
• Purification of soluble proteins was done using Ni-NTA affinity
chromatography.

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 Chapter 5

Enzymatic characterization of Entamoeba chitinases

90
5.1 Abstract

Enzymatic ability and efficiency of all four recombinant Entamoeba chitinases are
described in this chapter. All four recombinant chitinases demonstrate β (1-4) bond
cleavage activity against the soluble substrate, while both EiChit2 and EiChit3 are
found to be more efficient than other two. All Ei chitinases are able to bind the
insoluble substrate though two of them (EiChit2 and EiChit2) lacks the chitin binding
domain. All Entamoeba chitinase shows the ability of hydroxylation of insoluble
substrate and release dimers from the polymer substrate end as exochitinase. All Ei
chitinases get inhibited by methylxanthine derivatives at micro molar level and
inhibited in different manner which signifies the difference of their substrate binding
mode.

5.2 Introduction

Chitinases (EC 3.2.1.14) are glycosyl hydrolases that degrade the chitin, an insoluble
linear β (1-4) linked polymer of N-acetylglucosamine (GlcNAc) by cleaving its β (1-
4) glycosidic bonds. During the last few decades, chitinases have been considered as a
potential “bio-tool” because of their wide range of applications in different industries.
Applications of chitinases included the preparation of protoplasts from fungi, as
protective agent against plant-pathogenic fungi, and in the processing of biologically
active oligosaccharides. Enzymatically produced chito-oligomers have been in lime
light of interest in recent years due to their broad applications in medical and
agricultural fields as hypocholesterolemic, anti-hypertensive, antioxidative agents
(Chen et al., 2003) as well as for antibacterial and antifungal activity.

Different organisms produce chitinase for different purposes depending on their

biology, food habit, and parasitic invasion. Bacterial chitinases are probably used for
food processing and digestion, as well as play an important role in the marine ecology
by recycling the chitin pool (Bassler et al., 1991). In fungi, chitinases apparently help
to degrade and mobilize the organic matter and probably to antagonize the growth of
competitors, along with helping in cell separation. Plant chitinases seems to play a
defensive role against pathogenic or pestiferous organisms (Boller, 1986); while for
arthropods these enzymes were found to be involved during moulting, cuticle turnover
and host invasion. The exact role of chitinase in vertebrates specially in human is

91
uncertain, where its role may include chitinious food degradation to immune response
against fungal infections (Gooday, 1986).

In general, chitinolytic enzymes can be divided into three categories, depending on its
substrate cleaving pattern. They are categorized as exochitinase demonstrating
cleavage activity only for the end of the chitin chain and endochitinase hydrolyzing
internal β (1-4) glycosidic bonds. Another class β-N-acetylglucosaminidase cleaves
GlcNAc units sequentially from the non-reducing end of the substrate. Naturally the
enzyme cleaves the substrate in random fashion, at internal locations over the entire
length of the polymer, producing soluble, low molecular mass multimers of GlcNAc,
such as chitotetraose, chitotriose, and chitobiose, with the smallest oligosaccharides
being predominant.

All members of family 18 chitinases shows structurally similar catalytic domain, made
up of 8 folds of consecutive α and β domains. Conserved aromatic amino acid residues
along with several polar residues are present throughout the catalytic cleft to assist the
substrate binding. Polymeric chain of chitin binds with the catalytic domain, but at
once only 4-5 monomer units are accommodated within the catalytic cleft of family 18
chitinases. As per the substrate subsite nomenclature, +1 and +2 subsites are found to
be important during enzyme-substrate interaction (van Aalten et al., 2001).

Most of the well studied chitinases belongs to glycosidase family 18 and found to
degrade the chitin with retention of the stereochemistry at the anomeric carbon (Tews
et al., 1997) (Honda et al., 2000). The classical reaction mechanism of substrate
molecule retention was observed in lysozyme (Davies et al., 1998) and amylase
(Uitdehaag et al., 1999). In chitinase this reaction is presumed to be initiated by
distortion of the -1 sugar ring and protonation of the glycosidic oxygen by a
protonated acidic residue. The classic lysozyme mechanism differs from chitinases,
during the subsequent nucleophilic attack involving the N-acetyl group of the -1 sugar,
rather than a carboxylate side chain on the protein (Terwisscha et al., 1995; Kobayashi
et al., 1996). The first step of hydrolysis results in cleavage of the sugar chain and
formation of an oxazolinium ion intermediate, which get hydrolyzed in next step to
complete the reaction (Brameld et al., 1998). The intermediate oxazolinium group
helps in electron transfer and balancing the charge distribution, during the hydrolysis.

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As, chitinases are important in fungi and insect for maintaining their life cycle,
inhibition of chitinases was found to be a good target for pesticide design. Several
reported inhibitors of family 18 chitinase, mimic the oxazolinium intermediate
structure. Allosamidine, is one such inhibitor, isolated from Streptomyces sp
reportedly inhibits family 18 chitinases at nanomolar level (Sakuda et al., 1986). This
pseudotrisaccharide is commercially unavailable, which leads the search for potent
and synthetic inhibitors. Arfidin, Argadin and Cl4 are few such synthesized inhibitors
reported in recent past, though the high molecular weight, very low cLogP value
(logarithm of its partition coefficient between n-octanol and water log) and presence
of several stereocenters made them poor lead as drug (Rao et al., 2005). A
comparatively new avenue was discovered to identify potential drug molecule by
screening drug library. In one such study, few molecules belonging to methylxanthine
group of chemicals shows some interesting inhibitory effect (Rao et al., 2005). These
molecules have several advantage over others, and can act as a lead for more
refinement, which may increase their efficiency as inhibitors.

5.3 Results

5.3.1 β (1-4) bond cleavage efficiency

The natural substrate for chitinase is chitin or chitosan or the chito-oligomers. But due
to their crystalline structure and insolubility, they are not suitable to study the β (1-4)
bond cleavage efficiency of hydrolytic enzymes in vitro. To circumvent this problem
several artificial and soluble substrates were designed and made commercially
available for this purpose. These substrates contain small chito-oligomers, conjugated
with fluorescence or coloured compounds, which get cleaved and can be detected by
simple fluorogenic or colorimetric assay respectively.

In this study one of such artificial substrate, 4-methylumbelliferyl-tri-N-

acetylchitotrioside (Fig.5.1) was used for enzymatic study of EiChi1, EiChit2, EiChit3
and EhChit1, which contains a fluorogenic compound 4-methylumbelliferyl (4MU)
attached to chito-triose. Upon enzymatic degradation the fluorogenic compound gets
cleaved at the β (1-4) bond. The Released 4-methylumbelliferyl gets excited at 360 nm
and emits fluorescence at 450 nm.

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 Fig. 5.1: Chemical composition of 4-methylumbelliferyl-tri-N-acetylchitotrioside

The substrate was dissolved in pure DMSO to prepare 1 mM stock solution. Purified
proteins of required amount were added to a typical reaction mixture of 300 µl
containing 200 mM sodium phosphate buffer (pH-7.2) and 1-5 µM substrate. After 15-
30 min of incubation at 25˚C, the reaction was stopped by 200 mM of sodium
carbonate buffer. The product was quantified by Varian spectro-fluorometer, model
Cary-Eclipse equipped with a Xenon lamp. The concentration of the fluorescent 4-
methylumbelliferone product generated from these substrates was determined using
4MU as standards. One unit of chitinase activity was defined as the amount of enzyme
required to release 1 nmol of 4-methylumbelliferone (4MU) per second at optimum
temperature.

To identify the optimum pH for each Ei chitinases 200 mM sodium phosphate buffer
was used for pH range of 5-8 and 200 mM sodium acetate buffer was used for pH
range of 3-5. Optimum temperature was determined by repeating the assay within the
range of 4-45˚C.

To determine the enzymatic properties Michaelis–Menten equation was used which

relates the initial reaction rate v0 to the substrate concentration [S]. The corresponding
graph represented a hyperbolic function and the maximum rate was described as Vmax.
Hence the derived equation was v0 = Vmax [S]/(Km + [S]), where Km is Michaelis
constant. The equation, plotted using Lineweaver–Burk plot was widely used to
determine important terms in enzyme kinetics, such as Km and Vmax. Kinetics values for
recombinant chitinases were calculated by the Lineweaver–Burk plot, using the
enzyme kinetics module of Sigma plot (version 11) software, and tabulated in table 5.1.

EiChi2 and EiChit3 show comparatively high substrate affinity and high bond cleavage
efficiency than that of both EiChi1 and EhChi1. Optimum pH of all four chitinases was

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found to be same for this artificial substrate. The optimum temperature differs among
EiChi2, EiChit3 and EiChit1.

Table 5.1: Enzyme kinetics values of EiChit1, EiChit2 and EiChit3.

Protein Km[µM] Vmax Optimum pH Optimum

[nmol/ug/s] Temperature
EiChit1 6.31 0.47 7.20 37˚C
EiChit2 3.05 1.15 7.20 25˚C
EiChit3 1.91 1.07 7.20 25˚C
EhChit1 5.60 0.61 7.20 37˚C

5.3.2 Insoluble substrate binding efficiency

As a crystalline polysaccharide, chitin is completely insoluble in water or any other

kind of organic solvent. Chitosan, deacetylated form of chitin, is relatively more
soluble than its acetylated form. So, in order to degrade the natural polymeric
substrates chitinase needs to bind with the substrate at first, either by the N or C
terminal extra domain or by the catalytic domain. This extra domain was termed as
chitin binding domain (ChBD) due to its similarity with cellulose binding domain.
Presences of several aromatic and polar residues assist this non-covalent binding,
either by ionic or hydrophobic interaction. Substrate binding efficiency of chitinase is
important for its hydrolytic activity, as it influence the enzyme processivity.

To understand the substrate binding efficiency and the mode of ligand and enzyme
interaction, three insoluble substrates of different conformations were used in this
study. Spherical chitin beads and chitin dust were obtained commercially, while
amorphous colloidal chitin was prepared from chitin flakes (Hackman, 1962;
Jeuniaux, 1966).

Among the three substrates, spherical chitin beads has maximum exposed surface,
while the acid treated colloidal chitin have loosely bound fibrils (Fig. 5.2). Purified Ei
Chitinases were incubated with the substrates for 2 hr with constant agitation. The
bound and unbound fraction of proteins were separated by centrifugation, and
analyzed in 12% SDS PAGE (Fig. 5.3A).

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Fig. 5.2: Images of Chitin beads, colloidal chitin and chitin dust, 10 x magnifications in phase
contrast microscope.

To identify the nonspecific binding, BSA (fraction five) was used as control. The
protein bands were scanned by densitometric scanner to determine the percentage of
bound proteins and the values were plotted after deduction of blanks (Fig. 5.3 B).

All three chitinases show different level of binding efficiency, while EiChit1
demonstrate maximum binding efficiency with all three different substrates. EiChit2
and EiChit3 were found to bind efficiently with chitin beads and other insoluble
substrates though they lack of the specific chitin binding domain. From the
densitometric scan, it can be concluded that the chitinases bind maximum to chitin
beads, and least to chitin dust.

