362 Review TRENDS in Biotechnology
8 August 2003
Gone gene fishing: how to catch novel
marine antimicrobials
Aleksander Patrzykat and Susan E. Douglas
Institute for Marine Biosciences, 1411 Oxford Street, Halifax, Nova Scotia, Canada, B3H 3Z1
Medical or health-promoting products of marine origin
are often regarded with skepticism – some, such as
shark fins and cod liver oil, are frequently perceived as
low-tech ‘alternative treatments’ largely because they
have not been exploited to their full potential. The
marine environment is an enormous source of bio-
diversity – 80% of all life is found under the oceans’
surfaces – yet very little of this rich resource has been
utilized. Furthermore, most marine organisms rely
heavily on antimicrobial components of their innate
immune defenses to combat pathogens. The past three
years has seen a revolution in the methods used to
identify novel antimicrobials from marine sources;
among the most promising are marine cationic antimi-
crobial peptides (CAPs).
The oceans encompass an immense biodiversity (Box 1)
but, for many scientists, the idea of sorting through
biomaterial from marine depths to search for useful
compounds remains unattractive. Traditional screening
methods involve expensive and time-consuming sampling,
biochemical purification and extensive testing protocols.
However, with the power of molecular biology, it is no
longer necessary to gather and process tens of kilograms of
freshly obtained biomass. Today, using tissue banks,
samples from the most exotic fish species can be shipped
to laboratories, the nucleic acids extracted and genes of
interest ‘fished out’. Genomic screening offers an efficient
streamlined approach, providing opportunities for marine-
derived compounds to receive wider attention and rapidly
progress to the stage of clinical relevance. With the
emergence of antibiotic-resistant bacteria, and the scarcity
of new classes of useful antibiotics, there is an immediate
need for effective alternative therapeutants. Marine
cationic antimicrobial peptides (CAPs) represent a largely
unexploited resource that can follow the molecular route
from the ocean to the clinic.
General properties of CAPs and their distribution in
marine organisms
Fish and invertebrates rely heavily on their innate
immune defenses for protection against pathogen insults.
The protective role of mucous secretions of fish, such as
trout [1] and turbot [2], has been known for some time. The
antimicrobial protein, lysozyme, is found in trout leuko-
cytes in head kidney, skin, gills, gut and eggs [3].
Corresponding author: Susan E. Douglas (Susan.Douglas@nrc.ca).
http://tibtec.trends.com 0167-7799/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0167-7799(03)00145-8
Vol.21 No.
Bactericidal activities have been reported in hemocytes
of crustaceans including the shore crab, lobster and other
crustaceans (for review, see [4]) and blood of ascidians [5].
In addition, antimicrobial proteins of 31 kDa and 27 kDa
have been described in carp [6] and a steroid with
antimicrobial properties (squalamine) has been described
from shark [7]. Cleavage products of histone H2B from
catfish skin [8] and H1 from Atlantic salmon [9] also show
antimicrobial activity. Until recently, however, relatively
few CAPs have been described from marine organisms
(Table 1).
For the purpose of this review, CAPS are defined as
small (10 – 40 amino acid residues) peptides containing a
prevalence of positively charged residues (lysine and
arginine). However, some larger CAPs, such as those
from invertebrates, are also included. Most CAPs adopt an
amphipathic structure in which the charged residues are
separated in space from the hydrophobic residues. CAPs
are the products of single genes, are structurally diverse
and are broadly classified into four categories: a-helical,
b-sheet, loops with tails, and extended structures with a
Box 1. Why go fishinga?
† Oceans cover 71% of the Earth’s surface – 362 million square km
† The area of the Pacific Ocean exceeds that of all the land
† 80% of all life on Earth is found under the ocean surface
† Oceans contain 95% of the habitat space on the planet
† Oceans contain 97% of all the water on Earth
† Two-thirds of the phyla are exclusively or dominantly marine
† The number of ocean species known to man are 275 000; among
them:
W 58 species of sea grasses
W just under 1000 species of cephalopods
W 1000 species of sea anemones
W 1500 species of brown algae
W 7000 species of echinoderms
W 13 000 species of fishes
W 50 000 species of molluscs
† Global fish production exceeds that of cattle, sheep, poultry or
eggs, and is the biggest source of wild or domestic protein in the
world
† , 5% of antimicrobial peptides described to date are of
marine origin
a
Data compiled from the websites of Ocean 98 Foundation
(affiliated with UNESCO; http://www.ocean98.org) and Ocean
Voice International (http://www.ovi.ca), including referred
websites, as well as from the antimicrobial peptide database
(http://www.bbcm.univ.trieste.it/~tossi/pag1.htm).
 Review TRENDS in Biotechnology Vol.21 No.8 August 2003 363

