ANTIMICROBIAL AND CELL LYTIC PEPTIDES

2

1 . 1 INTRODUCTION

During the past fifteen years, proteins and peptides that have potent antimicrobial

and/or cytolytic activity have been isolated in pure form from organisms all along the

evolutionary scale. The ubiquitous presence of these molecules.implies a multitude of defensive

and offensive functions for them. Whereas the antimicrobial proteins and peptides kill the

invading microorganisms and provide protection to the host, peptide toxins perform offensive

functions. However, the size, structure and effects of many proteins and peptides are so

variable, - some of them act by interfering ~ith the metabolic activities of the cells while .

others by altering the membrane permeability - that common molecular determinants of

cytotoxic action and their target specificities have not become apparent, except perhaps

implicating the amphiphilic structural features in membrane-perturbing activity of many

cytolytic peptides. Some of the agents show very narrow target cell specificity and many are

indiscriminate in their cytotoxicity.

This chapter reviews the work on some of the well studied antibacterial and hemolytic

peptides that act by membrane permeabilization as well as the earli.er work on seminalplasmin

(SPLN), an antimicrobial peptide present in bovine seminal plasma. The structure-function

correlations of SPLN forms the subject of the thesis.

1 . 2 ANTIBACTERIAL PROTEINS AND PEPTIDES

Granular proteins and peptides from phagocytes:

Phagocytosis is one of the important mechanisms of the host defence network in animals.

The polymorphonuclear neutrophils (PMNs) are the most abundant phagocytic cells circulating

in the human body and these cells kill the engulfed bacteria by both oxidative and non-oxidative

mechanisms. The oxygen independent killing depends on several large proteins such as

bactericidal-permeability increasing protein (BPI) and cathepsin, as well as a group of small

peptides called defensins.

3

Bactericidal/permeability increasing protein

Bactericidal/permeability increasing protein (BPI) is a non-catalytic, lysine rich

protein with a mol. wt. of 50-60 kDa and has been purified from human and rabbit PMNs

(Weiss et al., 1978; Gray et al., 1989). The proteins from both the sources share similar

structure and function. On a molecular basis, BPI is the most potent of the mammalian

cytotoxins. BPI acts on numerous gram-negative bacterial species but does not seem to affect

gram-positive and eukaryotic cells. This remarkable specificity of BPI to gram-negative

bacteria arises due to its high binding affinity to the outer envelope lipopolysaccharide (LPS).

This property has also been exploited for its purification.

BPI seems to exert its activity in two stages. The first stage is characterized by a

reversible bacteriostatic activity involving discrete outer membrane alterations leading to

permeability changes. The second stage involves the irreversible growth arrest as a result of

impairment of biochemical functions linked to the intact cytoplasmic membrane (Mannion et al.,

1989).

The functional domains of human BPI have been dissected by limited proteolysis. All the

biological effects of BPI are reproduced by a 25 kDa N-terminal fragment. The C-terminal

portion of BPI is devoid of antibacterial activity but may serve as an anchor into the granule

membrane (Weiss and Olsson, 1987).

The action of BPI on its target cells is strikingly similar to that of intact PMN. This

suggests that BPI has a prominent role in the antimicrobial functions of PMN against BPI

sensitive gram-negative bacteria.

Defensins

Defensins constitute the most abundant (- 30%) of antibacterial peplides of the

cytoplasmic granules of the phagocytes (Greenwald and Ganz, 1987). The defensins are highly

conserved, disulfide linked, cationic peptides with a molecular weight < 4000. They exhibit

4

antimicrobial activity against gram-negative and gram-positive bacteria, fungi, mammalian

cells and enveloped viruses. They have been isolated from PMNs of humans, rabbits, guinea

pigs and rats and the lung macrophages of rabbits (Lehrer et al., 1991 ).

The amino acid sequences of fifteen of these, basic, arginine - and cystine -rich

peptides have been determined (Table 1.1 ). Defensin (HNP-3) has a dim eric ~-structure and

an architecture different from other lytic peptides. The structural aspects of defensins from

the point of view of their activities have been discussed in greater detail in Chapter 3.

Defensin-like peptides have recently been isolated from insects (Hoffmann and Hetru,

1992). Todate, seven defensins have been fully sequenced in species belonging to three insect

orders : (A) Diptera, (b) Calcoptera and (c) Odonata.

Bactenecins

Two antibacterial peptides named Bac-5 and Bac-7 (from the general term

bactenecins) have been purified from extracts of granules of bovine neutrophils. They have

molecular weights of 5000 and 7000 respectively (Gennaro et al., 1989). They are rich in

proline and arginine. Proline constitutes about 45% and arginine 20% of the amino acid

residues and the remaining are mainly hydrophobic amino acids like isoleucine, leucine and

phenylalanine. They exhibit a potent bactericidal activity towards several gram-negative

bacteria in vitro. They have been shown to exert their antibacterial activity by increasing the

permeability of both the outer and inner membrane of gram-negative bacteria (Skerlavaj et

al., 1990; Gennaro et al., 1989).