Fig. 5.3: SDS-PAGE analysis of different substrate binding efficiency of three Ei chitinases
[A]. Graphical representation of percentage of bound protein calculated from band intensity of
bound fraction obtained from Densitometry scan [B].

To identify the nature of ligand-enzyme interaction, equal amount of enzymes were

incubated with equal amount of chitin beads (100 µl bead volume) for 2 hr in presence

96
of binding buffer of different pH. EiChit2 and EiChit3 bind most at pH 5, while
EiChit1 binds most at pH 4.

5.3.3 Insoluble substrate cleavage efficiency

All recombinant Entamoeba chitinases show substrate binding affinity, which may
indicates their ability to degrade insoluble chitin. Colloidal chitin was used as
substrate as it contains more loose ends and comprises a relatively less compact
conformation. Fixed amount of colloidal chitin was incubated with fixed amount of
purified chitinases for 2 hr, in presence of 200 mM sodium phosphate buffer (pH-7.2).
The supernatant was collected by high speed centrifugation and analyzed in reverse
phase HPLC with C18 column and water-acetonitrile mobile phase. Elution was done
with water and product detection was done at 210 nm. Standard chito-oligomers were
used to identify the product structure. Product profile of colloid chitin was plotted
using multi-curve plotting option of Origin 8 software (Fig 5.4). All three chitinases
degrade colloid chitin and predominantly produce chitobiose, which classify them as
exochitinase. EiChit2 and EiChit3 show high colloid chitin degradation ability than
those of EiChit1 and EhChit1.

Fig. 5.4: Colloidal chitin cleavage patterns of EiChit1, EiChit2, EiChit3 [A] and EhChi1 [B].
Product absorbance intensity was quantified in arbitrary unit (A.U) and plotted in Y axis. The
standard Chitobiose peak is indicated with black arrow. Difference of Y axis scale should be
noted.

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5.3.4 Inhibitor assay using Methylxanthine compounds

Allosamidine mimics the oxazolinium ring and interact with the conserved aromatic
residues of catalytic cleft. Mostly all family 18 chitinase get inhibited by allosamidine,
as they share the common sequence and TIM barrel structure. Methylxanthine
derivatives were used as drug for treating asthma and inhibiting phosphodiesterase.
Pentoxifylline, Caffeine and Theophylline are such derivatives, with different no. of
attached methyl groups to a xanthine ring (Fig. 5.5)

These compounds were used as chitinase inhibitors and found to inhibit S.

marcenance chitinases at micromolar level (Rao et al., 2005). Interactions between the
aromatic residues and inhibitors were well studied and pentoxifylline was predicted to
be a potential inhibitor source molecule.

Fig. 5.5: Chemical structures of Pentoxifylline (A), Caffeine (B) and Theophylline (C)

These three compounds were analyzed in this study, to detect their binding property
and inhibitory effect on Ei chitinases. All three compounds were dissolved in water to
prepare 10 mM stock and used 50-150 µM range for each of the chitinases. 4-
methylumbelliferyl-tri-N-acetylchitotrioside was used as substrate with 200 mM
sodium phosphate buffer (pH-7.2). The product formation was quantified and used for
further calculation. Single substrate-single inhibitor option of Enzyme kinetic module
of Sigma Plot software was used to investigate the inhibition mode. Lineweaver-burk
plot was used to calculate the Ki value (Table 5.2). Pentoxifylline was observed to
show maximum inhibitory effect in general, while caffeine was found to show
minimum Ki values.

The mode of inhibition was determined by plotting the S and V values in competitive,
non competitive and un-competitive inhibition mode available in Sigma plot enzyme
kinetics module (version 9) (Table 5.3).

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 Table 5.2: Ki values (µM) of different inhibitors against Ei Chitinases

Protein Pentoxifylline Caffeine Theophylline

EiChit1 39 21 93
EiChit2 123 570 1448
EiChit3 33 22 25

The best fit inhibition model was selected by comparing the r2 values of different plots,
the accepted plots are presented in appendix C. Interestingly Pentoxifylline, Caffeine
and Theophylline behave in different way for these enzymes, though the three
dimensional structure of these three enzymes are exactly same.

Table 5.3: Inhibition modes of different methylxanthine derivatives against Ei Chitinases

Protein Pentoxifylline Caffeine Theophylline

EiChit1 Competitive Competitive Competitive

EiChit2 Uncompetitive Competitive Uncompetitive
EiChit3 Uncompetitive Non-competitive Non-competitive

5.4 Discussion

From the computational study it was predicted that all four Entamoeba chitinase
belongs to family 18 with same conserved residues within the catalytic cleft. To
ascertain the predictions, characterization of enzymatic properties of all four chitinases
was done by some well established assays.

Artificial substrate was used to identify the β (1-4) bond cleavage or hydrolytic
efficiency of these proteins. Full length EiChit1 and EhChit1 were found to be less
efficient than EiChit2 and EiChit3, by having a low specific activity and high Km. As
well as they require more incubation period in comparison to EiChit2 and EiChit3.
Binding of small chitotrioside substrate was facilitated by both EiChit2 and EiChit3,
probably due to the absence of the ChBD. The lower efficiency and lower turnover rate
of both EiChit1 and EhChit1 may be explained by the presence of extra domains
(ChBD and the heapta petide repeats).

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All the recombinant chitinases show optimum activity around the pH 7-7.2, for the
artificial substrate with resemblance to the physiological condition in vitro.

Optimum temperature range was determined between 5˚C to 45˚C. While for both
EiChit2 and EiChit3, the optimum temperature was found to be 25˚C, the EiChit1
showed its optimum activity at 37˚C and a reasonable amount of activity from 25°C-
40°C. The maximum activity of EiChit2 and EiChit3 around 25˚C can be explained by
the ambient temperature needed for E. invadens growth in vitro as well as within its
poikilothermal host. So, these enzymes probably get activated within the host body
during excystation. Interestingly, the different behaviour of EiChit1 might be explained
by its localization within the cyst and its two way function. As EiChit1 was detected in
cyst wall, it probably gets exposed to a wide range of temperature. Thus the stability of
EiChit1 within a long range of temperature is needed for its required enzymatic activity
during excystation. EhChit1 shows maximum activity at 37˚C, which resembles its host
temperature.

All recombinant chitinases were assessed to identify their insoluble substrate cleavage
ability, by using amorphous colloidal chitin dust as substrate. All chitinases
predominantly produce chitobiose as exochitinase. Among them, EiChit2 and EiChit3
demonstrate more processivity than both EiChit1 and EhChit1. So, it can be presumed
that, both recombinant EiChit2 and EiChit3 were better enzymes, and cleave their
natural substrate with an ease. The EhChit1 shows the lowest activity against colloidal
chitin.

EiChit1 showed low substrate binding affinity towards the soluble substrate, along with
very slow processivity, which describe it as a poor hydrolytic enzyme, which repeated
with colloidal chitin proving its slow and poor processivity again. Interestingly, the
poor performance of EiChit1 against insoluble substrate may be important, because
during encystation EiChit1 gets transported to the cyst wall, and reside there with chitin
fibrils. In that situation, its low hydrolytic activity against chitin seems to play an
important role without disturbing the cyst wall integrity.

During the substrate binding assay all three Ei chitinases demonstrate enough binding
ability irrespective of the physical difference of three substrates. The chitin binding
efficiency of EiChit2 was comparatively less than that of other two chitinase, as it is
evident from the SDS-PAGE, (Fig 5.3) that though equal amount of chitinases were

100
incubated with equal amount of insoluble chitin, in case of EiChit2 the amount of
bound and unbound enzyme is almost equal, where as in case of EiChit1 and EiChit3
there were no detectable protein in the unbound fractions. The occurrence of binding
ability could be due to the exposed aromatic residues of surface groove of catalytic
domain only, as reported earlier in other prokaryotic chitinases (Suzuki et al., 1999).
From the densitometry scan of band intensity, it was observed that, chitinases binds
most efficiently with chitin beads, which is comparable with colloidal chitin and far
better than chitin dusts. The enzyme binding ability probably depends on the three
dimensional aspects of the substrates and exposed surface also, as the spherical beads
with maximum exposed surface increased the bound protein. Colloidal chitin contains
rough and fibrilar surface which enhance the enzyme binding ability, while the
crystalline chitin dust have lowest amount of available surface and thus less amount of
protein gets bound to it.

It was observed that maximum binding for EiChit2 and EiChit3 occurs at pH 5, while
for EiChit1 it was pH 4. This indicates the involvement of hydrophobic interaction
between the catalytic domain of enzymes and the substrate (Rao et al., 2005).

Three derivatives were observed to inhibit the Ei chitinases, though the inhibition was
found to be around micro molar level as reported previously (Rao et al., 2005).
Pentoxifylline shows lowest Ki values against all three enzymes, which made it the
most potential inhibitor among these three. These derivatives were probably bound
with the aromatic residues of the catalytic domain and mimic the oxazolinium
intermediate.

All three derivatives inhibited each enzyme in more or less similar ways such as either
competitively or non-competitively. Pentoxifylline, Caffeine and Theophylline
inhibited EiChit1 competitively, in spite of their structural differences, which may
explain by the involvement of only the methylxanthine ring. The side groups attached
to the methylxanthine ring probably play no role in the enzyme binding. Among the
three derivatives, Caffeine inhibits two of them competitively, though the Ki values
were high. EiChit2 and EiChit3 were inhibited either non-competitively or
uncompetitively and thus separate themselves from EiChit1. Probably the substrate
binding mode of EiChit1 is different from EiChit2 and EiChit3 in spite of their

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exceptional similarity in primary sequence and three dimensional structures, and
reflected in the enzymatic kinetics and inhibitor assays of EiChit2 and EiChit3.

From the phylogenetic studies it was concluded that, EiChit3 and EiChit2 originated
before EiChit1, and have more similarity with each other, than with EiChit1. In the
light of enzymatic profile it can be concluded that both EiChit2 and EiChit3 originate
to serve different purpose than EiChit1. Native EiChit1 may have a lower ranking as a
hydrolytic enzyme and primarily work as a polymer connector. The main rationale
behind the evolution of EiChit2 and EiChit3 may explained by their hydrolytic
efficiency rather than their substrate binding ability.

Entamoeba histolytica contain single chitinase, which probably play the both roles as
connector and hydrolytic enzyme in vivo.

5.5 Conclusions

• All recombinant Entamoeba chitinases shows β (1-4) bond cleavage activity

against soluble artificial substrate. EiChit1 and EhChit1 were determined to be
the slow and least efficient enzyme among the four.
• All chitinases were able to degrade colloidal chitin and release chitobiose from
the chitin polymer end, which categorized them as exochitinase. In this case
also the EiChit1 and EhChit1 show lowest activity against the insoluble
substrate.
• All three E. invadens chitinases bound chitinious substrates, though EiChit2
and EiChit3 lack the specific chitin binding domain. EiChit1 and EiChit3
showed more affinity towards the insoluble substrates rather than EiChit2. The
binding force may generate through hydrophobic interaction.
• Three methylxanthine derivatives were able to inhibit the recombinant Ei
chitinases at micro molar range. Pentoxifylline was found to be more efficient
inhibitor than Caffeine and Theophylline.
• Different mode of enzyme inhibition probably indicate different substrate
binding mode within three Ei chitinases.
• Phylogenetic difference was supported by the enzymatic characterization,
which confirms the similarity between EiChit2 and EiChit3.