predominance of a single amino acid (usually Trp, Pro, Naturally occurring CAPs have been credited with many
His). CAPs that are cleavage products of larger molecules, roles in the host
such as histones and ribosomal proteins, represent CAPs are one of the most important effectors in innate
another class but will not be discussed here. For more immunity [14,15]. Being synthesized in epithelial and
information on the diversity of gene-encoded CAPs the mucosal cells and circulating immune cells, they are one of
reader is referred to several recent reviews [10– 12]. the first lines of defense in response to infection and
CAPs are synthesized as pre-pro-proteins, with the pre- participate in processes during both acute and chronic
pro regions generally being conserved and the antimicrobial inflammation (for review, see [16]). Given that CAP
portion being highly diverse. Post-translational modifi- production does not depend on prior exposure to patho-
cations, such as the formation of disulphide bonds, C-term- gens, local CAP levels can be increased rapidly (in the
inal amidation, N-terminal pyroglutamic acid formation order of minutes) through degranulation of phagocytes or
and, less frequently, glycosylation contribute to the stability via Toll receptor-mediated pathways [17].
and activity of some CAPs. In addition, multiple isoforms of a Interestingly, CAP activity is more complex than mere
peptide can be derived by N-terminal truncation, for killing of pathogens and extends into other aspects of
example hepcidins [13]. CAPs are widely distributed in immune response modulation. CAPs can interact with other
nature, occurring in a vast number of organisms, and .800 host immune molecules, such as lysozyme and lactoferrin, in
have been described (http://bbcm1.univ.trieste.it/~tossi/ a synergistic fashion to promote cell lysis. They also
pag1.htm). However, at the time of writing, marine fish modulate the host immune response and provide a link to
and invertebrates are poorly represented with only 11 fish, the adaptive immune system by stimulating production
three gastropod, two bivalve, five crustacean and two of immune mediators and signaling molecules (for review,
chelicerate species cited in this database. see [18]). Various CAPs are chemotactic for specific cells of
Table 1. Natural cationic antimicrobial peptides isolated from marine organisms
Group Species Peptide Classa Refsb
Teleost fish Pardachirus marmoratus Pardaxin a [69]
Pardachirus pavoninus Pardaxin a [70]
Misgurnus anguillicaudatus Misgurnin a [71]
Pseudopleuronectes americanus Pleurocidin a [21,72]
Hippoglossoides platessoides Pleurocidin a [32]
Hippoglossus hippoglossus Pleurocidin a [32]
Limanda ferruginea Pleurocidin a [32]
Glyptocephalus cynoglossus Pleurocidin a [32]
Morone chrysops/saxatilis Moronecidin a [39]
Morone chrysops Hepcidin b4 [22]
Pleuronectes americanus Hepcidin b3,b4 [33]
Salmo salar Hepcidin b4 [33]
Oncorhynchus mykiss Hepcidin b4 EST (AF281354_1)
Paralichthys olivaceus Hepcidin b3,b4 EST (C23298.1, C23432.1)
Oryzias latipes Hepcidin b4 EST (AU178966, AU179222, AU179314,
AU179768, AU180044)
Oncorhynchus mykiss Salmocidin nd partial sequence (P81369)
Eptatretus burgeri HFS-1 b [73]

Tunicates Styela clava Styelin a [26]

Styela clava Clavanin a [28]
Styela clava Clavaspirin a [27]
Halocynthia aurantium Dicynthaurin a(2) [74]

Chelicerates Tachyplesus tridentatus Tachyplesin I b2 [75]

Tachyplesus tridentatus Tachystatin b3 [76]
Tachyplesus tridentatus Tachycitin b5 [77]
Tachyplesus tridentatus Big defensin b3 [78]
Limulus polyphemus Polyphemusin b2 [79]
Limulus polyphemus Tachyplesin II b2 [79]

Crustaceans Carcinus maenas 6.5 kDa nd [80]

Callinectes sapidus Callinectin nd [81]
Penaeus vannemi Penaeidins Pro/b3 [25]

Gastropods Dolabella auricularia Dolabellanin nd [82]

Molluscs Mytilis galloprovincialis Mytilin b4 [83]

Mytilis galloprovincialis Myticin b4 [29]
Mytilis galloprovincialis MGD b4 [24]
Mytilis edulis Defensins b3 [84]
Mytilis edulis Mytilin b4 [84]
Mytilis edulis Mytimicin b6 [84]
a
Class designations are as follows: a, amphipathic alpha helix; b, structured; number refers to number of disulphide bonds; number in brackets refers to a dimer structure; nd,
structure not determined
b
Published literature or database accession numbers for unpublished data

http://tibtec.trends.com
364 Review TRENDS in Biotechnology Vol.21 No.8 August 2003

Table 2. Differences between the traditional and the genomic approach to isolating marine antimicrobial peptides
Genomics approach: isolate genes encoding peptides Traditional approach: isolate peptides

Isolation and Unknown peptides will not be identified Unknown novel peptides can be identified
assessment of Whole families of peptides can be isolated Generally a single peptide is purified to homogeneity
activity Can be done rapidly Requires time-consuming, labour-intensive protocols
Based on motif from known peptide or flanking sequence Based on activity
Unknown activity, peptide must be synthesized chemically Only peptides active against indicator strain isolated, but
to test range of activity still need to synthesize sufficient quantities chemically to
test range of activity
Expression studies Probes for in situ hybridization can be designed Need antibodies, or need to clone genes to design probes
immediately for expression studies
Primers for RT –PCR can be designed immediately

