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References 10 Hail% A. M. and Bruce, N. C. (1993) Appi Ewnvr. .\/i~-mhiol. 59,

2166-2170
11 Bruce, N. C., Wilmot, C. J., Jordan, K. N., Gray Stcphem, L. D. and
Lowe, C. R. (1991) Bio&m.J. 274, 875Gi80
12 Bruce, N. C., Caswell, D. A., French, C. E., H&s, A. M., Long,
M. 1. and Willey, D. L. (1994) At~n. 1vYAciul. Sti. 721, 85-100
13 Cameron, ‘2. W. W., Jordxl, K. N., Holt, P. J,, Raker, 1’. B.,
Lowe, C. 11. and Bruce, N. C. (1994) Appl. Ewirorr. ~!&mhiul.
60,3881-3883
14 Holt, 1’. J., Gray Stephens, L. I)., Bruce, N. C. and Lowe, C. R.
Riosors. Bioclecrm. m press)
15 Rathbonr, D. A., Holt, 1’. J., Bruce, N. C. and Lowe, C. R. .4rw
.
ihiY Atud. &I. (III j<ss)
16 Willey, D., C&well, D. A., Lowe, C. 11. and Bruce, N. C. (1993)
8 Kunz, 1). A., Reddy, G. S. and Vatvarc, A. (1985) Appl. Errvivon. Birch. 1. 290, 539-544
Mitn~brol. 50, X31-836 17 French, C. E. and Bruce, N. C. (1994) Biot/wm.~. 301, 97~103
9 Bruce, N. C., Wilma. C. J., Jordan, K. N., Trrbilcock, A. E., IS French, C. E., Rathbone, I). A., Hales, A. M., Long, M. T.,
Gray Stephem. L. D. and Lowe, C. R. (1990) flrc/l. Miuobiol. 154, W&y, D. L. dnd Btxcc, N. C. BiolTeclwwlqy (in press)
465-470 19 Jackson, J, 8. (199l)J. B’mnq. Rionrernbrmm 23, 715-741

Amphibian skin: a promising

resource for antimicrobial
peptides
Donatella Barra and Maurizio Simmaco

Amphibian skin is a rich source of biologically active compounds that are assumed
to have diverse physiological and defence functions. In addition to the range of
pharmacologically active peptides present, some of which have mammalian
homologues, skin secretions contain a broad spectrum of antimicrobial peptides.
As yet, such peptides from only a few species have been studied, and screening
of other species is expected to yield further new antimicrobial activities. Natural
antimicrobial peptides isolated from amphibian skin could provide lead structures
for either the chemical, or rDNA synthesis of novel antimicrobials.

It has been known for a long time that the granular as caeruleins, tachykinins, bradykinins, thyrotropin-
glands of amphibian skin produce many biologically releasing hormone, and bombesin-like and opioid pep-
active compoundslJ. These glands are controlled by tides (see Refi 45 for recent reviews). Vertebrate skin
sympathetic nerves, and discharge their content on the has the same embryonic-ectodermal origin as the brain,
dorsal surface of the animal in response to a variety of and many frog-skin peptides have been found to have
stimuli. The compounds secreted by the glands are counterparts in mammalian gastro-intestinal tract and
thought to play various roles, either in the regulation of brain’. This observation provided further stimulus to the
physiological functions of the skin, or in defence against study of hog-skin peptides.
predators or microorganisms3. Skin extracts of frogs are In addition to peptides related to mammalian hor-
a rich source of pharmacologically active peptides such mones and/or neurotransmitters, amphibian skin con-
tains numerous peptides with antimicrobial or
haemolytic activities. The antimicrobial peptide bom-
binin was first isolated more than 20 years ago from the
skin secretion of Bombina variqata (Ref. 6), but it was
only in the late 1980s that antimicrobial peptides from

TIBTECHJUNE 1995 (VOL13)

