Molecular Biology of

Infectious Disease

dr. Tri Wibawa, PhD

Dept. of Microbiology
Gadjah Mada Univ. Sch. of Med.

Application of Molecular Biology in

Infectious Diseases

Molecular epidemiology
Pathogenesis
Diagnosis
Treatment
Prognosis
Vaccine development

Molecular Epidemiology

Molecular epidemiology application:

Accessing the burden of recent transmission
False positive culture.
Risk factor for recent transmission (ethnic, ages, etc)
Reinfection case (endogenous or exogenous?)
Phenotyphic difference between strain: virulence,
growth, transmisibility
Relative transmisibility between drug sensitive and
resistance strain.

Molecular epidemiological markers

for M. tuberculosis
Molecular epidemiological markers used:
IS6110 restriction fragment length polymorphism
(RFLP).
Secondary markers:
Polymorphic guanin-cytosine-rich repetitive
sequence (PGRS) RFLP
Spolygotyping
Mycobacterial interspersed repetitive units (MIRUs)

IS6110 restriction fragment length polymorphism (RFLP):

Described initially in 1989
1,555 bp
IS3 family
Two ORF encoding transposition function
Number of copy range 0-25 (mean: 8-18)
Most widely applied.
Gold standard for other methods
High Stability over time
Problems arise when only small amount band appear
Restriction enzyme: pvuII
Probe: PCR product of Mtb IS6110

IS6110-based DNA
fingerprinting

Polymorphic guanine-cytosine-rich repetitive

sequence (PGRS) RFLP
Discriminatory power close to IS6110
Comprise many imperfect repeats
Encode some unknown function protein
Spoligotyping
PCR-based technique.
No need to culture the pathogen
Can be carried out directly to the clinical specimen
Repetitive 36 bp sequence were separated by non
repetitive sequence/spacer. Absent-present the
spacers in southern blotting is the individual
determinant.

Mycobacterial interspersed repetitive unit (MIRU)

VNTR (variable number tundem repeat)
The number of repeats at different loci varies between
strains.
PCR result : different length of amplified product.
Technical difficulty : sizing the PCR product.

Pathogenesis

Laminin - 2

Model of G domain of
laminin-M.leprae
interaction

Genome of S. enterica serotype typhi

(CT18)

Salmonella Virulence Genes

Clustered in certain area of chromosomes:
Salmonella pathogenicity islands (SPI)
SPI-1 and SPI-2 encode: Type III Secretion Systems (TTSS)
SPI-1:
Encoded TTSS translocates effector proteins into
the cytosol of host cells
Invasion of nonphagocytic host cells
Enteropathogenesis
SPI-2
Intracellular survival in murine macrophages

Model for the

structure of S.
typhimurium
SPI-2 TTSS

Translational arrest in rotavirus infectedeukaryotes cells

eIF : eukaryotic initiation factor

PABP: polyadenosine binding protein
GTP : Guanosine 5-triphosphate

Rotavirus
Rotavirus mRNA posses of 5 cap structure, but lack of
polyadenosine tail in its 3 end.
3 end mRNA contained a tetranucleotide motif.
Viral NSP3 protein bind specifically to the tetrancleotide
motif.
NSP3 bind to the binding site of PABP in the eIF4G.
NSP3 has higher affinity to the eIF4G than PABP.
Concequencies:
Increase the translation of viral mRNA
Disrupt the host cell translation process.

Diagnosis

Molecular Diagnostic Techniques of

Tuberculosis
Molecular Diagnostic Techniques are indicated:
Detection of organisms that cannot be grown in vitro or for
which current culture techniques are too insensitive.
Require complex media and prolong incubation time.
Molecular Diagnostic Techniques consideration:
high sensitivity
high specificity
speed
simplicity
clinical relevance.