Recombinant Ready-made genes available for transgenic production Need to clone genes to produce
production and Pre- and pro-sequences known – can be utilized to
transgenics optimize recombinant as well as transgenic production
Promoter sequences frequently known – can be utilized in
transgenic production

the immune system, such as T cells, monocytes and
neutrophils, and can influence the development of macro-
phages from monocytes. Defensins stimulate the pro-
duction of cytokine IL-8, which is responsible for
activating neutrophils and for degranulating mast cells,
causing histamine release and vasodilation. This enhances
transmigration of immune cells through the endothelium
to sites of infection. Microarray studies have shown that
treatment of macrophage cells with CAPs results in the
differential expression of several genes, many of which are
involved in immunity but some of which are unknown [19].
In addition, by binding and neutralizing LPS, CAPs
prevent the production of proinflammatory cytokines, such
as TNFa by macrophages, thus reducing sepsis. Certain
CAPs enhance apoptosis in macrophage cell lines and
activated lymphocytes, thereby clearing the system of
infected cells. Some CAPs inhibit proteases involved in
tissue degradation during inflammation and some effect
wound repair through the induction of proteoglycans.
Finally, CAPs are important for controlling the natural
flora and selecting and maintaining resident commensals
[15]. The use of CAPs as immune enhancers represents a
huge potential area of research as well as source of
therapeutants.
Genomics approaches
Much of our knowledge of CAPs has come from identifi-
cation of peptides through biochemical approaches and we
will continue to rely on such approaches for the identifi-
cation of novel peptide classes. However, genomics
approaches are now able to build on this information
and reveal families of related CAP sequences (Table 2).
The first molecular biological technique used to isolate
CAP-encoding sequences from marine organisms was
cDNA and genomic library screening using oligonucleotide
probes based on CAP amino acid sequences. This was
successfully used to isolate clones encoding pleurocidin
from winter flounder [20,21], hepcidin from bass [22],
mytilins [23] and MGDs [24] from mussels and penaeidins
from shrimp [25]. In addition, primers based on peptide
sequences have been used to amplify probes for isolating
styelin [26] and clavaspirin [27] sequences from tunicate
hemocyte and/or pharyngeal cDNA libraries [26], clavinin
http://tibtec.trends.com
sequences from tunicate hemocyte cDNA [28] and myticin
from a mussel hemocyte cDNA library [29].
Modern molecular technology offers the added benefit of
isolating whole families of peptides from the nucleic acids
that encode them. Given that many CAPs are encoded by
multigene families in mammals [30] and shrimp [31], PCR
approaches have been used to investigate if this is also true
in bony fish and other marine organisms. By using primers
based on conserved sequences in the N-terminal signal
peptide and the highly conserved anionic portion at the
carboxy terminus of the pre-propeptide, multiple pleur-
ocodin-like CAPs from both mRNA and genomic DNA of
winter flounder were amplified [21]. Recent studies
extended this work to the identification of additional
pleurocodin-like CAPs from other flatfish species [32].
Using a similar approach, we isolated multiple hepcidin-
like CAPs from both Atlantic salmon and various flatfish
species [33] using primers based on conserved sequences
revealed by expressed sequence tag (EST) surveys.
Extensive data-mining of dbEST using an EST encoding
a hepcidin-like peptide from Atlantic salmon [34] uncov-
ered several unannotated ESTs from Japanese flounder,
medaka and rainbow trout that, on closer scrutiny, indeed
encoded hepcidin-like peptides. Alignment of these
sequences shows that these CAPs fall into several
categories depending on the number of cysteine residues,
signature deletions and conserved amino acid residues
present. Recently, new members of the penaeidin family
have been identified as part of an EST survey of two species
of shrimp [35] (Fig. 1).
Another approach that has proved fruitful in identify-
ing additional CAP-encoding sequences is genomic
sequencing. Southern hybridization analysis indicates
that pleurocidins [21] and hepcidins [33] are organized
as gene clusters. All genes and pseudogenes investigated
have similar intron – exon features and careful scanning of
the conserved flanking sequences allowed presumptive
identification of promoters and transcription factor bind-
ing sites (S.E. Douglas, unpublished). The different genes
are expressed at different times during larval development
and show tissue-specific expression in adult fish. Genome-
walking upstream of genes encoding hepcidin-like pep-
tides is currently underway in our laboratory to identify
 Review TRENDS in Biotechnology
Flanking sequences
Known peptide
New peptide 1
New peptide 2
New peptide 3
New peptide 4
New peptide 5
New peptide 6
TRENDS in Biotechnology
Fig. 1. Strategy for isolating novel antimicrobial peptides using genomic technol-
ogy. Primers based on conserved portions of known peptides, or based on con-
served sequences flanking the peptides, are used to amplify novel cationic
antimicrobial peptides (CAPs).
promoters and sequence motifs involved in regulation of
members of this gene family. Elucidating the factors