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 Chapter 6

Characterization of N-terminal Chitin binding Domain of EiChit1

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6.1 Abstract

Molecular characterization of 60 amino acid long chitin binding domain of EiChit1 is

depicted in this chapter. This exclusive domain bears no resemblance with other
known ChBD and has conserved aromatic residues. Two fusion proteins were created
by replacing this domain with two lectin ChBDs of 60 amino acids (JacobChBD) and 60
amino acids (JessieChBD). Truncated EiChit1CAT was created by removing the wild
EiChit1ChBD. Removal of ChBD enhances the hydrolytic efficiency of truncated
EiChit1. Fusion proteins with different ChBD show different level of diminished
hydrolytic activity against soluble substrates. Affinity towards insoluble substrates
increased with increased enzymatic efficiency. Substrate binding efficiency found to
depends on the number and position of aromatic residues within the domain.

6.2 Introduction

Many polysaccharide hydrolases reported to contain discrete substrate-binding

domains, like the cellulose-binding domains (CBDs) of cellulases which have been
studied most extensively. The three-dimensional structures of CBDs of Trichoderma
reesei cellobiohydrolase I (Kraulis et al., 1989), Cellulomonas fimi β(1-4) glucanase
Cex (Johnson et al., 1996), Erwinia chrysanthemi endoglucanase Z (Brun et al., 1997)
and Clostridium thermocellum cellulosomal scaffolding subunit (Tormo et al., 1996)
have been determined and reported. From the structural analysis, CBDs suggested to
have a hydrophobic face constructed by linearly exposed aromatic amino acid
residues, for binding the sugar rings of the crystalline cellulose through hydrophobic
interactions (Bray et al., 1996; Mattinen et al., 1997). The exact role of CBDs in the
activity of cellulase is not clear, though two hypotheses have been proposed in defence
of their evolution and presence such as the CBDs simply increase the local enzyme
concentration on insoluble substrates (Tomme et al., 1988), or they facilitate the
disruption of cellulose fiber without enzymatic hydrolysis (Din et al., 1991).

On the same note the presence of Chitin binding domain (ChBDs) and putative ChBDs
have been suggested and observed in many chitinases, though detailed molecular and
biochemical analyses of these ChBDs have not yet been reported. Conserved 6-8
cysteine residues and few aromatic residues were found to be the signature sequence
of any ChBD. The penultimate residue, a tryptophan, is supposedly required for

105
interaction with the substrate, and at least two of these conserved residues are required
for binding activity and are predicted to participate in binding to glucosyl units through
hydrophobic or hydrogen bonding (Svensson et al., 1989).

To understand the degradation mechanisms of insoluble chitin by chitinases,

biochemical and structural studies of ChBDs are indispensable which can answer a
few questions about their origin. The detailed study with Bacillus circulans
ChBDChiA1 revealed that it bind specifically with the chitin and does not have an
array of aromatic residues within its surface as expected, suggesting a different way of
binding mechanism other than the cellulose binding domains (Hashimoto et al., 2000).
Removal of ChBD from Human chitinase is found to have no affect on the
hydrolyzing ability of the enzyme against the soluble substrate triacetylchitotriose, but
diminishes the hydrolytic ability. Mutation of any one of 6 cysteine of human ChBD to
serine results in complete loss of chitin binding activity (Tjoelker et al., 2000).

Most of the single or multiple C or N terminal chitin binding domains were further
classified in three domain types, such as ChtBD1 (SMART accession number:
SM00270), ChtBD2 (SMART accession number: SM00494) and ChtBD3 (SMART
accession number: SM00495) depending on their location, structure and amino acid
residues.

Till now 559 Chitin-binding domain type 1(ChtBD1) were identified among 402
proteins and contains approx 43 amino acid residues. This domain resides at the N-
terminus of the mature protein and may occur in one or more copies. A number of
plant and fungal proteins that bind N-acetylglucosamine (e.g. solanaceous lectins of
tomato and potato, plant endochitinases) contain this domain.

Chitin-binding domain type 2 (ChtBD2) generally belongs to insect and vertebrate

chitin binding proteins and contain six conserved cysteine. Few copies of this domain
were also found in some baculoviruses, peritrophic matrix proteins of insects and
animal chitinases. ChtBD2 domains contain 2219 members among 980 proteins.

Chitin-binding domain type 3(ChtBD3) was found to present in 521 proteins of

bacterial origin and represented by 818 members. It is a short domain found in
different glycosyl hydrolase enzymes, such as the C-terminal cellulose-binding

106
domain of endoglucanase Z. The domain has a core structure consisting of a 3-
stranded meander beta-sheet containing six aromatic groups.

Plant chitin-binding domain is mostly composed of 30–43 residues including eight

cysteines, three aromatic residues and glycines and frequently referred as hevein
domain, showing antimicrobial activity (Andersen et al., 1993). In invertebrates, the
65 residue long chitin-binding domain includes a high percentage of cysteine and
aromatic residues. An invertebrate chitin binding domain Tachycitin, was found in
Tachypleus tridentatus. This 73-residue polypeptide has several conserved aromatic
residues such as Cys, Pro, and Gly, having significant structural value (Suetake et al.,
2000). On the basis of such similarity between plant and invertebrate chitin-binding
proteins, a hypothesis was proposed that they are correlated by a rare evolutional
process, convergent evolution, i.e. proteins from different origins develop to construct
the same active site structure to acquire the same function (Shen et al., 1999).

Entamoeba hsitolytica and Entamoeba invadens were found to contain several chitin
lectins and chitinases containing chitin binding domains having conserved cysteines
which probably form disulphide bridges (Frisardi et al., 2000). Entamoeba chitin
binding lectins, Jacob contains several chitin binding domains with 6 conserved
cysteines residues, while the N terminal chitin binding domain of Entamoeba
chitinases and Jessie were found to have 8 cysteine (Van Dellen et al., 2002) (Fig.
6.1). Few of those putative lectins show several post translational modifications and
proteolytic cleavage sites probably for producing small fragments which required for
substrate binding (Van Dellen et al., 2006). ChBD of Entamoeba chitinases and other
chitin lectins differ in sequence and their activity may be different.

Fig. 6.1: Schematic diagrams of Ei Lectins

The ChBD of chitinases probably facilitates the accumulation of the enzymes, while
the lectins ChBDs may assist in the formation and maintenance of intricate cyst wall
structure [Van dellen et al, 2006; Chatterjee et al, 2009 (in press)]. As the catalytic

107
domain of all family 18 chitinases were found to be evolutionary conserved, it may not
act as a potential drug or antibody target in human system. Exclusive domain or ChBD
of Eh and Ei may be used as potential target due to its distinctive sequence. EhChBD
show very low structural similarity with EiChBD except of conserved 8 cysteine
residues and few aromatic residues.

6.3 Results

6.3.1 Entamoeba ChBD and fusion protein

Protein sequences of EiChit1ChBD, other lectin ChBD and other reported ChBDs of
different origin were aligned using Clustal W programme, to identify the probable
substrate binding aromatic residues. Characterization of this ~60 amino acid fragment
could be done by mutagenesis studies of aromatic residues to understand their role.
But the amino acid sequence of EiChit1 ChBD was found to be unique, without
having any similarity with any well studied ChBD, hinder the mutagenesis and
homology studies. So replacement and removal of ChBD is used here to study the role
of EiChit1ChBD in its enzymatic activity.

EiChit1 contains one N terminal 60 amino acid long ChBD, while seven Jacob and
three Jessie lectins contain multiple ChBD and other unidentified domain. Among the
lectins Jacob1 (11-14%) and Jessie3b (18-21%) found to be present in the cyst wall in
comparatively large amount, consisting about 70% of total cyst wall protein along
with chitinase1 (Van Dellen et al., 2006).

Jacob ChBD EiChit1R-CAT

Jessie ChBD EiChit1R-CAT

EiChit1CAT

Fig. 6.2: Schematic diagrams of fusion and truncated proteins

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ChBD of these two lectins (JacobChBD and JessieChBD) were selected due to their high
abundance and probable high substrate binding capacity. Complete removal of ChBD
was done to creating a truncated EiChit1 (EiChit1CAT) (Fig. 6.2).

To understand the expected morphological and other changes, which may occur due to
the modification, primary sequence analyzing tools from www.expasy.org were used
to gather few preliminary information regarding the truncated and fusion proteins.
Theoretical Mol. wt and pI of these three proteins were calculated and tabulated in
Table 6.1.

Table 6.1: Theoretical Mol. wt. and pI of truncated and fusion proteins.

Protein Base pairs Amino acid Mol. wt. (kDa) pI

EiChit1CAT 1098 ~365 41.25 5.53

JacobChBD-EiChit1R-CAT ~1476 ~493 54.55 4.80

JessieChBD-EiChit1R-CAT ~1506 ~502 55.25 4.75

6.3.2 Cloning of EiChit1CAT, EiChit1R-CAT, JacobChBD and Jessie ChBD

Nucleotide and protein sequences of EiJacob1 and EiJessie3b were obtained from
GenBank.

Primers were designed accordingly to amplify only 180 bp (JacobChBD) and 210 bp
(JessieChBD) long ChBDs of Jacob1 and Jessie3b respectively. Primers were designed for
EiChit1R-CAT (catalytic domain of EiChit1with hepta peptide repeat) and EiChit1CAT
(catalytic domain of EiChit1) also. Forward and reverse primers were designed
incorporating BamHI and BglII sites respectively for JacobChBD and JessieChBD. BglII
and SalI were used in EiChit1R-CAT forward and reverse primers respectively. Primers
for EiChit1CAT fragment were designed by incorporating BamHI and XhoI sites
respectively. Primer sequences were tabulated in materials and methods (Table 3.3).

PCR amplification was done using High Fidelity Expand Taq polymerase (Roche),
and PCR amplified products was verified in 0.8% agarose gel (Fig. 6.3).

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 Fig. 6.3: Amplicons of JacobChBD (180bp), Jessie ChBD (210 bp), EiChit1R-CAT (1296 bp) and
EiChit1CAT (1098 bp). M represents 1kb DNA molecular marker. The amplified products were
indicated by black arrows.

Amplified fragments of JacobChBD, JessieChBD and EiChit1R-CAT were purified from the
gel by gel elution kit (Qiagen, Germany) and cloned primarily in pTZ57R/T vector
(Fermentas) using the T/A overhangs. Truncated fragment EiChit1CAT was cloned in
pGEMT vector (Promega, USA) using the same basic principal. The ligated mixtures
were used to transform E. coli, Dh5α cells for clone selection. Recombinant clones
were selected preliminary by gel retardation study.

All TA clones of JacobChBD, JessieChBD and EiChit1R-CAT- pTZ57R/T were selected by

restriction enzyme digestion and PCR amplification, using respective sense and
antisense primers. Confirmed clones were sequenced and used further for fusion
fragment preparation.