A smaller bactenecin of molecular weight ·(2000 exhibiting bacteriolytic activity

against both E. coli and S. au reus at 1 o-7 - 1 o-5 M concentration has been purified and its

sequence (shown below) has also been determined (Romeo et al., 1988).

RLCRIVVIRVCR l I

Source

Human

Guinea

pig

Rabbit

Table 1.1

Comparison of defensin sequences

HNP-1

HNP-2

HNP-3

HNP-4

GPNP

NP-1

NP-2

NP-3a

NP-3b

NP-4

NP-5

Sequence

ACYCRIPACIAGERRYGTCIYQGRLWAFCC

CYCRIPACIAGERRYGYCIYQGRLWAFCC

DCYCRIPACIAGERRYGTCIYQGRLWAFCC

VCSCRLVFCRRTELRVGNCLIGGVSFTYCCTRV

RRCICTTRTCRFPYRRLGTCIFQNRVYTFCC

VVCACRRALCLPRERRAGFCRIRGRIHPLCCRR

VVCACRRALCLPLERRAGFCRIRGRIHPLCCRR

GICACRRFCPNSERFSGYCRVNGARYVRCCSRR

GRCVCRKQLLCSYRERRIGDCKIRGVRFPFCCPR

VSCTCRRFSCGFGERASGSCTVNGVRNTLCCRR

VFCTCRGFLCGSGERASGSCTINGVRHTLCCRR

Rat RatNP-1 VTCYCRRTRCGFRERLSGACGYRGRIYRLCCR

RatNP-2 VTCYCRSTRCGFRERLSGACGYRGRIYRLCCR

RatNP-3 CSCRTSSCRFGERLSGACRLNGRIYRLCC

RatNP-4 ACYCRIGACVSGERLTGACGLNGRIYRLCCR

5

6

lndolicidin

A potent and structurally novel antimicrobial peptide has been isolated from cytoplasmic

granules of bovine neutrophils (Selsted et al., 1992). 10 Jlg/ml of this peptide, named

indolicidin, has been found to virtuatty sterilize suspensions of gram-positive S. aureus and

gram-negative E. coli. The peptide was found to be very rich in tryptophan and amidated at the

C-terminal. Tryptophan residues have been thought to play an important role in the function of

this unique peptide. Th~ sequence of this peptide is shown below.

ILPWKWPWWPWRR-NH2

Antimicrobial peptides from amphibians

Magainins:

African clawed frog Xenopus laevis secretes a broad spectrum of oligopeptides, two of

which have been identified as strong antimicrobial agents and termed as magainins (Bevins and

Zasloff, 1990). The absence of infection during the healing of surgical wounds of these animals

prompted the search for these antimicrobial agents. The magainin peptides are each 23 amino

acids long and lack sequence identity with any other reported peptide (Table 1.2). They exert

antimicrobial activity against bacteria, fungi and protozoa at molar concentrations comparable to

other known antimicrobial agents.

Several other peptides having antimicrobial activity including Xenopsin precursor

fragment (XPF), caerulein precursor fragment (CPF) have also been identified (Bevins and

Zasloff, 1990) (Table 1 .2). All these peptides show antibiotic activity similar to magainins,

although they differ in their spectrum of activity. They are all 25 or 26 amino acid residues in

length and are lysine-rich basic polypeptides lacking tyrosine and lysine. Even though the

sequences display limited sequence identity except for the similarity between PGLa, XPF and

levitude precursor fragment (LPF), all of them can be configured as amphiphilic alpha helices

Source

Xenopus

laevis

Bombina

variegata

Bombina

orienta/is

Phylomedusa

sauvagii

Table 1.2

Antimicrobial peptides from amphibians

Name of

peptide

Magainin 1

Magainin 2

PGLA

XPF

LPF

CPF

Bombinin

Bombinin­

related pep­

tide ·(B RP)

Bombinin like-

Sequence

GIGKFLHSAGKFGKAFVGEIMKS

GIGKFLHSAKKFGKAFVGEIMNS

GMASKAGAIAGKIAKVALKAL-NH2

GWASKIGQTLGKIAKVGLKQLIQPK

GWASKIGQTLGKIAKVGLQGLMQPK

GFGSFLGKALKAALKIGANALGGSPQQ

GIGALSAKGALKGLAKGLAEHFAN-NH2

GIFFALLSAAKVGLKGLAKGLAEHFAN

peptide 1(BLP 1)GIGASILSAGKSALKGLAKGLAGHFAN-NH2

B L P- 2 GIGSAILSAGKSALKGLAKGLAGHFAN-NH2

B L P-3

8 L P-4

Dermaseptin

GIGAAILSAGKSALKGLAKGLAEHF-NH2

GIGAAILSAGKSIIKGLANGLAEHF-NH2

ALWKTMLKKLGTMALHAGLAALGAAADTISQ

GTQ-OH

7

8

which direct their surface activity and membrane affinity. Magainins form random coils in

aqueous solution but adopt a-helical structure in aqueous trifluoroethanol and upon interaction

with phosphatidyl serine vesicles and other micelles (Matsuzaki et al., 1989). Magainin 2 has

been shown to organise into macroscopic filamentous structures, presumably reflecting the

ability of the monomeric peptide to self aggregate (Urrutia et al., 1989). The antibacterial

activity of magainins is presumed to arise from their interaction with biomembranes.