controlling the expression of CAP genes will lead to a
better understanding of the various roles that these
peptides have in the host organism, and in turn, to a
better assessment of their therapeutic potential.
It appears that a given species can encode multiple
CAPs [32,33,36], each of which might have unique
properties such as spectrum of activity, inducibility in
response to disease, developmental expression and tissue-
specific expression. Having the nucleic acid sequences
encoding the peptides allows for the design of tailor-made
probes for comprehensive studies of temporal and tissue-
specific expression of the peptides (e.g. via in situ
hybridization or RT-PCR). Such studies are at the
forefront of peptide research providing information on
which peptides occur together, and which do not. This
information can be beneficial in designing therapeutic
peptides, or therapeutic mixtures of peptides.
Tissue-specific expression of hepcidin has been demon-
strated in bass [22] and Atlantic salmon [33]. Further-
more, we have shown tissue-specific expression of different
pleurocidin genes in adult winter flounder (S.E. Douglas,
unpublished). CAPs have been localized to circulating
granulocytes in bass gill [37] and we recently showed that
pleurocidin is present in granule-containing migratory
immune cells present in the gill of winter flounder [38] in
addition to the previously described location in skin
mucous cells [20].
http://tibtec.trends.com
Vol.21 No.8 August 2003 365
We demonstrated constitutive expression of one form of
hepcidin-like peptide and inducible expression of another
form in Atlantic salmon challenged with the bacterial
pathogen Aeromonas salmonicida [33]. Moronecidin [39]
and hepcidin [22] are also expressed by bass in response to
bacterial challenge. Expression of CAPs is constitutive in
adult shrimp [40], mussels [41] and horseshoe crab [42]
and has been localized to circulating large granule
hemocytes in shrimp [40] and phagocytic hemocytes in
mussel [41].
Transcripts of pleurocidin-like genes have been
detected as early as five days post-hatch in winter flounder
larvae [21] and different pleurocidin-like genes are
expressed at different times during development (S.E.
Douglas, unpublished). Recent work showed that CAP
expression occurs early in larval shrimp development [43].
In mussels, expression of mytilin B and MGD2 is also
developmentally regulated [23]. The early expression of
CAPs during development underscores both the import-
ance of these peptides in larval immunity and the potential
use of transgenic approaches in enhancing disease
resistance in these organisms.
Advantages of CAPs over other antibiotics
CAPs exhibit numerous activities that make them
promising candidates for therapeutants. They can display
broad-spectrum activity against a variety of bacteria,
selective activity against Gram-negative or Gram-positive
bacteria, or activity against fungi [44], enveloped viruses
[45,46], protozoa [47] and even some cancer cells [48,49].
They exhibit several desirable properties over many of the
antibiotics currently in use. First, CAPs kill rapidly (99.9%
within 20 min) [50] and exhibit synergy with conventional
antibiotics, lysozyme and each other [51,52]. Second, CAPs
are active against both antibiotic-susceptible and clinically
antibiotic-resistant strains of the same bacterial species.
Third, CAPs do not easily select resistant mutants [10],
although some bacteria survive by incorporating phos-
phorylcholine in their membranes to mimic host cells [53]
or alter other membrane characteristics (see [54]). Finally,
unlike many antibiotics that can indirectly cause sepsis
because of the release of endotoxin from dead bacterial
cells, CAPs bind endotoxin and reduce septic shock.
The large natural repertoire of marine CAPs uncovered
by genomic approaches provides a sizeable pipeline for
screening of potential therapeutants. To achieve the
required modality, whether it be to a specific bacterium,
a target range of bacteria, or a host-directed activity, we
can now screen entire libraries of naturally occurring,
closely related antimicrobial peptides, or combinations
thereof. At this stage of research, with limited consensus
as to the peptide mode of action, this seems to be a more
realistic approach than trying to design a ‘magic bullet’ by
systematically altering an existing peptide.
Mode of action
For CAPs to be considered as safe alternatives to
antibiotics, their mode of action must be better understood.
Although most researchers agree that CAPs interact with
membranes and can cause damage to bacterial membranes
at high concentrations, both the mechanism of membrane
366 Review TRENDS in Biotechnology
disruption and the actual cause of killing remain con-
troversial (for reviews, see [14,55,56]). In addition to
purely physical modes of action, CAPs might also interact
with internal targets (see [11]) such as heat shock proteins,
DNA and RNA, a feature that can be capitalized on in the
search for novel therapeutants.
Biotechnological approaches to CAP synthesis and
expression
Chemical synthesis of some classes of CAPs is possible but
those containing disulphide bonds are notoriously difficult
to synthesize and refold with the correct disulphide
connectivity. Although chemical synthesis is necessary
for initial screening protocols, the expense is substantial
for production of the amounts of peptide required for
