amit kumar sinha - antimicrobial peptide

8/14/2019 Amit Kumar Sinha - Antimicrobial Peptide

1/450 December 2009

Antimicrobial peptides: Possibletherapeutics in aquacultureaMit kuMar sinHa1, parisa norouzitallab2and kartik baruaH1

Disease outbreak has been the ma-

jor bottleneck in the successul arm-

ing o fsh and shellfsh. In recent

years, much attention has been given

to health management using various

orms o immunoprophylactic tech-

niques, such as vaccination, antibiot-

ics and immunostimulants. However,

these practices are now questioned be-

cause o issues, such as tissue residue,bacterial drug resistance and environ-

mental imbalances created by regular

use o antibiotics.

Vaccines, although commercially

available, generally cannot be used as

a universal disease control measure in

aquaculture. Conventional techniques,

such as selection and hybridization

have been used to produce a number

o resistant fsh or broodstocks using

existing resistant individuals in het-

erogeneous fsh stocks. However, such

techniques are restricted to within spe-cies or closely related species and do

not allow or the employment o po-

tentially eective resistance genes pos-

sessed by other organisms. In contrast,

modern approaches recombinant

DNA technology, or instance en-

able the transer o genes rom diverse

sources, such as insects, to improve the

natural resistance o fsh to inections

in a directed ashion. However, their

use at arm level is yet to be answered.

This indicated that the above-cited ap-

proaches were not able to solve every

problem alone. Hence, this called or

the development o new approaches

and technologies suited to improve

health o armed species and, at the

same time, not be harmul to the en-

vironment. In this article, the potential

o using natural antimicrobial agents

or immunoenhancers in eliminating

diseases in aquaculture is discussed.

It has long been known that the

innate (non-specifc) deense system

includes acute phase proteins such as

C-reactive protein and its homologues.

However, an important acet o this

deense mechanism was revealed two

decades ago by the discovery o ce-

cropins, inducible antimicrobial pep-

tides in the giant silk moth Hyalophora

cecropia. These biochemical deense

peptides possess antimicrobial activityand, thereore, are considered as natu-

ral antibiotics. Fish also possess anti-

microbial peptides as part o their de-

ense system. They are mainly located

in the mucus layer and help eliminate

pathogens beore they pass the skin

barrier. The so-called natural antibiot-

ics seem to be eective as a therapeutic

agent and an antimicrobial in enhanc-

ing innate or non-specifc immunity in

fsh.

What are Antimicrobial Peptides(AMPs)?

AMPs are low molecular weight

peptides (size


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World AquAculture 51

throughalternative modes o action or

that they may, in act, act uponmul-

tiple bacterial cell targets. Regardless

o their precisemode o action, the ac-

tivities o AMPs arealmost universally

dependent on interaction with the bac-

terialcell membrane.

Membrane-Permeabilizing

MechanismThe rst step in this interaction is the

initial attraction between the peptide

and the target cell.Electrostatic bond-

ing between the cationic peptide and

negatively charged components present

on the outer bacterial envelope is the

main driving orce or interaction. The

negatively charged components are the

phosphate groups withinthe lipopoly-

saccharides o Gram-negative bacteria

or lipoteichoicacids present on the sur-

aces o gram-positive bacteria.In case o Gram-negative bacteria,

peptides are inserted into the outer

membrane structure mediated by hy-

drophobic interactions and involve

preolding o the peptidesinto a mem-

brane-associated structure. This alters

the outer membrane structure and per-

meabilizes this membraneto other pep-

tide molecules in a process termed sel-

promoted uptake. This results in the

infux o peptides at the cytoplasmic

membrane where they enter the inter-

acial region o the membrane, betweenthe hydrophilic and hydrophobic por-

tionso the membrane. This step is car-

ried out by electrostatic and hydropho-

bicinteractions (Figure 1). The higher

proportion o negatively charged lipids

on the surace monolayer o the bacte-

rial cytoplasmic membraneplays an im-

portant role in the selectivity o AMPs

or bacterial cells over eukaryotic cells,

in whichuncharged lipids predominate

at the host cell surace.