Magainins have also been shown to rapidly depolarize the bacterial cell membrane and uncouple

oxidative phosphorylation. Magainin 2 has been shown to interact with synthetic lipid bilayers

to form anion selective channels (Duclohier et al., 1989).

Bombinin, bombinin-related peptides and bombinin-like peptides:

An antimicrobial peptide called 'bombinin' was characterzed by Csordas and Michl

(1969) from the European toad, Bombina variegata. This is a 24 amino acid residue peptide

(Table 1.2) with hemolytic and antibacterial properties and shows many of the general

structural feature of magainins.

A family of bombinin-related peptides with 27 amino acids has also been described by

Simmaco et al., (1991 ). One of them has been synthetically made and shown to possess

antibacterial activity.

Bombinin-like peptides having antimicrobial and hemolytic activity have been reported

from skin secretions of the Asian toad Bombina orienta/is. Three of the peptides BLP-1 to 3 have

been sequenced. All the three peptides share considerable but not complete homology with

bombinin (Gibson et al., 1991 ).

Dermaseptin

A highly potent antimicrobial peptide from skin extracts of the South American arboreal

frog, Phylomedusa sauvagii, which acts against pathogenic fungi has been reported. This peptide

called dermaseptin is 37 amino acid residue long. The peptide is non-hemolytic. The

9

sequence(Table 1.2)of this peptide has the potential to form amphiphilic a-helical structure

and probably acts by membrane permeabilization (Mor et al., 1991 ).

Antimicrobial peptides from insects

Cecropins:

Insects possess efficient immune systems, both cellular and humoral. The immune

systems of Hyalophora cecropia and other silkworms respond very effectively to bacterial

infections by induced synthesis of proteins and peptides consisting of a mixture of lysozyme and

two antibacterial protein families, named cecropins and attacins (Boman and Huttmark, 1987).

Cecropins are small, basic proteins with a broad spectrum of antibacterial activity against both

gram-positive and gram-negative bacteria. The primary structure of the three major cecropins,

cecropin A, cecropin B and cecropin D have been determined. Cecropin sequences from flesh fly

Sarcophaga, the silkworms Bombyx and the tobacco horn worm Manduca have also been reported

(Boman et al., 1991 ). During a search for mammalian antibacterial peptides, a cecropin

homolog was isolated from pig intestine and this shows 64-75% homology with insect cecropin

(Lee et al., 1989). The sequences of 11 known cecropins are given in the Table 1.3. The

sequences are characterized by a strongly basic N-terminal region and a long hydrophobic

stretch in the C-terminal half. Some other typical conserved features include, tryptophan at

position 2, single and double lysine at positions 5, 8 and 9 arginine at position 12. In the N­

terminal region, polar and hydrophobic amino acids are interspaced in a regular pattern. NMR

studies have demonstrated the presence of two amphipathic a-helices interrupted by a hinge

region composed of Ala-Giy-Pro-sequence (Holak et al., 1988).

The antibacterial activity of the three main forms of cecropin has been compared

(Hultmark et al., 1983). Cecropin A and B are highly active against several gram-positive and

gram-negative bacteria while the D form shows high activity against E. coli and Acinetobacter

calcoacitans and several other insect pathogens Cecropins have been shown to lyse bacteria but

not eukaryotic cells (Steiner et al., 1981 ). Cecropins also lyse vesicles composed of

10

Table 1.3

Comparison of 11 known cecropins

------------------------------------------------------------------Manduca B-2 W--NPFKEL ERAGQRVRDA VTSAAPAVAT VGQAAAIAR*

Hyalophora D W--NPFKEL EKVGQRVRDA VISAGPAVAT VAQATALAK*

Antheraea D W--NPFKEL ERAGQRVRDA IISAGPAVAT VAQATALAK*

Hyalophora A KW--KLFKKI EKVGQNIRDG IIKAGPAVAN VGQATQIAK*

Bombyx CMIV RW--KIFKKI EKVGQNIRDG IVKAGPAVAV VGQAATI*

Bombyx A RW--KIFKKI EKMGRNIRDG IVKAGPAIEV LGSAKAI*

Antheraea B KW--KIFKKI EKVGRNIRNG IIKAGPAVAV LGEAKAL*

Hyalophora B KW--KVFKKI EKMGRNIRNG IVKAGPAIAV LGEAKAL*

Drosophila A GWLAKKIGKKI ERVGQHTRDA TI-QGLGIAQ QAANVAATAR*

Sarcophaga lA GWLKKIGKKI ERVGQHTRDA TI-QGLGIAQ QAANVAATAR*

Porcine P1 SWLSKTAKKL ENSAK-KR---ISEGIAIAI QGGPR

* Indicates amidated C terminus

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phospholipids which are negatively charged .or zwitterionic in a stoichiometric way (Steiner et

al., 1988).