physiological investigation and clinical trials. In vitro
expression using recombinant DNA technology in bac-
terial [57– 59] and/or yeast [44,60] systems provides an
alternative method of cost-effective production. The
advantage of yeast expressions systems is that correct
post-translational modifications are performed. As yet,
there have been no reports of viral expression vectors
being used for CAP production.
PPL Therapeutics (http://www.ppl-therapeutics.com/)
has successfully produced a novel CAP in milk of
transgenically modified rabbits, with the aim of extending
this to cattle production. Special precautions must be used
to prevent toxic effects of the recombinant peptide on the
host cells. One option is the inclusion of the naturally
occurring anionic ‘neutralizing’ elements of the pre-
propeptide. Such sequences can be easily identified using
the genomic technologies described herein but would not
be identified in peptides isolated by protein purification
because the pre-pro-peptide would have already been
cleaved off.
In agriculture and especially aquaculture (in which few
approved antibiotics and chemicals are available and
vaccines are limited), genetically modified organisms
overexpressing CAPs might result in valuable disease-
resistant species. The use of intraperitoneal pumps [61] to
deliver protective CAPs to fish, as well as the engineering
of transgenic plants [62,63] expressing CAPs have
demonstrated the feasibility of this approach. Prelimi-
nary research with transgenic fish is underway and the
results are promising. Examples include medaka
transgenic for cecropin [64] and pleurocidin (T. Chen,
pers. commun.), zebrafish transgenic for pleurocidin
(C Hew, pers. commun.), as well as catfish [65] and
shrimp (J.K. Lu, pers. commun.) transgenic for cecropin.
In the context of producing organisms transgenic for
antimicrobial peptides from related species, there is a
great advantage to having the cDNA and genomic
sequences, because the pre-pro sequences can be ident-
ified, along with the natural promoters. This genetic
information can later be considered for use in the
transgenic organisms, accelerating the discovery-to-pro-
duct pipeline for marine CAPs. Once appropriate safety
considerations and containment issues are resolved,
consumer confidence and public acceptance of such
organisms should be obtained.
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Vol.21 No.8 August 2003
Efficacy testing and applications
By examining the peptide sequences potentially encoded
by genetic information and predicting which sequences are
likely to encode functional CAPs on the basis of charge,
hydrophobicity and similarity to known active peptides,
testing protocols can be highly focused. Furthermore,
assays of antimicrobial activity using chemically syn-
thesized peptides against a panel of Gram-positive and
Gram-negative bacteria as well as pathogenic fungi and
viruses can give a preliminary indication of which peptides
are most promising. Interestingly, even a peptide pre-
dicted from a putative pleurocidin pseudogene has been
shown to possess antimicrobial activity (S.E. Douglas,
unpublished), underscoring the advantage of scanning
genomic information for ‘dead’ genes. A similar approach
was used in the recreation of retrocyclin, a peptide with
activity against HIV-1, from an expressed pseudogene [45].
Cytotoxicity assays using a variety of host cells further
accelerates the screening, allowing identification of pep-
tides that could be tested in animal models. Animal testing
is generally the final step before clinical trials.
Several CAPs show great promise in a variety of
different applications (Box 2), and some are currently
undergoing clinical trials. Micrologix Biotech has two
cationic antimicrobial peptides in clinical trials (http://
www.mbiotech.com). The company’s first peptide, MBI
226, is being tested for its ability to prevent central venous
catheter-related bloodstream infections and has been
enrolled for Phase III clinical trials. The second peptide,
MBI594AN, is being tested for the treatment of acne and
has been enrolled for Phase IIb clinical trials.
Some issues generally associated with drug develop-
ment, such as drug stability, metabolism and delivery
a
Box 2. The varied applications of antimicrobial peptides
† Topical applications for skin infections, diabetic foot ulcers, burns
† Oral solution for prevention of mucosaitis
† Mouthrinse for treatment of oral candidiasis
† Inhalation treatment for respiratory infections in cystic fibrosis
patients
† Treatment of drug-resistant bacterial and fungal infections
† Prevention of venous catheter-related bloodstream infections
† Treatment of acne
† Treatment of Herpes simplex virus
† Treatment of Chlamydia
† Inhibition of bacterial growth in foods and packaged products
† Enhancement of conventional antibiotics through synergistic
activities
† Immune enhancers
† Resistance to phytopathogens through transgenic technology
W apples
W tobacco
W forestry industry
† Resistance to animal pathogens through transgenic technology
W catfish
a
Information compiled from publications referenced elsewhere in
this review as well as the following company websites: http://www.
genaera.com, http://www.intrabiotics.com, http://www.mbiotech.
com, http://www.inimexpharma.com, http://www.helixbiomedix.
com
 Review TRENDS in Biotechnology
systems required to administer the drug effectively and