6.3.3 Construction of JacobChBD-EiChit1R-CAT and JessieChBD-EiChit1R-CAT fused

fragments

Fusion of gene fragments was done by overlapping PCR. JacobChBD-pTZ57R/T,

JessieChBD-pTZ57R/T and EiChit1R-CAT-pTZ57R/T clones were digested with BamHI-
BglII and BglII-SalI combination respectively. Digested ChBD inserts with staggered
ends from BamHI-BglII digestion were ligated with BglII-SalI digested EiChit1R-CAT
fragment in presence of T4 Ligase. The staggered end of ChBD created by BglII was
supposed to ligate with the BglII end of EiChit1R-CAT. The chance of self ligation or
ligation in wrong orientation was detected and taken care of by further amplification
from the ligation mixture. Sense primers of ChBD fragments and antisense primers of

110
EiChit1R-CAT [JacChBD (forward), EiChi1R-CAT (reverse) and JessieChBD (forward),
EiChi1R-CAT (reverse) primers] were used to eliminate the incorporation of
homoduplex fragment. Amplified fragments of JacobChBD-EiChit1R-CAT (1476 bp) and
Jessie ChBD-EiChit1R-CAT (1506 bp) were analyzed in 0.8% agarose gel (Fig. 6.4).

Fig. 6.4: Amplified fragments of JacobChBD-EiChit1R-CAT (1476 bp) and Jessie ChBD- EiChit1R-
CAT (1506 bp). M represents 100 bp and 1kb DNA ladders. The amplified products were
indicated with black arrows.

The amplified products were directly ligated to pTZ57R/T for further cloning.
pTZ57R/T clones of JacobChBD-EiChit1R-CAT and JessieChBD-EiChit1R-CAT were
confirmed with restriction enzyme digestion (Fig. 6.5)

Fig. 6.5: Panel A-Restriction enzyme digested pTZ57R/T clones of JacobChBD-EiChit1R-CAT and
Jessie ChBD- EiChit1R-CAT. Lane 1- Undigested pTZ57R/T -JacobChBD-EiChit1R-CAT clone, Lane
2- Digested pTZ57R/T -JacobChBD-EiChit1R-CAT clone, Lane 3- Undigested pTZ57R/T -
JessieChBD-EiChit1R-CAT clone, Lane 4- pTZ57R/T -JessieChBD-EiChit1R-CAT clone, Lane M- 1kb
DNA ladder. Panel B- Restriction enzyme digested pGEMT-T clone of EiChit1CAT. Lane 1-
Digested pGEMT-EiChit1CAT clone, Lane M-1 kb plus DNA ladder. The inserts are indicated
with black arrows.

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6.3.4 Cloning, Expression and purification of EiChit1CAT, JacobChBD-EiChit1R-CAT
and JessieChBD- EiChit1R-CAT fragments in pQE30 vector

Cloned fusion fragments and truncated fragments were cloned into bacterial
expression vector pQE30 vector for expression purpose. Clone was confirmed by
BamHI and HindIII restriction enzyme digestion. Expression profiles were checked in
E. coli M15 strain after induction with 1 mM IPTG for 5 hr as post induction
incubation period at 37˚C (Fig. 6.6). Comparative analysis was done between un-
induced and induced bacterial culture. Total bacterial protein was boiled with 1X
sample buffer and analyzed in 12% SDS-PAGE with protein molecular wt. marker.
Over expressed protein band of expected mol. wt was observed in the induced lane
only. After separation of membrane bound and soluble protein fraction by sonication
and centrifugation, all over-expressed proteins were mostly found in membrane bound
fragments.

Further confirmation was done by western blot using monoclonal Anti His antibody,
protein A-HRP and DAB detection system (Fig. 6.7). To obtain maximum soluble
protein, optimization of protein expression was done by lowering IPTG concentration
and post induction incubation temperature. Different concentrations of IPTG (0.5-0.02
mM) along with increased incubation period were used for standardization. It was
observed that 0.2 mM IPTG and 16 hr incubation at 15˚C was able to produce
maximum protein in soluble fraction.

Fig. 6.6: Expression profile of JacobChBD-EiChit1R-CAT, JessieChBD-EiChit1R-CAT and EiChit1CAT

analysed in 15% and 12 % SDS-PAGE respectively. In panel A, Lane1-Uninduced JacobChBD-
EiChit1R-CAT M15 clone, Lane 2-Induced JacobChBD-EiChit1R-CAT M15 clone, Lane 3-
Uninduced JessieChBD-EiChit1R-CAT M15 clone, Lane 4-Induced JessieChBD-EiChit1R-CATM15
clone. In panel B, Lane 1-Induced EiChit1CAT M15 clone, M represent protein molecular
marker. The over expressed protein bands were indicated with black arrows.

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 Fig. 6.7: Western blots of EiChit1CAT, JacobChBD-EiChit1R-CAT and JessieChBD-EiChit1R-CAT
protein. Monoclonal Anti His Antibody used to detect the bands which are indicated with
black arrows. Approximate molecular weights are also given next to arrow.

Protein purification was done by using Ni-NTA agarose beads and elution was done
with 250 mM imidazole (Fig.6.8). The eluted fractions were analyzed in 12% SDS-
PAGE. Single step purification was found to be enough to purify the protein of interest
to ~80% homogeneity. Purified proteins were further concentrated using 30 kDa cut
off centricon to eliminate the contaminating proteins of low mol. wt. Protein
concentration were determined using Bradford reagent. Purified proteins were used for
further enzymatic studies.

Fig. 6.8: Purified fractions of fusion and truncated proteins. M represents protein molecular
ladder. The purified bands were indicated by black arrows.

6.3.5 β (1-4) bond cleavage efficiency of truncated and fusion protein

To identify the effect of subtraction/addition of ChBD on the hydrolytic ability of

EiChit1, the EiChit1CAT, along with JacobChBD-EiChit1R-CAT and JessieChBD-EiChit1R-
CAT were incubated with soluble substrates. Solution of 4-methylumbelliferyl-tri-N-
acetylchitotrioside (1-5 µM) was used with 200 mM sodium phosphate buffer (pH-

113
7.2) and incubated with the purified protein for 15-60 min. The reaction was stopped
by 200 mM sodium carbonate solution and the fluorescence product was measured and
quantified as mentioned before.

It was observed that EiChit1CAT is more active than its parent molecule and JacobChBD-
EiChit1R-CAT need more incubation period than EiChit1, while JessieChBD-EiChit1R-CAT
shows no detectable activity at all, even after 60 min incubation. Kinetics values for
these three fusion proteins were calculated by the Michaelis-Menten equation, using
the enzyme kinetics module of Sigma plot software, and tabulated in table 6.2. Both
EiChit1CAT and JacobChBD-EiChit1R-CAT demonstrate maximum activity against the
soluble substrate at 37˚C and pH 7.2, as their parent molecule.

Table 6.2: Enzyme kinetic properties of fusion and truncated protein.

Protein Km[ µM] Vmax[nmol/µg/s]

EiChit1CAT 4.32 3.57

JacobChBD-EiChit1R-CAT 30.60 0.0008
JessieChBD-EiChit1R-CAT N.D* N.D*

*N.D = not detected.

6.3.6 Insoluble substrate cleavage efficiency of JacobChBD-EiChit1R-CAT and

JessieChBD- EiChit1R-CAT

Both of these fusion proteins show very low/no activity against the soluble substrate,
which may be due to their modified three dimensional structures. To detect the
insoluble substrate cleavage efficiency colloidal chitin and chitin dust was used and
incubated with these fusion proteins. During the enzymatic studies of recombinant
chitinases, colloidal chitin was observed to act as a more accessible substrate due to
the presence of more open ends. Crystalline structure of chitin dust was found to be
more compact in nature and need long incubation period.

JacobChBD-EiChit1R-CAT and JessieChBD-EiChit1R-CAT were incubated with colloidal

chitin and 200 mM sodium phosphate buffer (pH 7.2) for 24 hr at room temperature
with constant agitation. The oligosaccharide products were separated by centrifugation
and the supernatant was analyzed by reverse phase HPLC as mentioned earlier.
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Oligosaccharide standards were analyzed and the predominant product was found to
be the dimers (chitobiose) (Fig. 6.9).

The elution profiles were plotted using multi-curve option of Origin 8 software and
plotting the absorbance values at Y axis. Both JacobChBD-EiChit1R-CAT and JessieChBD-
EiChit1R-CAT demonstrate cleavage ability against colloidal chitin. JacobChBD-EiChit1R-
CAT was found to produce comparatively more oligomer product than JessieChBD-
EiChit1R-CAT from colloidal chitin within same incubation period.

Fig. 6.9: HPLC profile of colloidal chitin degradation by JacobChBD-EiChit1R-CAT and

JessieChBD-EiChit1R-CAT. Product absorbance intensity was quantified in arbitrary unit (A.U)
and plotted in Y axis. The black arrow indicated Chitobiose standard.

JacobChBD-EiChit1R-CAT and JessieChBD-EiChit1R-CAT were incubated with Chitin dust

to identify their hydrolytic ability against a relatively more composed substrate, where
the accessible ends are not available.

A time dependent study was done by studying the supernatant obtained from reaction
mixtures of different incubation period (2-24 hr). Product analysis was done as earlier
(Fig. 6.10).

Increasing amount of product was observed in both cases, though JessieChBD-EiChit1R-

CAT was found to degrade the dust particle more quickly and after 24 hr, accumulates
more product than JacobChBD-EiChit1R-CAT.

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Fig. 6.10: Time dependent profiles of chitin dust degradation by JacobChBD-EiChit1R-CAT [A]
and JessieChBD-EiChit1R-CAT [B]. Product absorbance intensity was quantified in arbitrary unit
(A.U) and plotted in Y axis. Chitobiose standard was marked by black arrow. Difference in
scales should be noted.

6.3.7 Substrate binding efficiency of JacobChBD-EiChit1R-CAT and JessieChBD-

EiChit1R-CAT

Though both fusion protein JacobChBD-EiChit1R-CAT and JessieChBD-EiChit1R-CAT

exhibit insoluble substrate cleavage activity, their enzymatic profile differ from each
other. This difference depends to some extent on the substrate specificity. To identify
their substrate specificity, both of the fusion proteins were incubated with different
insoluble substrates such as chitin beads, colloidal chitin and chitin dust.

Binding assay was performed as described before and the bound and unbound
fractions were analysed in 12% SDS-PAGE, followed by staining and destaining (Fig
6.11A). Densitometry scan of SDS-PAGE was done to calculate the binding efficiency
and values are plotted on a graph (Fig 6.11 B). It was observed that, both proteins bind
preferentially to colloidal chitin, while the least binding occur with chitin dust.
Maximum substrate binding occur at different pH range, which coincide with their pI
values.

To recognize the maximum substrate binding affinity of the ChBD, both fusion
proteins and the native one were incubated with fixed amount (100 µl of suspension)
of chitin beads for 2 hr with constant agitation. Free and bound protein was separated
by centrifugation, and the free protein content of the supernatant was measured. Bound
protein fraction was calculated by subtracting the free protein amount from initial

116
amount. The values were plotted using Langmuir equation, using single site ligand
binding mode of Sigma plot software. Dissociation constant was calculated to identify
the best ChBD. It was observed that JessieChBD-EiChit1R-CAT bound most efficiently to
chitin beads. EiChit1 was found to be a little less competent than JessieChBD-EiChit1R-
CAT, but perform far better than JacobChBD-EiChit1R-CAT at substrate binding.