Antibacterial peptides from horseshoe crabs

Tachyplesins I and II and polyphemusin I and II, cationic peptides isolated from the

hemocytes of horseshoe crabs show antibacterial activity with similar efficiencies for both

gram-negative and gram-positive bacteria (Miyata et al., 1989). Tachyplesin I, a 17 residue

peptide was found in the hemocytes of Japanese horseshoe crab Tachypleus tridentatus as an

inhibitor of the bacterial lipopolysaccharide (LPS)-mediated activation of factor C, the

initiation factor in the limulus clotting cascade. Tachyplesin Ill was isolated from the south east

Asian horseshoe crab (Muta et al., 1990), Carcinoscorpins rotundacauda and Tachyp/eus gigas.

Polyphemusins I and II were isolated from the American horseshoe crab Limulus polyphemus.

The sequences of these related peptides are shown in Table 1.4.

These peptides are characterised by two intramolecular disulfide bridges and three

tandem repeats of a tetrapeptide, hydrophobic amino acid-Cys-hydrophobic amino acid-basic

amino acid, suggesting an amphiphilic nature of the peptide. A two dimensional NMR study of

tachyplesin I has shown it to form a rigid antiparallel ~- sheet structure.

Tachyplesins exhibit broad spectrum antimicrobial activity (Nakamura et al., 1988;

Miyata et al., 1989). The strong binding of these peptides to LPS cannot explain activities for

gram-positive bacteria and fungi. The mechanism of action thus appears to be different from

that of polymyxin B. Tachyplesin I has been shown to interact with lipid bilayer membranes

(Matsuzaki et al., 1991 ). The interaction induces permeability changes and results in the

leakage of entrapped calcein suggesting that the peptide~induced membrane permeability changes

may bring about its antibacterial activity. Tachyplesin indeed causes a rapid efflux of K+ ions

from E. coli cells. Subinhibitory concentration of the peptides was shown to sensitize gram­

negative bacteria to the bactericidal action of novobiocin and nalidixic acid showing that

tachyplesins permeabilise the outer membrane of bacteria. As polymyxin B resistant bacteria

Tachyplesin

Tachyplesin II

Tachyplesin Ill

Polyphemusin

Polyphemusin II

Table 1.4

Antimicrobial peptides from horseshoe crabs

KWCFRV<(YRGI<(YRKtR - NH2

RWCFRVCYRGICYRRtR - NH2 l l

KW&;RV<(YRG1fYRR6R - NH2

RRW6FRVCYRGFCYRKCR - NH2 ' l

RRWtFRVCYKGFCYRK~R - NH2 h ,

l2

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are also susceptible to the action of tachyplesins, the target of its action seems to be

constituents in the outer membrane other than LPS (Ohta et al., 1992).

Plant antimicrobial peptides

Two antimicrobial peptides designated as Mj-AMP1 and Mj-AMP2 have been isolated

from seeds of Mirabilis jalapa (CamrrJUne et al_11992). These peptides are highly basic and 37

and 36 residues in length. Both peptides contain three disulfide bridges and differ from one

another only by 4 amino acids.

The peptides appear to associate into dimers in the native form. Mj-AMPs exhibit broad

spectrum antifungal activity and also act on gram-positive bacteria. They are non-toxic to gram

-negative bacteria and cultured human cells. Although Mj-AMPs show sequence homology to J.L­

agatoxins a class of insecticidal neurotoxic peptides isolated from venom of spiders, they do not

possess any neurotoxic effect.

Mj-AMPs, have structural similarities to defensins and sequences are shown below.