safely still need to be addressed in the context of most
CAPs. However, these factors are being researched, with
tantalizing results in some cases. For instance, it is known
that peptide precursors can be protease inhibitors [66],
which could possibly protect the peptides from destruction.
Further development along these lines could yield an
effective method for stabilizing antimicrobial peptide
drugs, especially for systemic applications. Specialized
delivery systems, such as liposomes, are also being
considered for CAPs [67].
Finally, CAPs are also being considered for non-medical
applications, such as disease control in aquaculture [68]
and agriculture, in which regulatory approval is easier to
obtain, and in which high costs of peptide production can
be circumvented by introducing peptide transgenes. The
application of components of non-specific immunity,
including CAPs, to aquaculture was the subject of a recent
workshop entitled the ‘Biotechnology – Aquaculture Inter-
face: The Site of Maximum Impact Workshop’ sponsored by
the United States Department of Agriculture (http://nps.
ars.usda.gov/static/arsoibiotecws2001/contributions/).
Conclusions
The marine environment, with its enormous biodiversity,
provides a largely untapped reservoir of previously
undescribed CAPs. As can be seen from Table 1, most of
the CAPs isolated from marine sources are highly
disulphide-bonded b structures; only a few are amphi-
pathic a-helices and these have been isolated mostly from
teleosts. It is becoming evident that many CAPs are
encoded by multigene families, a feature that makes
genomics approaches to identification of novel sequences
particularly attractive. Recombinant DNA technologies
are being developed for cost-effective production of CAPs
and information is beginning to accrue on mode of action
and regulation of gene expression, both of which are
aspects that require a thorough understanding if CAPs are
to be used as therapeutants.
Finally, one must keep in mind that CAPs are only one
group of useful compounds offered to us by the sea and that
other as yet unidentified classes of CAPs almost certainly
exist. The exploration of marine biodiversity has yielded
crops ranging from sponges to antifreeze proteins and
specialized detoxification systems. With the molecular
technology of the 21st century it is highly likely that
potent anti-infective drugs, anti-cancer drugs, and
drugs to treat diseases yet to be discovered will also
come from the sea.
Acknowledgements
Antimicrobial peptide research in S.E.D’s laboratory in supported by the
National Research Council of Canada, the Genomics and Health
Initiative, and MedInnova Partners Inc. We thank Dr Neil Mattatall for
critical review of this manuscript before submission. This is NRCC
publication 42387.
References
1 Austin, B. and McIntosh, D. (1988) Natural antimicrobial compounds
on the surface of rainbow trout, Salmo gairdneri Richardson. J. Fish
Dis. 11, 275 – 277
http://tibtec.trends.com
Vol.21 No.8 August 2003 367
2 Fouz, B. et al. (1990) Antibacterial action of the mucus of the turbot.
Bull. Eur. Assoc. Fish Pathol. 10, 56 – 59
3 Grindle, B. (1989) Lysozyme from rainbow trout, Salmo gairdneri
Richardson, as an antibacterial agent against fish pathogens. J. Fish
Dis. 12, 95 – 104
4 Smith, V.J. and Chisholm, J.R. (2001) Antimicrobial proteins in
crustaceans. Adv. Exp. Med. Biol. 484, 95– 112
5 Findlay, C. and Smith, V.J. (1995) Antimicrobial factors in solitary
ascidians. Fish Shellfish Immunol. 5, 645 – 658
6 LeMaitre, C. et al. (1996) Characterization and ion channel activities of
novel antibacterial proteins from the skin mucosa of carp (Cyprinus
carpio). Eur. J. Biochem. 240, 143 – 149
7 Moore, K.S. et al. (1993) Squalamine: an aminosterol antibiotic from
the shark. Proc. Natl. Acad. Sci. U. S. A. 90, 1354– 1358
8 Robinette, D. et al. (1998) Antimicrobial activity in the skin of the
channel catfish Ictalurus punctatus: characterization of broad-spec-
trum histone-like antimicrobial proteins. Cell. Mol. Life Sci. 54,
467 – 475
9 Richards, R.C. et al. (2001) Histone H1: an antimicrobial protein of
Atlantic salmon (Salmo salar). Biochem. Biophys. Res. Commun. 284,
549 – 555
10 Devine, D.A. and Hancock, R.E. (2002) Cationic peptides: distribution
and mechanisms of resistance. Curr. Pharm. Des. 8, 703 – 714
11 Tossi, A. and Sandri, L. (2002) Molecular diversity in gene-encoded,
cationic antimicrobial peptides. Curr. Pharm. Des. 8, 743 – 761
12 Lehrer, R.I. and Ganz, T. (2002) Defensins of vertebrate animals. Curr.
Opin. Immunol. 14, 96 – 102
13 Krause, A. et al. (2000) LEAP-1, a novel highly disulfide-bonded
human peptide, exhibits antimicrobial activity. FEBS Lett. 480,
147– 150
14 Hancock, R.E. (2001) Cationic peptides: effectors in innate immunity
and novel antimicrobials. Lancet Infect. Dis. 1, 156 – 164
15 Boman, H.G. (2000) Innate immunity and the normal microflora.
Immunol. Rev. 173, 5 – 16
16 Scott, M.G. and Hancock, R.E. (2000) Cationic antimicrobial peptides
and their multifunctional role in the immune system. Crit. Rev.
Immunol. 20, 407 – 431
17 Hoffman, J.A. and Reichart, J.M. (2002) Drosophila innate immunity:
an evolutionary perspective. Nat. Immun. 3, 121 – 126
18 Yang, D. et al. (2001) The role of mammalian antimicrobial peptides
and proteins in awakening of innate host defenses and adaptive
immunity. Cell. Mol. Life Sci. 58, 978– 989
19 Scott, M.G. et al. (2000) An a-helical cationic antimicrobial peptide