 Xenopus laevis
antinlicrobial skin peptides studied to date are cationic,
MagaininiPGS it is already clear that large differences exist in features
PGLa such as size, the prcscnce of disulphide bonds, and
XPF
other structural niotifs.
Bombina species
Mechanism of action
Bombinin A large nunlber of antimicrobial peptides frorm
Bombtnin H
aniphibian skin can adopt an anlphipathic a-helical
Phyllomedusa species structure in hydrophobic environments, suggesting
that oligomers ofsuch helices would forni pores in the
Dermaseptin phospholipid bilayer of target niembranes. Inhibition
Adenoregulin
of cell growth and cell death may then result frorm the
Litoria species disturbance of rnenlbrane functions. This hypothesis
is confirmed by the results of experiments perfonled
Caerin-1.1
Caeridin-1 with synthetic peptides containing only I)-amino acids:
an amphipathic left-handed a-helix is fornled, but the
Rana species biological activity of the natural I counterpart is
retained. Therefore, an interaction with chiral bind-
Brevinin-1
Brevinln-l E ing sites of receptors, enzynles or other melnbranc
Gaegurin-6 proteins can be ruled out. The selectivity of sonle of
Ranalexin these peptides for bacterial membranes rmay be related
to the number, and distribution, of positive charges. It
Brevinin-2
Brevinin-2E is known that bacterial nienibranes contain higher
Gaegurin-2 anlounts of anionic phospholipids than the erythro-
cyte niembrane, for example. However, nluch less is
known about the mechanisnl by which other types of
antimicrobial peptides, which do not fornl amph-
pathic helices, inhibit cell growth or cause cell death.
The interaction of peptides sucl~ as bornbinin H and
brevinill-IE (Fig. 1) with bacterial and niodel men-
Figure 1
branes reniains to be investigated.
Amino acid sequences of antimicroblal peptldes from different amphibian species.
Representative members of the different peptide families are presented: peptldyl-
Peptide structure and antimicrobial activity
glyclne-senne (PGS); peptldyl-glyclne-leucine carboxyamide (PGLa); xenopsin precur-
In recent years, antinlicrobial peptides have been
sor fragment (XPF). Amidated C-terminal residues are represented by d; o-alloisoleucine
isolated and characterized front the skin of Xerro~~rrs
is represented by 1*. The bars represent disulphlde bridges.
Iacvis, Bmbirra varicpfa and B. wimtali~, W~yl1orrrc~1rr.w
ralrqri and I? h/w, Litovia spkrdida and L. cacvrrlcn,
and several species of Rorritlac~. In the original studies
different amphibian species were studied in more on X. lawis, the skin secretion was investigated in great
detail. The production ofantimicrobial peptides is part detail, and was found to contain the magainins/pep-
of the innate immune system and is widespread in tidyl-glycine-scrine (PGS) peptides”,“‘, peptidyl-
Nature’,“. This system was first discovered in insect glycine-leucine carboxyarnide (PGLa), and a multi-
haemolymph when the synthesis of antimicrobial pep- tude of fragments derived from the precursors of,
tides, such as cecropins and dofensins, is induced in among others, caerulein, xenopsin and laevitide.
response to lllicrobial infection. A similar systenl was These peptides, each of which contains nlore than 20
subsequently shown to operate in the lung and gastro- anlino acids, and can form amphipathic helices, have
intestinal tract of nlanmlals; indeed, the system shows a broad spectrum of antimicrobial activity against
striking similarities to the vertebrate acute-phase Grant-positive and Gram-negative bacteria, fungi and
imnunc response. In alnphibians, the production of protozoa 1I. In addition, Imixtures of these peptides
antinlicrobial pcptides appears to be constitutivc, but show strong synergistic effects, which lead to
they are released in response to an external stimulus; an enhancement of the antimicrobial action of the
this is probably mediated by epinephrine. Although secretion.
this innate immunity lacks the ‘memory and speci- Two different families of skin peptides from Bornbina
ficity of acquired inumunity based on itmmunoglobu- species have been described: the bonibinins”,‘” and
lins directed against specific antigens, it has at least two the type H bombinins 14. The bombinins are a group
advantages: the peptides are inimediately available of antibacterial peptides with identical C-terminal
front a reservoir in the cutaneous granular glands, and sequences, but with variable N-terminal sequences
they usually have activity against a broad spectrunz of related to the original bonlbinin4,“,‘“. Type H
niici-oorgaliisiiis. bonlbinins are Imore hydrophobic molecules, possess-
So far, relatively few frog species from a limited ing both antibacterial and haernolytic activities1z-‘4.
number of fanlilies have been studied. While all the Surprisingly, it was found that sonle type H bonibinin