Mj-AMP1

Mj-AMP2

OCIGNGGRCNENVGPPYCCSGFCLRQPGGYGYCKNR

CIGNGGRCNENVGPPYCCSGFCLROPN.QGYGVCRNA

1.3 HEMOLYTIC PEPTIDES

Mel itt in

Structural aspects

Melittin is the main toxic component of the venom of the honey bee, Apis me/litera

(Haberman, 1972). It is a short, 26 residue peptide and has multiple effects on membrane

structure and functions. It is perhaps the best studied membrane perturbatory peptide. Even

then the mechanism of membrane lysis still remains to be proven and no consensus exists on the

nature of its interaction with membrane lipids. The sequence (Table 1.5) of melittin appears to

have two structural regions (De Grado et al., 1982). The No-terminal 20 amino acids have the

Peptide

Melittin

Mastoparan

Bombolitin

Bombolitin II

Bombolitin Ill

Bombolitin IV

Bombolitin v

o-toxin

(human ·strain)

Table1.5

Sequences of cytolytic peptides

Sequence

GIGAVLKVLTTGLPALISWIKRKRQQ-NH2

INLKALAALAKKIL-NH2

I KITTM LA K LG KV LAH V-Nti2

SKITDILAKLGKVLAHV-NH2

rKIMDILAKLGKVLAHV-NH2

INIKDILAKLVKVLGHV-NH2

INVLGILGLLGKALSHL-NH2

formyl MAQDilSTIGDLVKWIIDTVNKFTKK

o-t ox in formyl MAADIISTIVEFVKLIAETVEKFIKK

(canine strain)

14

15

potential to form an amphiphilic a-helix with a proline link at position 14 and the C-terminal

hexapeptide is highly cationic with 2 lysines1 2 arginines and amidated o- terminus. Neither the

peptide fragment 1-20 nor the C-terminal!hexapeptide shows hemolytic activity (Dawson et

al., 1978; Schroder et al., 1971 ).

Melittin exists as a monomer both at low concentration and low ionic strength but

aggregates at high concentration and high ionic strength, forming tetramers as the only

detectable form (Brown et al., 1980). The structure of melittin has been investigated by X-ray

diffraction (Terwilliger and Eisenberg, 1982), NMR (Pastore et al., 1989), CD and many

other techniques. The monomer has been found to be in a-helical conformation with a bend at

proline 14. The crystal structure of the tetramer contains pairs of dimers facing each other

along the hydrophobic faces thus shielding them from the aqueous environment. Within each

dimer the monomers are arranged antiparallel to each other , minimising the electrostatic

repulsion between the cationic C-termini.

Hemolytic activity of melittin

Melittin is known for its marked cytolytic activity. Its hemolytic activity has been

extensively studied. Although considerable controversy exists regarding the aggregation state of

melittin bringing about hemolysis, it has been well established that binding of the peptide to the

membrane is essential for bringing about lysis. The apparent dissociation constant has been

found to be 3 x 10-8M and a maximum binding capacity of 1.8 x 107 molecules/cell (Tosteson

et al., 1985). Melittin induced hemolysis follows reproducible biphasic kinetics - a fast phase

of hemoglobin release triggered by binding of melittin to the membrane followed by a slow steady

state release.

Studies done by Tosteson et al.1suggest that the hemolysis by melittin occurs by a colloid

osmotic mechanism. The evidence for such a conclusion include (i) hemoglobin release from the

melittin treated cells is preceded by 200-2000 fold increase in permeability to Na+ and K+

ions (ii) erythrocytes show swelling before the release of hemoglobin and (iii) osmotic

protectants of higher molecular weights are capable of providing protection to lysis by melittin.

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In order to understand the structure-function correlations of melittin, various synthetic

analogs have been used. Studies done with chemically modified or synthetic analogs have shown

that hemolytic activity and surface activity are not directly correlated. Studies done by De Grado

et al., (1982) using a model peptide which is homologous to melittin only at five positions (6,

9, 13, 16 & 19) in the N-terminal hydrophobic stretch and the C·terminal hexapeptide showed

that neither the sequence nor the presence of proline in the centre of the helix are required for

the hemolytic activity. The model peptide had been designed to have a higher potential to form an

amphipathic a-helix than melittin and composed solely of leucine on the hydrophobic side of the

helix and of serine and glutamine as hydrophilic residues. These results showed that the N­

terminal 20 residues have only a structural role which can be duplicated by non homologous

sequence having a potential to form a amphiphilic a-helix. A synthetic analog of melittin

(Ala 14 melittin) containing a Pro to Ala substitution at residue 14 also showed 2.5 times

higher hemolytic activity than melittin confirming the lack of importance of the helix bend for

the hemolytic activity of melittin (Dempsey, 1990). But this analog had poorer voltage

dependent ion channeJ activity than native melittin suggesting the importance of the central

flexible region for the stability of voltage-gated channels. This also supports the idea that

hemolytic activity of melittin is not directly related to its channel forming property.

Formylation of N-terminal methionine did not result in the loss of hemolytic activity of

melitrin (Lubke et al., 1971 )indicating that free N-terminal a-amino group is not essential

for activity. Photooxidative destruction of tryptophan or replacement of tryptophan with

leucine resulted in reduced hemolytic activity suggesting an important role for tryptophan in its

hemolytic activity (Haberman and Kowallek, 1970; Blonde lie and Houghton, 1991 a).