Basic principle of any Molecular Diagnostic

Techniques is the detection of specific nucleic
acid (DNA) sequence of the pathogens.
Example:
Polymerase chain reaction (PCR)
Southern blot hybridization
Nested PCR
Multiplex PCR
Reverse transcriptase PCR (RT PCR)
Trancription-mediated amplification (TMA)
Ligase chain reaction (LCR)
Nucleic acid sequence-based amplification (NASBA)

Technical aspects: (for MTB)

Target:
Insertion sequences/repetitive element: IS986; IS6110
Antigens : 32 kDa; 38-kDa; 65-kDa.
Genes: dnaJ; groEl; mtb-4
Sample preparation:
Boiling; freezing-boiling; shaking with glass bead;
sonication; chloroform; proteinase K; resin treatment; more
complicated techniques (kit).
PCRs techniques
Internal controls

Studies using in house PCR techniques with IS6110 as a

targets from sputum specimen

Resume
None of the studies observed a statistically significant
difference between culture and PCR
Specificity : 85-100%
Sensitivity : 74-97%
Discrepancy of sensitivity in smear neg culture pos
Human resources dependent.

Treatment

Rifampisin (RIF)
Action: Inhibition of DNA-dependent RNA polymerase.
RNA polymerase: 4 subunit: ; ; ;
Genes : rpoA; rpoB; rpoC; rpoD
Target: bind to the subunit resulting in transcription
inhibition
Mutation in the rpoB gene responsible to rifampisin
resistance.

Identified mutations in rpoB genes of MTB

Isoniazid (INH)
Action and target: Not clearly known
Candidate; INH or INH metabolite block the synthesis
of mycolic acids.
Genes : katG. Encoding catalase-peroxidase enzym
INH resistance MTB had decreased catalase activity.
Mutation in the katB gene responsible to isoniazid
resistance.

Isoniazid (INH)
Genes : inhA. Encoding protein for fatty acid
biosynthesis.
inhA has correlation with resistance to INH and ETH
Polymorphisms were found in the upstream of orfI

Ethambutol (EMB)
Action and target: Not clearly known
Candidate;
Inhibition of RNA metabolism
Inhibition of phospholipid synthesis
Inhibition of transfer of mycolic acid
Inhibition of spermidine synthesis
Inhibition of first step of glucose conversion.
Genes : No genes were identified.

Pyrazinamide (PZA)
Action and target: Not clearly known
Candidate; Pyrazinamidase convert PZA to pyarzinoic
acid. PZA-resistance MTB lack of the pyrazinamidase
activity.
Genes : No gene were identified

Prognosis

T7809C polymorphism of the LAMA2 gene study

GTA GCA

T/T

T/C

C/C

Val Ala

2 =
8.07;
p < 0.025

Tuberculoid group
Lepromatous group

T/C genotype is strongly

associated with the TT (OR =
6.73). (2 8.73; p < 0.005).

SIRS/
SEPSIS

Vaccine Development

Subunit vaccine (recombinant)

Advantages

No risk of pathogenicity
Defined composition
Various delivery system available
Simplified large-scale production
Possibility of further engineering

Disadvantages
Multiple doses typically required
Adjuvants needed

Recombinant Subunit Vaccine

Reduction side effect of other components/epitope

Often poorly immunogenic
Short in vivo half lives
Often elicit only strain-specific production

Recombinant Subunit Vaccine

Protein immunogens production in heterologous
hosts
(bacterial, yeast, insects, mammalian system
vector)
Live delivery systems
Construct recombinant live viral and bacterial
carrying
foreign immunogens
- Vaccinia virus of rabies,
- Attenuated recombinant vacinia virus
vector for
plasmodium
- Adenovirus
- Bacteria gram negative (Salmonella sp)
- Bacteria gram positives

Live delivery system

S typhi TY21a as a vector for vaccine antigens

Antigen

plasmi
d

Bacterium A

S typhi TY21a

S typhi TY21a
Expressing antigen
of bacterium A

Reassortment
Quadrivalent
Vaccine
Common strain in human:
P[8]G1
P[8]G3
P[8]G4;
P[4]G2

P= VP4
G= VP7