Fig. 6.11: SDS-PAGE analysis of substrate binding ability of fusion protein JacobChBD-
EiChit1R-CAT and JessieChBD-EiChit1R-CAT [A]. Graphical representation of percentage of bound
protein from densitometry scan values [B].

6.4 Discussion

All three Ei Chitinases show chitin binding ability in presence or in absence of specific
chitin binding domain, as the catalytic domain also bind to chitinious material to some
extent. But from the point of identifying new drug target, the catalytic domain of was
not found to be of good choice because of their conserved sequences throughout the
evolution. The ChBD of Ei chitinase 1 was found to be an exclusive domain, without
having any similarity with human ChBD. EiChit1ChBD doesn’t have any striking
similarity with EhChit1ChBD though the key cysteine and aromatic residues were found
to be placed strategically.

As the aromatic residues were believed to play an important role in substrate binding,
it was crucial to identify the important residues within EiChit1ChBD. To recognize these
key aromatic residues, wild EiChit1ChBD was replaced with two other lectins ChBDs
having six (JacobChBD) and eight cysteine (JessieChBD) residues. Two fusion proteins
JacobChBD-EiChit1R-CAT and JessieChBD-EiChit1R-CAT were created by restriction

117
enzyme digestion and PCR amplification. Expression of fusion proteins were done in
bacterial system.

Previously it was observed that EiChit1 show slow hydrolytic activity as well as low
substrate affinity towards soluble substrate which was probably due to the presence of
extra domain. So to reveal the role of this extra domain, a truncated EiChit1 was
expressed. Both fusion proteins are found to have lowered pI in comparison their
parent protein EiChit1, while truncated portion retain the same pI. So it can be
presumed that the removal of 60 N-terminal amino acids does not have any significant
effect in the total charge of the protein, while the addition of extra 60 amino acids
change the total charge towards the positive values probably making the fusion
proteins little more basic.

All fusion and truncated proteins were tested against soluble substrate and kinetic
profiles were calculated. Truncated EiChit1CAT shows high hydrolytic activity and
high substrate affinity as expected. Absence of ChBD was found to be helpful for
proper substrate binding and enzymatic processing in case of soluble substrate. Native
EiChit1 show lower activity against insoluble substrates and found on the cyst wall of
encysting and mature cyst, probably helping the binding of chitin fibrils, which
evidently doesn’t require high hydrolytic activity against the fibrils. It is tempting to
conclude that, EiChit1 was evolved not only for cleaving the chitin fibrils at large but
also to assemble them in a tightly knitted structure.

Against the soluble substrate JacobChBD-EiChit1R-CAT shows comparably low activity

and low affinity while JessieChBD-EiChit1R-CAT demonstrates no detectable activity at
all. These discrepancies can be explained by lowered substrate binding ability,
possibly due to changed morphology. So probably the native EiChit1ChBD was
optimized evolutionary to balance the substrate binding ability and hydrolytic
efficiency.

As the soluble substrate doesn’t resemble the natural condition, both the fusion
proteins were tested against colloidal chitin and chitin dust. Long incubation period
was needed to produce sufficient product, which belongs to chitobiose group. Both
fusion proteins were efficiently cleave loosely integrated colloidal chitin and compact
chitin dust. JacobChBD-EiChit1R-CAT cleaves colloidal chitin more in comparison with
JessieChBD-EiChit1R-CAT. But in the case of chitin dust, JessieChBD-EiChit1R-CAT were
118
found to be more competent than JacobChBD-EiChit1R-CAT by releasing more dimers
within 2 hr. After a long incubation period JessieChBD-EiChit1R-CAT release more
chitobiose than JacobChBD-EiChit1R-CAT. In both cases, preliminary profiles were
shown to contain high molecular weight product, which later masked by accumulating
chitobiose.

Substrate binding affinity of these two fusion protein increased at their pI, indicating
hydrophobic interaction as the interacting force. Substrate binding ability of these two
fusion proteins were compared with native EiChit1. JacobChBD-EiChit1R-CAT and
JessieChBD-EiChit1R-CAT were found to exhibit lowest and highest binding affinity
respectively towards chitin beads, while EiChit1 show intermediate affinity.

The alignment of all three ChBD (Fig. 6.12) reveal some interesting points, such as
EiChit1ChBD contain more number of aromatic residues than JacobChBD which
strengthen the relationship between aromatic residue number and substrate binding
affinity (Kikkawa et al., 2008). Few conserved aromatic residues were identified
between EiChitChBD and JessieChBD. After aligning the EhChit1ChBD sequence, those
aromatic residues were again found to be conserved. Alignment with Human ChBD
shows no significant sequence similarity with EhChit1ChBD, which increase its
potential as a drug target.

It can be observed that three ChBD sequences bear few conserved aromatic residues,
though they were more conserved between EiChit1ChBD and JessieChBD and placed
strategically. Among the residues, Phe8, Try18, Try49 and Try51 probably play the
most important role in substrate binding ability.

Fig. 6.12: Entamoeba chitinase and lectin ChBD aligned and compared with human ChBD.
Conserved cysteine were coloured in red and aromatic residues coloured in yellow. Aromatic
residues responsible for substrate binding are indicated with black arrows.

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6.5 Conclusions

• Fusion protein containing different chitin binding domain were designed and
expressed in bacterial system.
• Truncated EiChit1CAT was created by removing the ChBD and expressed in
bacterial system.
• Truncated EiChit1CAT exhibit more hydrolytic ability indicating that the ChBD
is not required to hydrolyze small substrates.
• Both fusion proteins were able to hydrolyze insoluble substrates though they
behave differently with soluble substrate.
• Number of Cysteine and aromatic residues and their placement within the
ChBD probably play some important role in maintaining the structural integrity
and substrate binding ability respectively.

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 Chapter 7

Transcriptional profiling and localization of Ei chitinases during

encystation
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7.1 Abstract

Transcriptional and translational profiling of chitinases during encystation determined

by RT-PCR and immuno-localization study is depicted in this chapter. Transcription
of all three chitinases begins within 18 hr of encysting in E. invadens (Ei) cells.
Around 48 hr of encystation no mRNA was detected. Translation of chitinase protein
found to occur within the same time frame as transcription. Within 18 hr of
encystation initiation all chitinases were found within numerous small vesicles.
Further localization with two different antibodies defines two different destinations for
EiChit1 and EiChit2, EiChit3. Chitinase 1 was observed in cyst wall only, while
chitinase 2 and 3 may occur in the vesicles surrounded by the cyst wall.

7.2 Introduction

Free living or parasitic prokaryotic life such as bacteria surround themselves by a

carbohydrate wall to avoid the harsh and fluctuating environmental condition as well
as to survive the nutrition shortage. Similar strategy is also used by different free
living and parasitic protozoa. Among the parasitic protozoa such as Entamoeba and
Giardia, encystation and subsequent release of trophozoites were studied in detail, to
understand the changes at a molecular level. As it was reported earlier, Giardia and
Entamoeba enter the encystation procedure within the host gastro-intestinal system, by
covering themselves with a thick wall. Identification and characterization of
physiological signals responsible for encystation initiation within the host is an
emerging field of research. For Giardia, identification of encystation initiation sites
within the host intestinal tract (Gillin et al., 1988) along with the recognition of other
factors were studied extensively in last few years (Lujan et al., 1996), while for
Entamoeba, the knowledge is partial (Eichinger, 2001). Different conditions such as
removal of carbon source, lowered nutrition level along with increased osmotic
pressure induce the encystation by aggregating the Ei cells into clumps (Sanchez et al.,
1994).

Extensive ultra-structural studies involved with encystation and excystation of E.

invadens were done in past few years. Encystation was observed with the deposition
of microfibrilar structures above the cell membrane followed by the formation of a 30-
50 nm thick layer of polysaccharide and protein (Chavez-Munguia et al., 2003). Along

123
with the visible morphological changes, many genes were observed to get regulated at
transcriptional level during this process (Sanchez et al., 1994). Several encystation
specific genes such as Jacob (Frisardi et al., 2000), chitinase (Villagomez-Castro et al.,
1992) and chitin synthase (Das and Gillin, 1991) were found to be up regulated in Ei.
Only 15% of Ei genes were predicted to be regulated developmentally during this
transformation. All these genes have their respective homologues in Eh, and they are
also found to up-regulated in isolated clinical strains of xenic culture of E. histolytica,
grown in complex diphasic media (such as Robinson’s medium with an agar slant)
(Ehrenkaufer et al., 2007).

In encysting Eh and Ei up-regulation of chitinase gene was categorised as a well

established marker for encystation and its inhibition by allosamidine was able to
inhibit the encystation in Ei (Villagomez-Castro et al., 1992). Presence of multiple
chitinases in Ei was reported earlier (Villagomez-Castro and Lopez-Romero, 1996).
Both Ei Chitinase 2 and Ei Chitinase 3 were not detected in mass spectrometric
analysis of mature cyst wall protein and in the early transcript (22 hr) by RT-PCR
from encysting m RNA (Van Dellen et al., 2006). Occurrence of chitinase filled
vesicles within the encysting Ei and transfected Eh was reported concluding the
presence of encystation specific transport system (Ghosh et al., 1999)

In this report, an effort was made to characterize the transcriptional and localization
profile of all three Ei chitinases.

7.3 Results

7.3.1 Standardization of axenic culture and Encystation of E. invadens

E. invadens (IP1strain) were cultured in TYIS-33 medium at 25˚C (Diamond et al.,

1978). Sub culturing was done thrice a week in 15 ml glass tube. Large amount of
culture was maintained in 50 ml flask (Fig 7.1 A and B).

Encystation was standardized using log phase E. invadens cell. Cells were washed
thrice and resuspended in 47% LG (low glucose) medium (Sanchez et al., 1994). After
36-48 hr clumping of Ei cells occur with appearance of spherical cyst (Fig. 7.1 C and

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D). Within 60-72 hr of encystation 60-70% encystation was observed, though the
encystation was not found to be synchronic.

Fig.7.1: Confocal micrograph of E. invadens trophozoites and cyst. Fluorescence (A) and
phase contrast (B) image of Ei trophozoites with DAPI stained single nucleus. The bar in A
and B represents 5 µm. Fluorescence (C) and phase contrast (D) image of Ei cyst with DAPI
stained four nuclei. Nuclei are indicated with white arrows. The bar in C and D represents
2 µm.

7.3.2 Transcriptional profile of three Ei chitinase in encysting cells

Transcriptional profiling was performed in this study by doing qualitative RT-PCR.

To detect the presence of three Ei chitinases, within the mRNA pool of encysting
cells, log phase Ei cells were washed and incubated in LG medium at 5x107 cells/ml
concentration. Encysting cells were collected at different time point (0-48 hr) by
chilling the flask.