Two models have emerged from the structural and functional studies for the molecular

mechanism of action. In one type of model, melittin forms aqueous channels by spanning the

membrane forming channel oligomers exposing its hydrophobic side to the lipid and its

hydrophilic face to the aqueous pore (Tosteson and Tosteson, 1981 ). The pore size of these

channels will not be big enough to allow the hemoglobin to pass through. The model predicts that

17

these channels produce ionic imbalance which leads to colloid-osmotic lysis. Some of the

experimental data which support such a mechanism have been mentioned earlier. An analog of .

melittin lacking Lys7 (Biondelle and Houghton, 1991b) lacksits activity. The omission of

lysine 7 in the analog is likely to cause formation of smaller pore channels and make it

selecNve for anions over cations. Replacement of lysine 7 with leucine also results in loss of

activity. These two observations also are in agreement with the melittin pore theory.

The second set of models proposes that melittin produces lysis by disrupting the

phospholipid structure of the membrane by forming a wedge like conformation (Dawson et al.,

1978). The resulting weakening of the overall membrane structure allows the leakage of

hemoglobin.

Several specific models of this type have been proposed (Dawson et al., 1978; DeGrade et

al., 1982; Terwilliger et al., 1982). The observations suggesting that melittin lies parallel to

the membrane plane or that melittin penetrates only a few angstroms into the bilayer tend to

support this type of model (De Grado et al.11982; Terwilliger et al, 1982).

Mastoparan

Mastoparan is a toxic tetradecapeptide (Table 1.5) isolated from wasp venom (Hirai et

al., 1979). It stimulates exocytosis from several mammalian cells, of histamine from mast

ceils, serotinin from platelets and insulin from pancreatic islet via a mechanism that involves

activation of GTP-binding regulatory proteins (G-proteins) (Higashijima et al., 1988).

Mastoparan was first isolated from Varpula lewisii and chemically synthesised by Hirai and co-

workers in 1979 (Hirai et al., 1979).

Mastoparan has also been found to bind to calmodulin with high affinity. Like melittin,

mastoparan is also potent stimulator of phospolipase activity (Male nick and Anderson, 1983).

However melittin is a more potent hemolytic agent than mastoparan. Mastoparan exists in a

random coil conformation in aqueous solution but takes up a a-helical conformation in

hydrophobic environment (Higashijima et al., 1983). Mastoparan was shown to increase the

18

cation permeability of planar lipid bilayers, indicating its possible ionophoric properties

(Okumura et al, 1981 ). Mellor and Sansom (1990} have demonstrated the existence of two

types of voltage dependent ion channel property of mastoparan in planar lipid bilayers. In the

presence of 0.3 - 3.0 J..LM mastoparan, type I activity was characterized by discrete channel

openings exhibiting multiple conductance levels in the range of 15-700 ps. Type II activity was

characterized by transient increases in the bil~yer conductance upto a maximum of about 650

ps. Mastoparan channels are weakly cationic in character. A twelve residue analog of

·mastoparan lacking the N-terminal dipeptide 1-N also showed Type I form of ion channel

activity.

Bombolitins

Five structurally related heptadecapeptides which are hemolytic have been discovered in

the venom of bumblebee, Megabombus pennslyvanicus . The sequences of these peptides, named

bombolitins, have also been determined (Argiolas and Pisano, 1985) (Table 1.5).

In addition to their structural similarity they have functional similarities also. They

lyse erythrocytes and liposomes, release histamine from rat peritoneal mast cells and stimulate

phospholipase A2 from different sources. Bombolitin V has been found to be the most potent of

hemolytic agents and its activity compares very well with that of melittin. Its mast cells

degranulating activity is also five times higher than mastoparans.

The conformationsof bombolitin I & Ill have been determined by using circular dichroism

(CD) nuclear magnetic resonance (NMR) and computer simulations (Bairaktani et al., 1990).

Bombolitin was found to lack an observable secondary structure in aqueous solution independent

of temperature, pH and concentration. In striking contrast, bombolitin Ill adopts a well defined

a-helix at concentrations greater than 3 mM. The a-helix of bombolitin was found to be pH

dependent, with a great decrease in the observed secondary structure at pH values below 3.5.

This was probably due to protonation of the Asp residue which could prevent the formation of

salt bridges and consequent molecular aggregation and secondary structure. In the presen-ce of

19

SDS micelles, the characteristic bands in CD spectra indicated an a-helix content of 60% for

bombolitin Ill and 70% for bombolitin I. When the sequences of bombolitin are depicted in the

a-helical conformation they appear amphiphilic. The amphiphilic nature of bombolitin

probably determines their biological activities.

Delta toxin [a-toxin, a-hemolysin]

Staphylococcus aureus secretes several peptide toxins that are membrane active. Delta

lysin is the smallest of them with 26 residues and has been extensively studied. Delta lysins

from two different strains of S. aureus have been sequenced (Table 1.5).