Non-Membrane-PermeabilizingMechanism

There are several AMPs that show

their action without permeabilizing the

membrane. They translocateacross the

membrane and accumulate intracellu-

larly, where they interere with several

essential cellular processes and mediate

cell destruction. Their modes o action

include inhibition o nucleic acid syn-

Fig. 1. Pictorial description o the sel-promoted uptake o cationic peptides across

the outer membrane. The peptide monomers bind to the cell membrane in a -helicalconfrmation (A), this is ollowed by the localization o more peptide molecules on the

cell membrane (B) ater which the peptide helices insert themselves into the hydro-

phobic core o the membrane (C). Progressive recruitment o additional monomers

increase the pore size causing leakage o cytoplasmic material (D) and, hence, death

o the cell (Hancock 2001 and Reddy et al. 2004).

thesis, proteinsynthesis, enzymatic ac-

tivity and cell wall synthesis. Buorin II,

an AMP present in rogs, translocates

acrossthe bacterial membrane without

causing permeabilization andbinds to

both DNA and RNA within the cyto-plasm oE. coli(Park et al. 1998).Simi-

larly, pleurocidin, a sh-derived AMP

and dermaseptin isolated rom rog

skin, causes inhibition o DNA and

RNA synthesis without destabilizing

the membrane oE. colicells(Patrzykat

et al. 2002). Inhibition o nucleic acid

synthesis has also been demonstrated

or AMPs, such as human deensin,

HNP-1 and the extended-structure bo-

vine peptide indolicidin(SubbalakshmiandSitaram 1998). Additionally, someo these peptides have been shownto

interere with protein synthesis. Pleuro-

cidin and dermaseptincan block tritiat-

ed leucine uptake in E. coli, and PR-39-

andindolicidin-treated cells also exhibit

reduced rates o proteinsynthesis (Sub-

balakshmi and Sitaram 1998)

Classifcation o AMPAMPs are broadly classied into

ve groups

1. Helical AMPs: Linear, mostly

helical peptides without cystine

residue, with or without a hinge

region. Cecropins,magainins and

bombinins are in this group.

2. Cysteine rich AMPs:They are richin cysteine residues that are joined

by with two or more disulde

bridges. Deensins are well known

representatives o this group.

3. -Sheet AMPs: They generallyorm a single -hairpin structure

and are approximately 20 residues

long, containing one or two disul-

de linkages. They include horse-

shoe crab peptides, tachyplesins

and polyphemusin II.

4. AMPs rich in regular amino acids:These includelinear peptides with-out cysteine residue but with a high

number o regular amino acids. For

instance, attacins and diptericins

rom this group contain glycine-

rich and proline-rich domains, while

indolicidins and tritripticin are rich

in tryptophan. Bactenecins Bac-5

and Bac-7 isolated rom bacteria

are rich in proline residue.

5. AMPs with rare modied amino ac-


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52 December 2009

ids:These are the molecules derivedrom larger peptides by posttrans-

lational modication or alternative

splicing. Nisin is one such peptide

produced by bacteria Lactococcus

lactis and is composed o rare

amino acids, including lanthionine,

3-methyllanthionine, dehydroala-

nine and dehydrobutyrine.

AMPs Isolated rom Fish

In sh, AMPs are mainly conned

to the mucus layers. The early discov-

ered AMPs rom sh, such as the red

sea moses sole, Pardachirus marmoratus,

was pardaxin. It is a 33-amino-acid pore

orming peptide with a helix-hinge-helix

structure similar to cecropin and mel-

litin. It is an excitatory toxin that pos-

sesses high antibacterial activity and

low hemolytic activity towards human

red blood cells compared with mellitin.Lemaitre et al. (1996) isolated two novel

antibacterial proteins, 31 kDa and 27

kDa, rom the skin mucosa o the com-

mon carp, Cyprinus carpio, and ound

strong bactericidal activity against sh

pathogens. Cole et al. (1997) reported

pleurocidin, a novel 25-amino acid

residue linear AMP, ound in the skin

mucous o the winter founder, Pleu-

ronectes americanus. Park et al. (1997)

isolated misgurin rom mudsh, Misgur-

nus anguillicaudatus. It is 21-amino-acid

compound having strong antimicrobialactivity against various microorganisms

and slight hemolytic activity. Addition-

ally, three antimicrobial proteins were

isolated rom catsh skin in 1998. The

molecular masses o the proteins were

15.5, 15.5, and 30 kDa. These proteins,

which are closely related to histone

H2B proteins, can inhibit the growth

o Aeromonas hydrophila and Sapro-

legnia spp. An antimicrobial compound,

squalamine, was isolated by Zaslos

group rom the stomach as well as rom

various other organs o the dog shark,Squalus acanthias. Although a handul

o antimicrobial steroids have been iso-

lated rom plants, squalamine is the rst

antimicrobial steroid isolated rom an

animal.

AMPs Isolated rom Shellfsh

Recently, it was demonstrated that

the AMPs are the widespread mecha-

nism o host deense in shrimp, mussels

and crabs. Penaeidins is an AMP docu-

mented in shrimp, such as hemocytes

o Penaeus vannamei. The molecular

weight o penaeidins is 5.5-6.6 kDa.