TIETECH JUNE 1995 (VOL 13,

 207

f oats
molecules contain a I)-alloicoleuci~~e as the second the same f&nily. Apart from ranalexin, these all COW
amino acid of their sequence. r)-alloisoleucine is not tain 24 amino acid residues (ranalexin has only 30) and,
essential for the cytolytic activity of these peptides, but except for the pipinins, for which the antimicrobial
it may increase their biostabilitylJ. To date, the pres- activity ha5 not been tested, they are highly active against
ence of 3 I)-amino acid in peptidcs of animal origin &an-positive bacteria. In addition, brevinim1E was
ha\ only been demonstrated for opioid peptides, such found to be highly hacnmlytic, being more potent
as the dermorphins and deltorphins, that were isolated than melittin, a cytolytic peptide from bee venonP.
from South American frogs of the Phyllomedusinae Brevinin-2 and brevinin-2E from R. bwviyod~~
subfamily’ ?. The presence of the I>-amino acid in these (Ref. 23) and R. c~r~lr~tn (Ref. 34), respectively, and
pcptidrs is essential for biological activity. Since a nor- gaegurin-1 to gaegurim3 from R. rrgosa (Ref. 26)
mal codon is present in the cI1NAs encoding those f&m a class of peptides, containing 33-34 amino acid
peptides that contain this I)-amino acidlZ.lC1, a post- residues, that are active against G-an-positive and
translational reaction is suggested to occur, whereby &an-negative bacteria and fungi. Gaegurin-4 from
the configuration at the a-carbon atom is changed. At R. IW~CISOis related to esculentin-2 from R. cwrdcrrta,
present, nothing is known about the mechanism of this and both contain 37 amino acid residues. R. a~~r/crztn
post-translational modification. also secretes esculentin-1, a 46residue peptide that
Several related peptides, called the dernmeptinc, is a highly potent antimicrobial agentZJ. More than
have been isolated from the skin of P sarrvqr.i; these ten peptides belonging to four different families were
are particularly active against pathogenic fungi17. found to be present in a single specimen of R. cwrlrrrtu
Adenoregulin, which is related to the dcrrnaseptins, is (Ref. 24).
present in the skin of I! />i&r, and was initially iso- The amino acid sequences of representative n~cn-
lated because of its ability to enhance the binding of bers from the different peptide families have been eluci-
agonists to the A3 adenosine receptorlx. However, it dated (Fig. 1). The structural diversity of the various
was subsequently found to possess a spectrum of anti- families, combined with the presence of molecules dif-
Inicrobial activity similar to the other dernmcptins”‘. ferillg from each other in a few positions within each
Cacrills and caeridins have been isolated from the family, apparently provides the basis for a wide range
parotoid and rostra1 glands of the Australian frogs of antimicrobial agents. However, a comparison of the
L. .y/urdida and L. cm~rlrcl (Refs 20,21). Some ofthrse selectivity for the target microorganism, and of the
peptides have been shown to possess potent anti- potency of these peptides, has been hampered by
microbial and antiviral activity’“. the diversity of assays and test strains used, as well as
A large variety of antimicrobial pcptides has been by the intrinsic variability of some of these tests. The
isolated from Ram species. These peptides, grouped antibacterial activities ofsome ofthese amphibian pep-
in several families on the basis of differing length and tides, for which a direct comparison is possible, are
distinct activity, were found to have one structural reported in Table 1; activity was determined using an
motif in conm~on: an intramolecular disulphide bridge inhibition-zone assay, and expressed as lethal concell-
located at the C-terminal end, forming a scvcmmen- tration (as described in Ref. 28).
ber ring. The pipinins, from R. pipicw (Ref. 22), In many cases, the sequence of the precursors of
brevinin-1 and brevinin-1 E from R. bvc~vipfn (Ref. 23) these antimicrobial peptides was established using
md R. ~-rr/c~rfa (Ref. 24), respectively, ranalexin from cDNA cloning techniques. One puzzling feature of
K. mt&~iarm (Ref. 2J), and gaeguriti-5 and gae~~rin-6 the precursors of diverse antimicrobial peptides such
from R. JYI~LW (Ref. X), all appear to be members of as esculentin and the brcvinins’d, ranalexi+,