The structural requirements of a-toxin to show its activity have been investigated by

Alouf et al. (1989} by synthesizing the sequence identical to the natural toxin from the human

strain along with five 26 residue analogs and two shorter peptides corresponding to residues 1-

11 and 11-26. The 26 residue analogs were designed in such a way that they maintain (a) the

hydrophobic/hydrophilic balance, (b) the a-helical conformation and, (c) common epitopic

structure. These analogs were, (i) deformylated toxin, (ii) an idealised amphipathic sequence

for residues 1-20, (iii) an analog which closely resembles (ii) but with the tryptophan moved

from position 16 to position 5, (iv) unformylated peptides of the above analog, (v) F-moc

analog.

All 26-residue analogs were hemolytically active. The peptide 11-26 bound weakly to

lipids and was weakly hemolytic. The peptide 1-11 did not bind to lipids and was not hemolytic.

a-hemolysin shares many of its membrane-modifying properties with melittin. Both the

peptides are 26 residues in length with a single tryptophan residue and lacking cystine. Both

peptides ·have potential to form amphipathic helices with a cationic tail. They lyse erythrocytes

and many other mammalian cells including fibr:oplasts, intracellular organelles and bacterial

protoplasts. They bind to phospholipids and penetrate phospholipid monolayers and induce

permeability changes in phospholipid vesicles. Both of them activate phospholipase A2 of 3T3

mouse fibroblasts. Both of them have similar solubilities in organic solvents, aggregate to

multimeric forms and are heat stable.

However, there are many differences between these and toxins. Their amino acid

sequences do not show any homology and differ in the distribution of charges. a-toxin does not

have a long hydrophobic sequence. The functional differences include, (i) melittin is more

hemolytic than a-toxin, (ii) melittin shows a preference for negative lipids whereas o-toxin

has no such preference, (iii) melittin causes aggregation of gel phase liposomes unlike o-toxin.

Specificity of antibacterial and hemolytic peptides

Although amphiphilic a-helix or amphiphilic p-structure motif has been identified as

an essential feature of antibacterial and cell lytic peptides which act by membrane

permeabilization, nothing is known as to what determines their target specificity. For example,

cecropins and magainins are relatively less active against mammalian cells but more specific on

bacterial cells. Defensin on the other hand has broad cytotoxicity which include bacteria, fungi

and tumour cells. No clear cut rules exist to explain the broad cytotoxicity of melittin. o-lysin

for example is highly cytotoxic but relatively less antimicrobial (Dhople and Nagaraj, 1993).

A large number of functional properties have been attributed for amphiphilic helices.

1 . 4 SEMINALPLASMH'J

Seminalplasmin (SPLN) is a protein present in bovine seminal plasma that is known to

exert antimicrobial effect (Reddy and Bhargava, 1979). The presence of an inhibitory factor of

spermatozoal RNA synthesis in bovine seminal plasma had been postulated by Bhargava and

colleagues from the observation that bovine seminal plasma inhibited the synthesis of RNA by

spermatozoa. Preliminary studies had also indicated this factor to be macromolecular and

susceptible to proteolytic activity. As the RNA synthesis in spermatozoa was found to be

exclusively mitochondrial (Premkumar and Bhargava, 1972& 1973) and susceptible to the

same inhibitors as the corresponding synthesis in prokaryotes, it was reasoned that the

inhibitory factor in seminal plasma would act as an antibacterial agent. Bovine seminal plasma

21

indeed inhibited the growth of E. coli. Using this as the assay system, a protein named

seminalplasmin (SPLN) was purified from dialysed bovine seminal plasma (Reddy and

Bhargava, 1979).

Extensive work has been done by Bhargava and co-workers on SPLN Following are some

of the properties of SPLN.

( i ) It has been shown to be a potent antimicrobial agent acting on a variety of gram-positive

and gram-negative bacteria as well as yeast (Bhargava, 1981, Reddy and Bhargava,

1979; Scheit et al., 1985; Scheit and Bhargava, 1985). It is bacteriostatic at low

concentration and bactericidal at high concentrations. It appears to activate autolysins in

E. coli at higher concentrations (Chitnis et al., 1987). The effect of SPLN on the growth

of various microorganisms is tabulated in Table 1.6.

( i i ) The effects of SPLN on the synthesis of macromolecules have been studied in whole cells,

protoplasts, mitochondria and cell free systems (Table 1.7). The authors have concluded

that SPLN is likely in to penetrate into susceptible cells and inhibit RNA synthesis

(Reddy and Bhargava, 1979; Scheit and Bhargava, 1985; Scheit et al., 1979 & 1985).

(iii) Studies on the interaction of SPLN with bovine spermatozoal plasma and acrosomal

membranes have shown that SPLN binds to both of these membranes .increasing their

fluidity (Shivaji, 1986).