Fig.7.2: RT-PCR analysis of Ei Chitinase 1, Chitinase 2 and Chitinase 3 at different time point
of encystation. Lane DNA in each panel represent the chitinase amplicons amplified from
genomic DNA (+ve control). Hours are representing the encystation time period. Ei Chitinase
2 PCR product was loaded in double amount with respect to other chitinases (indicated by 2x)
to get a cooperative image. Actin amplification was used as internal control.

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Total RNA was isolated using TRI Reagent and was used for cDNA synthesis using
oligo (dT) primers by RETRO script first strand synthesis kit (Ambion). PCR was
carried out with cDNA using chitinase gene specific primers, designed to amplify only
~400 bp region. Actin was used as an internal control and all the amplicons were
analyzed in 1.5% agarose gel (Fig.7.2).

Amplicons of all three Ei chitinase were detected after 18 hr of encystation continuing

for approximately 48 hr. Ei Chitinase 2 express less than that of other two chitinase in
transcriptional level, and its expression was not detected 36 hr, while both Ei
Chitinase 1 and Ei Chitinase 3 are expressed at least up to 48 hr of encystation.

7.3.3 Raising polyclonal antibody in rabbit against EiChit2

All three Ei chitinase proteins bear a very high sequence similarity within their
catalytic domain. So, it was presumed that a single antibody raised against the
catalytic domain may identify three chitinases in vivo. Polyclonal antibody in rabbit
was raised by following the method described in Method and Material. Briefly,
EiChit2 (only the catalytic domain) was purified using Ni-NTA bead and analyzed in
12% SDS-PAGE. Electro-elution of purified EiChit2 from the sliced gel piece was
done for further purification. Lyophilized protein was used for subsequent
intramuscular injections. Finally 12 ml blood was collected by heart bleed and used
for IgG purification. Western blot and ELISA were done against recombinant EiChit2
to detect antibody specificity and antibody titre. Anti EiChit2 antibody was observed
to identify all three recombinant Ei chitinases.

7.3.4 Localization of chitinases in encysting E. invadens

In a previous study Ei Chitinase 1 was localized within numerous small vesicles of

encysting cells. The localization was performed using Ei Chitinase 1 specific antibody
raised against the heptapeptide linker region of Ei Chitinase 1 (Ghosh et al., 1999). In
this study in vitro and in vivo localization was done using the anti EiChit2 antibody
against total protein and encysting cell respectively. Total protein from mature Ei
cysts were isolated at 60-72 hr and used for western blot. 1X Protease inhibitor
cocktail was used to prevent the proteolytic degradation. Anti EiChit2 antibody was
used as primary antibody and anti rabbit goat IgG conjugated with HRP was used as

126
secondary antibody. Detection of bands was done with DAB system. Two distinct
bands of approximately 35 and 50 kDa were identified after 60 hr of incubation.

Fig.7.3: Western blot of total protein isolated from mature Ei cysts of 60-72 hr. Anti EiChit2
antibody was used as primary antibody. Panel A- Lane1-3 represents silver stained SDS-
PAGE of 0 hr, 60 hr and 72 hr respectively. Lane M represents the protein mol wt markers.
Panel B - Lane 1-3 represents developed blot of 0 hr, 60 hr, and 72 hr respectively. The
developed bands were indicated with black arrows. Two distinct bands of 35 and 50 kDa were
observed.

For microscopic study encysting Ei cells of different time period (0- 72 hr) was chilled
and fixed with 2% PFA. Permeabilization was done by 0.01% Triton X-100.
Permeabilized and non-permeabilized cells were incubated with anti chitinase
antibodies for 1 hr (1:200 dilution), followed by the washing with chilled 1X PBS for
three times. Secondary staining was done with TRITC conjugated anti rabbit IgG
(1:500 dilution). Stained cells were visualized with Olympus FluoView FV1000
confocal microscope with TRITC channel. All the images were taken in Z-stacking
mode and the middle slice was used for representation. Image analysis and
representation was done using FluoView software (Olympus) (Fig.7.4 and Fig.7.5).

Ei Chitinase 1 was detected after 12 hr of encystation within small vesicles, and

observed in cyst wall also. With the increasing time of encystation period
accumulation of fluorescence signals around the cyst wall indicates the localization of
chitinase in cyst wall. Ei Chitinase1 signal decreased with encystation time period and
noticed only in cyst wall (Fig. 7.4).

Anti EiChit2 antibody also detect chitinase in maturing cyst wall, which might be due
to the presence of Ei Chitinase 1 as this is also detected by anti EiChit1 antibody and
Ei Chitinase 1 only has the chitin binding domain.

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Fig.7.4: Fluorescence (bottom panel) and phase contrast (upper panel) image of Ei Chitinase 1
localization in encysting E. invadens at different time point. Encysting cells were fixed,
permeabilized and incubated with Ei Chitinase 1 primary antibody and TRITC conjugated anti
rabbit IgG secondary antibody. Panel A-F represent cells of 0 hr, 24 hr, 36 hr, 48 hr, 60 hr and
72 hr incubation. The bar represents 5µm.

Anti EiChit2 antibody detects all three chitinases to some extent, and identifies Ei
Chitinase 1 in cyst wall (Fig. 7.5). EiCht2 and EiChit3 were not found in cyst wall
analyzed by Mass spectrometry (Van Dellen et al., 2006). In mature cyst small
vesicles within the cyst wall were detected by anti EiChit2 antibody, which was not
localized by anti Ei Chitinase 1 antibody that indicates the presence of only Ei
Chitinase 2 and Ei Chitinase 3 in those vesicles of mature cysts. Much better signal
obtained by anti EiChit2 antibody over anti EiChit1 antibody is probably due to high
affinity and better quality.

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 Fig.7.5: Fluorescence (bottom panel) and phase contrast (upper panel) image of Ei chitinase
localization in encysting E. invadens at different time point. Encysting cells were fixed,
permeabilized and incubated with EiChit2 primary antibody and TRITC conjugated anti rabbit
IgG secondary antibody. Panel A-H represent cells of 0 hr, 12 hr, 18 hr, 24 hr, 36 hr, 48 hr, 60
hr and 72 hr incubation. The bar represents 5µm.

7.3.5 Identification of EhChit1 and Eh cyst with anti EiChit2 antibody

From the protein sequence alignment of all Entamoeba chitinases it was observed that
catalytic domain of EhChit1 has high resemblance with EiChit2. So it was assumed
that the polyclonal antibody raised against EiChit2 catalytic domain may detect the Eh
counterpart also. Western blot was done to identify the cross recognition level, and
anti EiChit2 antibody was found to recognize recombinant EhChit1 (Fig.7.6). Eh cyst
in clinical stool samples was collected from symptomic patients of surrounding

129
hospitals and clinics. Collected samples were diluted with 1X PBS and filtered
through steel mesh to discard the debris.

Fig.7.6: Western blot analysis of recombinant EhChit1 using anti EiChit2 antibody. EiChit2
and EhChit1 bands are detected and indicated by the black arrow. M represents the pre-stained
protein marker and mol wt of only visible bands is represented here.

Filtrate fractions was collected and washed with 1x PBS for twice. After confirming
the presence of mature Eh cyst by phase contrast microscopy, the samples was
incubated with anti EiChit2 antibody for 1 hr followed by 1hr incubation with TRITC
conjugated anti rabbit IgG. Counter staining of chitin wall was done by incubating the
samples for 30 min at room temperature with 10 µl of 100 mM Calcoflour White
(CFW) stain. Stained samples were visualized with Olympus FluoView FV1000
confocal microscopy using TRITC and DAPI channel for TRITC and CFW stain
respectively (Fig.7.7). Images were taken in Z-stacking mode and the middle slide was
represented in TRITC panel.

Anti EiChit2 antibody was able to recognize the Eh Chitinase 1 in mature Eh cyst
wall, and can be used for clinical detection of Eh cyst.

Fig.7.5: Fluorescence image of Eh cyst stained with Calcoflour White (A) and Anti EiChit2
antibody (B). The bar represents 2 µm.

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7.4 Discussion

Human gastro-intestinal tract parasite such as Giardia and Entamoeba, live a very
simple yet opportunistic life within the host. E. histolytica enter the host body in a
chitin walled cyst form, through contaminated food or water and gives rise to four
trophozoites. The excystation occur within the alkaline environment of small intestine.
Trophozoites invade the mucous and sub mucous layer of the small intestine and cause
the well known symptoms such as irritable bowel syndrome, and bloody mucous.
Though very few cyst bearers ever become cyst passers, at a certain time point the
existing trophozoites in large intestine enter the encystation cycle triggered by some
unknown stimulants. For Giardia most of the physical conditions such as cholesterol
starvation, responsible for such changes were discovered (Lujan et al., 1996) while for
E. histolytica the triggers are unknown (Eichinger, 2001).

E. invadens, a reptilian parasite was used widely as a model for encystation study, due
to its capacity to encyst in vitro. Low nutrition, high concentration of parasite and high
osmotic pressure were found to trigger the encystation in vitro for E. invadens
(Sanchez et al., 1994) however the same conditions were not found enough to induce
the changes in E. histolytica. Very complex procedure of E. histolytica encystation
was reported several times, despite the fact that none of them were able to encyst
axenic culture (Ehrenkaufer et al., 2007).

After the initiation, the exact order of cyst wall formation or the appearance of
different encystation specific protein are not clear, though most important players of
this change are being characterized. Jacob, a multi-domain chitin binding lectin was
discovered to appear first (Frisardi et al., 2000), followed by a string of other chitin
binding proteins such as chitinase and Jessie. Chitin and chitosan were found as the
main ingredients of the polysaccharide constituent (Das et al., 2006). A complex
meshwork of carbohydrate and protein are formed to cover the cyst structure. As a
part of the protein complex, multiple chitin binding lectins such as Jacob and Jessie
with different domain structure along with the chitinase play an important role to
complete the meshwork (Chatterjee et al., 2009) (in press).

E. histolytica contain single chitinase while in E. invadens three chitinases were

evolved to play the same role (de la Vega et al., 1997). In E. invadens the longest

131
chitinase with an extra chitin binding domain detected in the cyst wall, while the trace
of other two chitinases were not detected either in cyst wall or in m RNA pool of
previous studies (Van Dellen et al., 2006)

In this study, encysting cells of different time period shows the presence of all three
chitinases from 18 hr onwards which continued up to 48 hr. All three chitinases get
transcribed in more or less similar fashion, though the transcription of Ei Chitinase 2
stopped first at 36 hr and expressed in lesser quantity than that of other two chitinases
at least in transcription level.

Polyclonal anti EiChit2 antibody was raised and found to detect all three recombinant
Ei chitinases during immunoblotting. Total protein from mature Ei cysts of 60-72 hr
was collected to detect the presence of native chitinases. Two distinct bands of 35 and
50 kDa (approx) were noticed after 60 hr of encystation. Previous studies with native
Ei chitinases reports same range of mol wt. for Ei Chitinase 1 (approx 50 kDa) and Ei
Chitinase 2 and 3 (both around 35 kDa) (Villagomez Castro and Lopez-Romero,
1996). Though the signals were found to be poor, it can be concluded that anti EiChit2
antibody recognize other two native chitinases as well.