They have an N-terminal proline-rich

domain and a C-terminal domain con-

taining six cysteine residues. Penaeidins

have several isoorms and are classied

into penaeidin 2, penaeidin 3 and pe-

naeidin 4 according to their similarityo amino acid sequence. Penaeidin 3

is the most abundant at both levels o

peptide and mRNA in Litopenaeus van-

nameihaemocytes. Moreover, dierent

members o penaeidins amily, includ-

ing penaeidin-1, -2, -3a, -3b and -3c

have been described. These molecules

have chitin-binding properties as well as

antimicrobial activities against Gram-

positive bacteria and lamentous ungi.

They also have the property to opsonise

bacteria o the genus Vibrio.

Another AMP, crustin, was rst iso-

lated rom the shore crab Carcinus mae-

nas. It is cysteine-rich 11.5-kDa pep-

tide with antimicrobial activity against

Gram-positive bacteria. However, the

presence o homologues o crustin in

L. vannamei and the Atlantic white

shrimp L. setierus was also document-

ed. Vargas-Albores et al. (2004) urther

divided penaeid crustins into crustins

I and P. These AMPs are dominantly

synthesized and stored in haemocytes

and their release rom haemocytes is in-duced by bacterial inection. Also, nu-

merous AMPs have been characterized

rom mussels recently. They are orga-

nized into three groups. The rst group

comprises the deensins. Charlet et al.

(1996) reported two deensins, contain-

ing six cysteines residues rom Mytilus

edulis. The second group o molecules,

the mytilins, consists o ve isoorms

(A, B, C, D and G1). The isoorms A

and B were isolated rom M. edulis and

isoorms B, C, D and G1 rom M. gal-

loprovincialis. The third group o pep-tides contains myticins A and B, which

were characterized rom hemocytes and

plasma oM. galloprovincialis.

Limitations

Certain technical diculties exist

when using AMPs as therapeutics in

aquaculture. The initial binding o the

positive charged peptide to the nega-

tively charged microbial membrane is

a simple electrostatic interaction and

thereore very sensitive to the ionic

strength o the medium. The sensitivity

to ionic strength conditions has serious

implications or proper evaluation o

the antimicrobial potency o peptides.

Peptides rom marine animals that are

adapted to living in a salt-rich envi-

ronment are generally less sensitive to

ionic strength. Such peptides have beenound in marine organisms such as

molluscs, tunicates, shrimp, crabs and

various nsh. Little is known o the

structural eatures that are responsible

or the insensitivity to salt. Physiologi-

cal implication o salt sensitivity could

aect the invivo activity o the peptide.

One should be very careul to extrapo-

late in vitro phenomena to physiological

processes and always bear in mind that

the situation in vivo is much more com-

plex than a simple test in vitro.

Conclusion and Future Role in

Aquaculture

This discussion indicates that

AMPs, which have a startling range o

antimicrobial activities against most

Gram-negative and Gram-positive

bacteria, ungi, enveloped viruses

and eukaryotic parasites as well as

an immuno-modulation eect, might

be useul as promising antimicrobial

agents in aquaculture. Hitherto, there

have been ew examples o the appli-cation o AMPs as therapeutic agents

in aquaculture though many are an-

ticipated. Jia et al. (2000) observed

cationic AMPs protect coho salmon

(Oncorhynchus kisutch) against inec-

tion caused by Vibrio anguillarum.

Japanese medaka injected with ce-

cropin B homolog transgene AMP

showed more resistance to pathogenic

bacteria such as P .fuorescens and V.

anguillarum compared to control sh

(Sarmasik 2000). Hence, all o thesepeptides seem to be extremely eective

therapeutic agents in aquaculture but

elucidation o their biological impor-

tance in innate immunity and realiza-

tion o their ull clinical potential will

require much more eort.

Beore these new agents can be ap-

plied in aquaculture, there are some

important topics that should be ex-

plored. First, the impact o AMPs on

the virulence o several aquatic patho-


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World AquAculture 53

gens should be studied in detail to de-

termine i they are valid antimicrobial

agents or aquaculture. Secondly, the

eect o such agents on the health o

the nal consumer o the aquaculture

product and also on the aquaculture

system should be determined. More-

over, some practical problems, such as

the cost o treatments and the delivery

system o such agents to the site o ac-tion, should be considered. Finally, the

problem o eventual resistance devel-

opment should not be neglected.

Notes1Laboratory o Aquaculture and Artemia

Reerence Center, Department o Ani-

mal Production, Ghent University,

Gent, Belgium2Department o Fisheries and Environment,

Faculty o Natural Resource Engineer-

ing, Tehran University, Karaj, Iran*

Corresponding author: [email protected]

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amit kumar sinha - antimicrobial peptide

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