( i v) SPLN incorporates into lipid bilayers of phosphatidylcholine and phosphatidic acid

vesicles and as a consequence of such an interaction the a-helical content of SPLN increaseS

(Galla et al., 1985).

( v ) SPLN binds to calmodulin and acts as a powerful calmodulin antagonist (Gietzen and Galla,

1985). SPLN forms a ca2+ dependent 1 :1 complex with calmodulin which is resistant to

urea (Comte et al., 1986).

Table 1.6

Effect of SPLN on the growth of microorganisms

Microorganism

E. coli B

E. coli K -1 2

E. coli 1 6 0 . 3 7

Euterobacter aerogenous

Streptococcus faccalis

Cryptococcus neoformans

Salmonella typhemurium

Candida albicans 1

Candida a1bicans (din)

Bacillus subtilis

Saccharomyces cerevisiae SM202

S. cerevisiae Y V 11 0 6

Proteus vulgaris

Pseudomonas aeruginosa

Minimum inhibitory concentration of

SPLN J.Lg/ml

25-40

2 5-4 0

40

100

100

25

400

4 0

1 0 0

20

240

14

5

20

22

23 .

Table 1.7

Summary of the effects of SPLN on growth and macromolecular synthesis in various systems

System

Whole cells of E. coli

Cell free system from E. coli

Purified E. coli RNA polyme-

rase

Whole cells from C. albicans

Cell free system from C. albi-

cans

Proto:p.last of C. albicans

Wh-o~e ceiJs o-f S. cerevisiae

Protoplasts of S. cerevisiae

Nuclei of S. cerevisiae

Mitochondria of S. cerevisiae

Growth

NA

NA

NA

NA

NA

NA

NA

Cell free system from S. ceresiae NA

Purified RNA polymerase NA

from S. cerevisiae

Purified RNA pol 1-1 from NA

S. cerevisiae

Adult rat liver cells in NT

suspension

Synthesis of

RNA DNA Protein

Nl Nl

Nl

NA NA

NA Nl

NT

NT NA

Nl

Nl

NA Nl Nl

NA NA

NA NA

NT NT

----------------------------------------------------------

(vi) SPLN binds strongly to DNA and unwinds it (Reddy and Bhargava, 1979; Gopal et al.,

1988). DNA carrying as much as 1 mol. of SPLN per 8 base pairs can however be

transcribed normally by E. coli RNA polymerase (Scheit et al., 1979).

Many of the properties of SPLN appear to be membrane related. Most of the above

studies have been done when the primary structure of SPLN was unknown and hence no

attempts could be made to correlate its activities with its structure.

Objectives of the work discussed in the thesis

The presence of antimicrobial proteins/peptides in organisms all along the

evolutionary scale emphasizes their importance in defence. The primary target of many of

these peptides like defensins, magainins and cecropins is the bacterial membrane. They act by

permeabilizing the membrane. In spite of the broad antimicrobial spectrum of the above

peptides, their use as therapeutic agents has not been thoroughly investigated due to their

relatively long length or complex structure and consequent difficulties in large scale

synthesis. Hence it would be desirable to get short-antimicrobial peptides without cytolytic

activity. One of the pre-requisites for synthesizing short and simple antimicrobial peptdies

without cytotoxic properties is to understand their structure-activity relationship. ln the

case of long peptides, it would be desirable to define the minimal requirements for biological

activity.

SPLN is one of the first antimicrobial peptides to be isolated and characterized from

mammalian sources (Reddy and Bhargava, 1979). SPLN has been postulated by Reddy and

Bhargava to possibly protect the male and/or female reproductive tract against

microorganisms and retroviruses. Recently, a male specific antimicrobial peptide 'andropin'

has been detected in the extracts of male reproductive tract of Drosophila (Samakovlis et al.,

1991 ). Thus, antibacterial peptides may play an important role in protecting the male

reproductive tract. In fact, caltrin (which was later shown to be identical to SPLN) like

24

peptides have been shown to be widely distributed in seminal plasma of various animals and

have been purified and characterized from guinea pig, rat and mouse.

Since SPLN is a noncytotoxic peptide with potent, broad spectrum antimicrobial

activity, determining its structure will conceivably help in studying its structure-activity

relationship. Once the primary structure is known, it can possibly be analysed to understand

the structural basis for its antibacterial activity and delineate regions responsible for it In

this study, SPLN has been used as a model peptide to study structure-activity correlations of

cytolytic and antibacterial peptides. SPLN has been sequenced and its antibacterial activity

further characterized. Two putative segments responsible for its antib~cterial activity have

been identified by analysing its sequence. These segments have been chemically synthesized

and their antibacterial and cell lytic activities characterized. In order to further understand

the structure and charge requirements for their biological activity, several active and

inactive analogs have been synthesised. Interaction o.f SPLN and several of these peptides with

model membranes has also been studied in an effort to rationalize the basis of their biological

activity.

25