Localization of Ei Chitinase 1 specifically was done by the antibody raised against the
repeat region; while anti EiChit2 antibody identify all three chitinases as all three of
them share a similar catalytic domain. During the time based study it was observed
that, Ei Chitinase 1 translated and appear along with other two chitinases. Translated
proteins were accumulated within numerous small vesicles and distributed throughout
the cytoplasm of encysting cells. Within 24 hr of encystation Ei Chitinase 1 was
observed to reach the cyst wall, which indicates that it might play a role in chitin
binding and cyst wall formation. Vesicles containing Ei Chitinase 2 and 3 were also
observed to reach the area adjacent to cyst wall. Large vesicles were detected by this
antibody around the cyst wall, which may contain Ei Chitinase 2 and 3, as they were
not separated in this study. Vesicles containing electron dense material surrounding
the cyst wall were reported previously (Chavez-Munguia et al., 2003), concluded to
contain wall dissolving enzymes. From the present study it can be concluded that
those vesicles contain chitinase which may help to degrade the chitin wall from inside
during excystation. No vesicular localization of Ei Chitinase 1 was not detected in
mature cyst, which indicates that the Ei Chitinase 1 presents only in the cyst wall and

132
play the major role during the encystation to made the tailor made chitin fibrils. Which
was not possible to conclude from this study is whether Ei Chitinase 2 or Ei Chitinase
3 is present in the cyst wall or not. Different chitinase specific antibody might resolve
this question.

Due to the high similarity of protein sequence Anti EiChit2 antibody was able to
identify other two recombinant as well as native Ei chitinases also. As EiChit2 share a
common catalytic domain with EhChit1 also, it was presumed that, the antibody may
identify the recombinant EhChit1 also. From the western blot it was discovered that,
indeed the antibody recognize the Eh chitinase. Eh Chitinase 1 in mature Eh cyst was
also detected by the anti EiChit2 antibody, confirming the presence of Eh Chitinase 1
on the cyst wall. This cross recognition may be used to identify clinical sample.

7.5 Conclusion

• Encystation of E. invadens in 47% LG medium was standardized.

• Transcription of all three Ei chitinases starts within 18 hr of encystation, and
continue at least up to 36 hr. Ei Chitinase 2 stops its expression between 36-48
hr.
• Expressed chitinases accumulated in vesicles during first 36-48 hr of
encystation, as well as transported towards the cyst wall.
• Ei chitinase 1 specific antibody detect the presence of Ei Chitinase 1
throughout the cyst wall.
• Anti EiChit2 antibody detect all three chitinases and notified their presence in
cyst wall and within small vesicles surrounding the cyst wall.
• Anti EiChit2 antibody was able to detect recombinant EhChit1, due to their
high sequence similarity.
• Anti EiChit2 antibody was able to identify mature Eh cyst from clinical
samples.

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134
Chapter 8

Discussion
136
Chitin as a structural polysaccharide placed 2nd most abundant polymer on Earth after
cellulose and play an important role in bacterial, fungal, insect and higher vertebrate
life, by creating the their cell wall or exoskeleton. Natural pool of chitin remains same
though it is insoluble in most of the available solution. Chitinase, a member of
glycosyl hydrolase group is responsible for the biodegradation of chitin and found to
occur in most of the chitin bearing organism, as well as in the non-chitinious
organisms also. Chitinases are assigned many roles ranging from food processing to
immuno-modulation depending on the system. Occurrences of multiple chitinases
were observed in many bacterial and fungal species, where they act synergistically to
degrade the polymeric chitin into soluble oligomers. Origin of multiple chitinase and
chitinase like proteins were assumed due to domain shuffling and gene duplication.
Phylogenetic analysis of chitinases depicts their conserved structural aspects
throughout the evolutionary pathway.

Chitinase was detected in several human parasites such as Plasmodium, Leishmania

and Entamoeba playing important role in their life cycle (Shahabuddin et al., 1993;
Rogers et al., 2008; Villagomez et al., 1992) Inhibitor or antibody mediated inhibition
of chitinase was found to inhibit these parasitic transmissions, emphasising the role of
chitinase as a potential drug target (Jarroll and Sener, 2003). Human chitinase and
chitinase like protein bear high resemblance with these parasitic chitinases, leading the
search for an exclusive domain as a probable target.

Among the human protozoan parasites, E. histolytica is responsible for more than 48
million new infections and more than 100,000 deaths per anum (Walsh, 1986). Current
treatment system of amoebiasis largely depends on a nitro-imidazole drug
“Metronidazole” (Mtz) only. Occurrence of Mtz resistant bacterial and amoebic strains
in last few years initiated the search for new drug targets in Entamoeba, which was
accelerated by the release of E. histolytica genome project (Loftus et al., 2005).
Among other plausible targets, encystation and excystation specific molecules were
found to be of heightened interest, as their inhibition can restrain the infection of new
hosts as well as invasion of host gastro-intestinal tract respectively. E. invadens, a
reptilian parasite resemble the human parasite and used worldwide as a model of
encystation study. Advancement in genomic studies of E. histolytica, disclose the

137
presence of a single chitinase gene, similar to Entamoeba dispar, though the reptilian
counterpart was reported to contain three chitinases (Wang et al., 2003).

In this study we report the molecular characterization of all three E. invadens

chitinase in their recombinant form to understand their necessity and role during
encystation and excystation. E. histolytica chitinase (EhChit1) and E. invadens
chitinase (EiChit1, EiChit2 and EiChit3) sequences were obtained from the Gene
Bank. EiChi1 and EhChit1 have similar structural conformation and contain three
different domain named as chitin binding domain (N-terminal ChBD), linker and
catalytic domain. EiChit2 and EiChit3 have somewhat different structure lacking the
ChBD and linker region. Respective chitinase ORF were amplified from the genomic
DNA and cloned in TA vector. All the ORFs were sub-cloned in pQE30 vector and
the expressed proteins were observed to be of higher molecular weight, when
examined in 12% SDS-PAGE, probably due to the presence of extra amino acids.
Expressed soluble proteins were isolated and purified to homogeneity.

Protein homology modelling study of Ei Chitinases was done using Human

chitotriosidase as template. Superimposition of predicted Ei Chitinase models shows
very high similarity within the (β/α)8 TIM barrel structure of catalytic domain, except
some loop region. Several aromatic residues were identified and expected to play the
key role in substrate binding. Enzymatic characterization of all expressed protein was
done by some well established assays using soluble and insoluble substrates. EiChit1
and EhChit1 demonstrated similar hydrolysis pattern against both soluble and
insoluble substrates. Both enzymes were predicted to have same range of pI and
similar distribution of secondary structural motif confirming their structure and
conformational similarities. Phylogenetic analysis by minimum evolution method
explains their evolutionary relation as both of them were presumed to evolve in same
time period. On the other hand EiChit2 and EiChit3 confirmed their closeness by
having same enzymatic profile against soluble and insoluble substrates. All three Ei
Chitinases were found to bind insoluble substrates efficiently, though EiChit2 and
EiChit3 lack of any specific chitin binding domain. Probably their catalytic domain
structure was enough to bind the substrates (Suzuki et al., 1999). Binding to insoluble
substrate possibly occur due to hydrophobic interaction. All recombinant chitinases
were characterized as exochitinase as they release dimers from the non reducing end
138
of chitin polymer, which was also predicted from their molecular docking study.
Docking with NAG5 molecule emphasize the substrate binding at +1 and +2 subsites
from the non reducing sites, and release of dimers by hydrolysis of the β (1-4) link
between +1 and -1 subsites. In all aspect EiChit3 was found to be the most efficient
chitinase which may be explained by its secondary structure analysis, where it shows
different distribution of secondary motifs than other three, and bound to the substrate
molecule more efficiently.

Inhibitor assay was done using methylxanthine derivatives as lead inhibitor molecules.
Methylxanthine derivatives were supposed to mimic the oxazolinium intermediate
within the catalytic domain of family 18 chitinases as allosamidine, the most potent
chitinase inhibitor (Rao et al., 2005). It was observed that all three derivatives such as
Pentoxifylline, Caffeine and Theophylline inhibit EiChit1 in competitive way, in spite
of their different side chain structure. It can be assumed then, that the side chains are
not important for enzyme binding or inhibition. Both EiChit2 and EiChit3 were found
to be inhibited in uncompetitive or in-competitive way, exhibiting their different
substrate binding mode. From the different inhibition profile and Ki values, it was
found that pentoxifylline was the most potential lead molecule among the three and
EiChit1 get inhibited maximum by following the family 18 substrate binding mode
more efficiently.

Among three Ei Chitinases, EiChit1 exhibits maximum similarity to EhChit1, in

respect of structure and activity. But the catalytic domain of both Eh and EiChit1 bear
structural similarity with Human chitinase also, which obstruct their use as drug target.
In search of exclusive domain within EiChit1 and Ehchit1, N-terminal ChBD of
EiChit1 was also characterized in this study.

ChBD was discovered in human chitinase without any significant similarity with
Entamoeba ChBD. This amino acid long fragments were assumed to enhance the
substrate degradation by increasing enzyme concentration on polymeric substrate
surface. To identify the role of 8 cysteine rich ChBD of EiChit1, it was replaced by
two other E. invadens lectin ChBD of 60 (JacobChBD) and ~70 (JessieChBD) amino
acids. Truncated EiChit1 (catalytic domain only) was created to detect the role of
EiChit1ChBD on its own enzymatic profile. Fusion proteins were created by restriction

139
enzymatic digestion and PCR amplification. The truncated protein exhibits increased
hydrolytic activity against soluble substrate, explaining the lowered efficiency of
EiChit1, due to the presence of extra domain. Both fusion proteins exhibited somewhat
diminished/no activity against soluble substrate, which may be due to different
substrate binding mode. But hydrolysis of insoluble substrate was not found to be
impeded due to the replacement of ChBD. Both fusion and native proteins exhibit high
substrate binding ability and JessieChBDEiChit1R-CAT shows maximum binding ability,
followed by EiChit1 and JacobChBDEichit1R-CAT. Analysis of ChBD sequence reveals
that, EiChit1ChBD contains maximum aromatic amino acids, which may directly
influence the substrate binding ability (Kikkawa et al., 2008). Multiple alignment of
all three ChBD identifies few strategically placed aromatic residues in both JessieChBD
and EiChit1ChBD, which are assumed to play important role in substrate binding. It was
further observed that, EhChit1ChBD also contains those specific residues, probably
playing the same role. So, it can be concluded that ChBD might be to EiChit1 and
EhChit1 moderate its hydrolytic activity but to increase its substrate binding activity.
The sequence of EiChit1ChBD might be tuned evolutionarily to optimize the substrate
binding, without obstructing the substrate hydrolysis.