selectively modulates macrophage responses to lipopolysaccharide and
directly alters macrophage gene expression. J. Immunol. 165,
3358– 3365
20 Cole, A.M. et al. (2000) Characterization of a fish antimicrobial
peptide: gene expression, subcellular localization, and spectrum of
activity. Antimicrob. Agents Chemother. 44, 2039– 2045
21 Douglas, S.E. et al. (2001) Cloning and developmental expression of a
family of pleurocidin-like antimicrobial peptides from winter flounder,
Pleuronectes americanus (Walbaum). Dev. Comp. Immunol. 25,
137– 147
22 Shike, H. et al. (2002) Bass hepcidin is a novel antimicrobial peptide
induced by bacterial challenge. Eur. J. Biochem. 269, 2232 – 2237
23 Mitta, G. et al. (2000) Mytilin B and MGD2, two antimicrobial peptides
of marine mussels: gene structure and expression analysis. Dev. Comp.
Immunol. 24, 381 – 393
24 Mitta, G. et al. (1999) Mussel defensins are synthesised and processed
in granulocytes then released into the plasma after bacterial
challenge. J. Cell Sci. 112, 4233 – 4242
25 Destoumieux, D. et al. (1997) Penaeidins, a new family of antimicrobial
peptides isolated from the shrimp Penaeus vannamei (Decapoda).
J. Biol. Chem. 272, 28398 – 28406
26 Zhao, C. et al. (1997) cDNA cloning of three cecropin-like antimicrobial
peptides (Styelins) from the tunicate, Styela clava. FEBS Lett. 412,
144– 148
27 Lee, I.H. et al. (2001) Clavaspirin, an antibacterial and haemolytic
peptide from Styela clava. J. Pept. Res. 58, 445 – 456
28 Lee, I.H. et al. (1997) Clavanins, a-helical antimicrobial peptides from
tunicate hemocytes. FEBS Lett. 400, 158 – 162
29 Mitta, G. et al. (1999) Myticin, a novel cysteine-rich antimicrobial
368 Review TRENDS in Biotechnology
peptide isolated from haemocytes and plasma of the mussel Mytilus
galloprovincialis. Eur. J. Biochem. 265, 71 – 78
30 Harder, J. et al. (1997) Mapping of the gene encoding human beta-
defensin-2 (DEFB2) to chromosome region 8p22-p23.1. Genomics 46,
472 – 475
31 Destoumieux, D. et al. (2000) Penaeidins, a family of antimicrobial
peptides from penaeid shrimp (Crustacea, Decapoda). Cell. Mol. Life
Sci. 57, 1260– 1271
32 Patrzykat, A. et al. Novel natural antimicrobial peptides from genes of
selected flatfish species. Antimicrob. Agents Chemother. (in press)
33 Douglas, S.E. et al. (2003) Identification and expression analysis of
hepcidin-like antimicrobial peptides in bony fish. Dev. Comp.
Immunol. 27, 589 – 601
34 Tsoi, S.C.M. et al. Identification of immune-relevant genes from
Atlantic salmon using suppression subtractive hybridization. Mar.
Biotechnol. (in press)
35 Gross, P.S. et al. (2001) Immune gene discovery by expressed sequence
tag analysis of hemocytes and hepatopancreas in the Pacific White
Shrimp, Litopenaeus vannamei, and the Atlantic White Shrimp,
L. setiferus. Dev. Comp. Immunol. 25, 565 – 577
36 Cuthbertson, B.J. et al. (2002) Diversity of the penaeidin antimicrobial
peptides in two shrimp species. Immunogenetics 54, 442 – 445
37 Silphaduang, U. and Noga, E.J. (2001) Peptide antibiotics in mast cells
of fish. Nature 414, 268 – 269
38 Murray, H.M. et al. Cellular localization of pleurocidin expression in
winter flounder gill using immunohistochemistry and in situ hybrid-
ization. Cell Tissue Res. (in press)
39 Lauth, X. et al. (2002) Discovery and characterization of two isoforms
of moronecidin, a novel antimicrobial peptide from hybrid striped bass.
J. Biol. Chem. 277, 5030– 5039
40 Destoumieux, D. et al. (2000) Penaeidins, antimicrobial peptides with
chitin-binding activity, are produced and stored in shrimp granulo-
cytes and released after microbial challenge. J. Cell Sci. 113, 461– 469
41 Mitta, G. et al. (2000) Original involvement of antimicrobial peptides
in mussel innate immunity. FEBS Lett. 486, 185– 190
42 Iwanaga, S. and Kawabata, S. (1998) Evolution and phylogeny of
defense molecules associated with innate immunity in horseshoe crab.
Front. Biosci. 3, D973– D984
43 Munoz, M. et al. (2003) Expression of penaeidin antimicrobial peptides
in early larval stages of the shrimp Penaeus vannamei. Dev. Comp.
Immunol. 27, 283 – 289
44 Destoumieux, D. et al. (1999) Recombinant expression and range of
activity of penaeidins, antimicrobial peptides from penaeid shrimp.
Eur. J. Biochem. 266, 335– 346
45 Cole, A.M. et al. (2002) Retrocyclin: a primate peptide that protects
cells from infection by T- and M-tropic strains of HIV-1. Proc. Natl.
Acad. Sci. U. S. A. 99, 1813 – 1818
46 Daher, K.A. et al. (1986) Direct inactivation of viruses by human
granulocyte defensins. J. Virol. 60, 1068 – 1074
47 Huang, C.M. et al. (1990) Magainin analogs effective against
pathogenic protozoa. Antimicrob. Agents Chemother. 34, 1824 – 1826
48 Cruciani, R.A. et al. (1991) Antibiotic magainins exert cytolytic activity
against transformed cells through channel formation. Proc. Natl.
Acad. Sci. U. S. A. 88, 3792 – 3796
49 Baker, M.A. et al. (1993) Anticancer efficacy of magainin 2 and
analogue peptides. Cancer Res. 53, 3052– 3057
50 Piers, K.L. et al. (1994) Improvement of outer membrane permeabi-
lization and lipopolysaccharide-binding activities of an antimicrobial
cationic peptide by C-terminal modification. Antimicrob. Agents
Chemother. 38, 2311 – 2316
51 Hancock, R.E.W. and Scott, M.G. (2000) The role of antimicrobial
peptides in animal defenses. Proc. Natl. Acad. Sci. U. S. A. 97,
8856 – 8861
52 Yan, H. and Hancock, R.E. (2001) Synergistic interactions between