During encystation of E. invadens, occurrence of only EiChit1 was previously

reported in mRNA pool as well as in cyst wall (Van Dellen et al., 2006), without any
trace of EiChit2 and EiChit3. In this study transcriptional profiling of all three Ei
Chitinases was done at some point is 0-48 hr of encystation by RT-PCR. All three
chitinases start to transcript within 18 hr of encystation and continue at least up to 48
hr. Among three chitinases, EiChit2 expression stops between 36-48 hr of encystations
and the level of expression at transcriptional level is also much less that of other two
chitinases. Localization of all three chitinases in encysting E. invadens were done by
using two antibodies raised against the heptapeptide repeat region of EiChit1 (Ghosh
et al., 1999) and catalytic domain of EiChit2. Anti EiChit2 antibody detect all three
recombinant Ei and Eh chitinases due to the similarity of primary sequence. Within 18
hr of encystation all three chitinases were observed in small vesicles, which further
transported towards the cyst wall. Within 48 hr most of the vesicles containing all
three chitinases reached the wall. After 60 and 72 hr EiChit1 only detected in cyst
wall, while anti EiChit2 antibody detects signals from cyst wall and vesicles within the

140
wall. From the presence of chitinase containing vesicles surrounded by the cyst wall
and the absence of both EiChit2 and EiChit3 in cyst wall protein (Van Dellen et al.,
2006), it can be concluded that the EiChit1 exist on the wall and the EiChit2 and
EiChit3 probably reside within those vesicles and not on the cyst wall, as both of them
lack the ChBD. It can also be assumed that, EiChi1 exist on the cyst wall, while
EiChit2 and EiChit3 stay within the vesicles adjacent to cyst wall waiting for the
excystation specific signals.

Finally we conclude that all three Ei Chitinases were evolved to play different roles
during the encystation and excystation. EiChit1, EiChit2 and EiChit3 simultaneously
get transcribed and translated during encystation. They get transported through
vesicles towards the cyst wall, where EiChit1 reach the cyst wall, while other two
remain within the vesicles. During encystation EiChit1 trim the chitin fibrils and help
in cyst wall formation, while other two more efficient enzymes help during
excystation by cleaving the chitin wall.

Evolutionarily they evolved in different time point, following the order of EiChit3,
EiChit2 and EiChit1. The most currently evolved protein EiChit1 coincide with
EhChit1 in every way. So, it may be assumed that, previously E. invadens used to
have more efficient single chitinase which get multiplied either by gene duplication or
domain shuffling through evolution, parallel to EhChit1.

141
142
 Chapter 9

Conclusions and future scopes

144
9.1 Conclusions

1. One Eh and three Ei chitinases were characterized in silico.

2. All four Entamoeba chitinases were amplified and sub-cloned in pQE30

vector.

3. All four chitinases were expressed in soluble fraction and purified by

affinity chromatography.

4. Recombinant Entamoeba chitinases were characterized enzymatically and

all of them belong to family 18 exochitinase.

5. Methylxanthine derivatives were found to inhibit three Ei chitinases in

different mode at micro molar level.

6. Two Fusion proteins were created using lectin ChBD and catalytic domain
of EiChit1 and cloned along with EiChit1CAT followed by bacterial
expression using pQE30 vector.

7. EiChit1CAT was able to hydrolyze soluble substrate more efficiently,

signifying the absence of N-terminal ChBD of EiChit1.

8. Fusion proteins were found to have lower/no hydrolytic ability against

soluble substrate, though cleaves insoluble substrate in exochitinase
manner.

9. Aromatic residues of Entamoeba ChBD plays important role in substrate

binding.

10. Transcription of all three Ei chitinase initiated within 18 hr of encystation

and found to continue up to 36-48 hr.

11. All three Ei chitinases were localized in vesicles of encysting cells.

12. EiChit1 detected very feebly on the mature cyst wall, while anti EiChit2
antibody detects chitinases in cyst wall and small vesicles.

13. Anti EiChit2 antibody was able to identify recombinant and native
EhChit1, and detect mature Eh cyst in clinical samples.

145
9.2 Future Scopes

1. Mutagenesis study of catalytic domain and Entamoeba ChBD to identify the

role of identified aromatic residues in enzymatic ability.

2. Crystallographic studies with Entamoeba chitinases.

3. Design and identification of new chitinase inhibitors.

4. Inhibition of Entamoeba encystation by those inhibitors.

146
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 Appendices

Appendix A

1. >gi|1685364|gb|AAB52724.1| chitinase 1[Entamoeba invadens]

2. >gi|91983531|gb|ABC59330.2| chitinase 2 [Entamoeba invadens]

3. >gi|84620156|gb|ABC59331.1| chitinase 3 [Entamoeba invadens]

4. >gi|67472835|ref|XP_652205.1| chitinase [Entamoeba histolytica HM-1:IMSS]

5. >gi|8895221|gb|AAF80859.1|AF175527_1 cyst wall-specific glycoprotein Jacob1

[Entamoeba invadens]

6. >gi|91983529|gb|ABC59329.2| Jessie 3b [Entamoeba invadens]

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 Appendix B

1. >gi|1685364|gb|AAB52724.1| chitinase 1[Entamoeba invadens]

2. >gi|91983531|gb|ABC59330.2| chitinase 2 [Entamoeba invadens]
3. >gi|84620156|gb|ABC59331.1| chitinase 3 [Entamoeba invadens]
4. >gi|67472835|ref|XP_652205.1| chitinase [Entamoeba histolytica HM-1:IMSS]
5. >gi|167380579|ref|XP_001735375.1| chitotriosidase-1 precursor [Entamoeba dispar
SAW760]
6. >gi|73909055|gb|AAI03696.1| Chitinase 1 (chitotriosidase) [Homo sapiens]
7. >gi|114571914|ref|XP_514112.2| PREDICTED: chitotriosidase isoform 6 [Pan
troglodytes]
8. >gi|46240808|dbj|BAD15061.1| chitinase3 [Paralichthys olivaceus]
9. >gi|219437504|ref|XP_002216528.1| hypothetical protein BRAFLDRAFT_
280438 [Branchiostoma floridae]
10. >gi|213625929|gb|AAI71633.1| Zgc:173927 [Danio rerio]
11. >gi|219689080|ref|NP_001137269.1| chitinase 1 (chitotriosidase) [Equus caballus]
12. >gi|195374614|ref|XP_002046098.1| GJ12709 [Drosophila virilis]
13. >gi|33325189|gb|AAQ08200.1| chitinase [Helicoverpa armigera]
14. >gi|212509328|gb|EEB12743.1| conserved hypothetical protein [Pediculus
humanus corporis]
15. >gi|119120779|ref|NP_001073157.1| chitinase1 (chitotriosidase) [Rattus
orvegicus]
16. >gi|157107967|ref|XP_001650020.1| brain chitinase and chia[Aedes aegypti]
17. >gi|158286667|ref|XP_308858.4| AGAP006898-PA [Anopheles gambiae
str.PEST]
18.>gi|115400105|ref|XP_001215641.1| predicted protein [Aspergillus terreus
NIH2624]
19. >gi|75759179|ref|ZP_00739282.1| Endochitinase [Bacillus thuringiensis serovar
israelensis ATCC 35646]
20. >gi|47092241|ref|ZP_00230033.1| chitinase B [Listeria monocytogenes str. 4b
H7858]

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 Appendix C

Fig. I: Lineweaver Burk Plot of EiChit1 with 1/S along X-axis and 1/V along Y-axis.
These graphs display the inhibition mode of EiChit1 by Pentoxifylline, Caffeine and
Theophylline. Increasing concentrations of inhibitors (50-150 µM) were used with 1-5 µM
range of 4-methylumbelliferyl-tri-N-acetylchitotrioside as substrate. The kinetic data were
plotted in single substrate-single inhibitor mode of Enzyme kinetics module of Sigma plot (9)
software. Every data set was checked for full competitive, full non-competitive and full un-
competitive inhibition mode and the plot with highest r2 value is represented here.

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 Fig. II: Lineweaver Burk Plot of EiChit2 with 1/S along X-axis and 1/V along Y-axis.
These graphs display the inhibition mode of EiChit2 by Pentoxifylline, Caffeine and
Theophylline. Increasing concentrations of inhibitors (50-150 µM) were used with 1-5 µM
range of 4-methylumbelliferyl-tri-N-acetylchitotrioside as substrate. The kinetic data were
plotted in single substrate-single inhibitor mode of Enzyme kinetics module of Sigma plot (9)
software. Every data set was checked for full competitive, full non-competitive and full un-
competitive inhibition mode and the plot with highest r2 value is represented here.

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 Fig. III: Lineweaver Burk Plot of EiChit3 with 1/S along X-axis and 1/V along Y-axis.
These graphs display the inhibition mode of EiChit3 by Pentoxifylline, Caffeine and
Theophylline. Increasing concentrations of inhibitors (50-150 µM) were used with 1-5 µM
range of 4-methylumbelliferyl-tri-N-acetylchitotrioside as substrate. The kinetic data were
plotted in single substrate-single inhibitor mode of Enzyme kinetics module of Sigma plot (9)
software. Every data set was checked for full competitive, full non-competitive and full un-
competitive inhibition mode and the plot with highest r2 value is represented here.

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 Curriculum vitae
Tuli Dey
Microbiology and Immunotechnology Laboratory
Department of Biotechnology
Indian Institute of Technology, Kharagpur
India 721302
E mail - tulidey@yahoo.com
Phone no. +91-3222-255226 (Res)
+91-9474143894 (Mob)
Educational Qualifications
• Doctoral studies from 2005 in Molecular Parasitology in the Department of
Biotechnology, Indian Institute of Technology, Kharagpur West Bengal India.
• Masters in Science (Zoology) from Vidyasagar University, West Bengal, India
(2000).
• Bachelor in Science (Zoology, chemistry and physiology) from Raja N.R.L.
Khan Women’s college. West Bengal, India (1998).
Awards
• Awarded Senior Research fellowship from CSIR, India in 2007
• Qualified National Eligibility Test in Life Sciences and awarded Junior
Research fellowship from CSIR, India in 2004.
• Qualified Graduate Aptitude Test in Engineering (GATE) in 2003.
• Qualified Graduate Aptitude Test in Engineering (GATE) in 2002.
Publications
• Dey, T., Basu, R., Ghosh, S.K., Entamoeba invadens: Molecular
characterization of chitinases (under review).
• Pal, T.K., Dey, T., Ghosh, S.K., Pathak, T., First synthesis and antiprotozoal
activities of Divinyl sulfone-modified carbohydrates (communicated)

Conference presentation
• Dey, T., Basu, R., Pathan, M., Ghosh, S. K.
Molecular characterization of Entamoeba invadens Chitinase 2
Organized by 19th Molecular Parasitology Meeting 2008 MBL, Woods Hole,
Boston.
• Dey, T., and Ghosh, S. K.
Cloning and molecular modelling of Entamoeba Chitinases.
Oral presentation: “National Seminar on “Microbes in Pharmaceuticals, Food
& Agriculture” Organized by Department of Microbiology Vidyasagar
University, Midnapore-721102, West Bengal. December 20 – 21, 2006.
• Pal, D., Dey, T., Sameulson, J., Ghosh, S. K.
Molecular Characterization of Entamoeba histotytica Nitroreductase.
Poster presentation: 74th Annual meeting of Society of Biological Chemists
[SBC], Central Drug Research Institute, Lucknow, India. November 7-10,
2005.

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