mammalian antimicrobial defense peptides. Antimicrob. Agents
Chemother. 45, 1558 – 1560
53 Lysenko, E.S. et al. (2000) Bacterial phosphorylcholine decreases
susceptibility to the antimicrobial peptide LL-37/hCAP18 expressed in
the upper respiratory tract. Infect. Immun. 68, 1664 – 1671
54 Andreu, A. and Rivas, L. (1998) Animal antimicrobial peptides: an
overview. Biopolymers 47, 415 – 433
55 Hancock, R.E. and Rozek, A. (2002) Role of membranes in the activities
http://tibtec.trends.com
Vol.21 No.8 August 2003
of antimicrobial cationic peptides. FEMS Microbiol. Lett. 206,
143– 149
56 Shai, Y. (2002) Mode of action of membrane active antimicrobial
peptides. Biopolymers 66, 236– 248
57 Piers, K.L. et al. (1993) Recombinant DNA procedures for producing
small antimicrobial cationic peptides. Gene 134, 7 – 13
58 Hwang, S.W. et al. (2001) A simple method for the purification of an
antimicrobial peptide in recombinant Escherichia coli. Mol. Biotech-
nol. 18, 193– 198
59 Lee, J.H. et al. (2000) High-level expression of an antimicrobial peptide
mediated by a fusion partner reinforcing formation of inclusion bodies.
Biochem. Biophys. Res. Commun. 277, 575 – 580
60 Reichart, J.M. et al. (1992) Expression and secretion in yeast of active
insect defensin, an inducible antibacterial peptide from the fleshfly,
Phormia terranovae. Invertebr. Reprod. Dev. 21, 15– 24
61 Jia, X. et al. (2000) Antimicrobial peptides protect coho salmon from
Vibrio anguillarum infections. Appl. Environ. Microbiol. 66,
1928– 1932
62 Huang, Y. et al. (1997) Expression of an engineered cecropin gene
cassette in transgenic tobacco plants confers disease resistance to
Pseudomonas syringae pv. tabaci. Phytopathology 87, 494 – 499
63 Osusky, M. et al. (2000) Transgenic plants expressing cationic peptide
chimeras exhibit broad-spectrum resistance to phytopathogens. Nat.
Biotechnol. 18, 1162 – 1166
64 Sarmasik, A.A. et al. (2002) Production of transgenic medaka with
increased resistance to bacterial pathogens. Mar. Biotechnol. 4,
310– 322
65 Dunham, R.A. et al. (2002) Enhanced bacterial disease resistance of
transgenic channel catfish Ictalurus punctatus possessing cecropin
genes. Mar. Biotechnol. 4, 338 – 344
66 Verbanac, D. et al. (1993) Chemotactic and protease-inhibiting
activities of antibiotic peptide precursors. FEBS Lett. 317, 255 – 258
67 Ahmad, I. et al. (1995) Liposomal entrapment of the neutrophil-
derived peptide indolicidin endows it with in vivo anti-fungal activity.
Biochim. Biophys. Acta 1237, 109– 114
68 Bachere, E. (2000) Shrimp immunity and disease control. Aquaculture
191, 3 – 11
69 Lazarovici, P. et al. (1986) Purification and pore-forming activity of
two hydrophobic polypeptides from the secretion of the Red Sea
Moses sole (Pardachirus marmoratus). J. Biol. Chem. 261,
16704 – 16713
70 Thompson, S.A. et al. (1986) Melittin-like peptides from the shark-
repelling defense secretion of the sole Pardachirus pavoninus. Science
233, 341 – 343
71 Park, C.B. et al. (1997) A novel antimicrobial peptide from the loach,
Misgurnus anguillicandatus. FEBS Lett. 411, 173 – 178
72 Cole, A.M. et al. (1997) Isolation and characterization of pleurocidin,
an antimicrobial peptide in the skin secretions of winter flounder.
J. Biol. Chem. 272, 12008 – 12013
73 Hwang, E.-Y. et al. (1999) Purification and characterization of a novel
antimicrobial peptide from the skin of the hagfish, Eptatretus burgeri.
J. Food Sci. Nutr. 4, 28 – 32
74 Lee, I.H. et al. (2001) Dicynthaurin: an antimicrobial peptide from
hemocytes of the solitary tunicate, Halocynthia aurantium. Biochim.
Biophys. Acta 1527, 141 – 148
75 Nakamura, T. et al. (1988) Tachyplesin, a class of antimicrobial peptide
from the hemocytes of the horseshoe crab (Tachyplesus tridentatus).
J. Biol. Chem. 263, 16709 – 16713
76 Dimarcq, J.L. et al. (1998) Cysteine-rich antimicrobial peptides in
invertebrates. Biopolymers 47, 465 – 477
77 Kawabata, S. et al. (1996) Tachycitin, a small granular component in
horseshoe crab hemocytes, is an antimicrobial protein with chitin-
binding activity. J. Biochem. (Tokyo) 120, 1253– 1260
78 Saito, T. et al. (1995) A novel big defensin identified in horseshoe crab
hemocytes: isolation, amino acid sequence, and antibacterial activity.
J. Biochem. (Tokyo) 117, 1131 – 1137
79 Miyata, T. et al. (1989) Antimicrobial peptides, isolated from
horseshoe crab hemocytes, tachyplesin II, and polyphemusins I
and II: chemical structures and biological activity. J. Biochem.
(Tokyo) 106, 663 – 668
80 Schnapp, D. et al. (1996) Purification and characterization of a proline-
rich antibacterial peptide, with sequence similarity to bactenecin-7,
 Review TRENDS in Biotechnology Vol.21 No.8 August 2003 369

from the haemocytes of the shore crab, Carcinus maenas. Eur.
J. Biochem. 240, 532 – 539
81 Khoo, L. et al. (1999) Callinectin, an antibacterial peptide from blue
crab, Callinectes sapidus, hemocytes. Mar. Biotechnol. 1, 44 – 51
82 Iijima, R. et al. (2003) A novel antimicrobial peptide from the sea hare
Dolabella auricularia. Dev. Comp. Immunol. 27, 305 – 311
83 Mitta, G. et al. (2000) Involvement of mytilins in mussel antimicrobial
defense. J. Biol. Chem. 275, 12954 – 12962
84 Charlet, M. et al. (1996) Innate immunity. Isolation of several cysteine-
rich antimicrobial peptides from the blood of a mollusc, Mytilus edulis.
J. Biol. Chem. 271, 21808 – 21813

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