Table 1. Antimicrobial and lytic activity of some amphibian peptides”

Lethal concentration (PM)

Red
E. co/i 8. magaterium S. aureus P. aeruginosa blood
Peptide D21 Bmll Cowan1 ATCC15692 C. albicans S. cerevisiae cells Refs

Magainin2-NH, 4 -b 300 30 - 300 38

Bombinin 3 - 14 - - - >50 14
Bombinin H3 4 2.4 15.7 14
Brevinin-1 E 1.8 0.4 0.6 30.6 4.7 3.8 0.5 24
Brevinin-2E 0.5 0.2 2.0 >30 Inactive lnactwe >lOO
Esculentin-1 0.2 0.1 0.4 0.7 0.5 0.9 >lOO 22:
Esculentin-2a 0.4 0.2 0.8 3.5 - - - 24

“Abbreviations: E. coli, Escherichla colr; 6. magaterium, Bacillus magaterrum; S. aureus, Staphylococcus aureus; P. aeruginosa, Pseudomonas
aeruginosa; C. albrcans, Candida albicans; S. cerevislae, Saccharomyces cerevisrae.
bEntr~es marked (-) have not been tested for peptlde actnity.

TIBTECHJUNE 1995WOL13)
 308
f ecus

Cecropin SUPPlY
K~~MLFKKIEKVGQNI~IIKAGPAVA~~GQATOI~K;I
In the past, peptides have mostly been obtained by
Apidaecin (~~RPVYI7XX'KPPHPRI methanol or acid extraction from sun-dried amphib-
Tachyplesin KbX:FKVCTRCTCYFSCR
r ian skins. In some cases, more than 1000 specimens
have been sacrificed in order to obtain sufficient yuanti-
Defensin ACYCRIPA(:lACERRY(:lTIY~RLWAFCC
ties of the compounds for identification. More
Bactenecin RFRPPIKRPPIRPPE'YPPFRPPlRPPIFPlKPPFRPPFRPPLGPFPa recently, and because of concern about the worldwide
PR-39 RRRPRPPYl~PRPRPPPFE‘PPRLPPRlPK;FPP~PPRFPa decline in many frog species, noninvasive techniques
have been developed. One such method involves the
lndolicidin ILE%JKWl?hWF%JRRa
collection of skin secretion after a mild electrical
stimulation of the frog. The animal is able to tolerate
Figure 2 this, and after 2-4 weeks the secretion can be collected
Representatives of antimicrobial peptides of diverse origin: cecropin from Hyalophora
again, once the glands have been replenished.
cecropia, apidaecin from Apis mellifera, tachyplesin from Tachypleus tridentatus,
Short peptides and peptide analogues resistant to
defensin from phagocytic cells, bactenecin and indolicidin from bovine neutrophils,
proteolytic degradation and containing I>-amino acids,
and PR-39 from pig intestine. Amldated C-terminal residues are represented by a..
can be produced in large quantities by chemical syn-
thesis. Larger peptides can be obtained by expressing
cloned cDNAs encoding a fusion protein in prokary-
dermaseptin and adenoregulin’“, is that they all con- otic host?“. A method for the efficient synthesis of
tain signal sequences similar to those found in the pre- therapeutic peptides in the erythrocytes of transgenic
cursors ofseveral opioid peptides from the skin of Wlyllo- mice has been described”‘; the desired peptide is
nledusu spcciesl6. This similarity has not been observed obtained after selective cleavage of a fusion protein in
for other peptide precursors, such as those of the which the peptide is located at the C-terminal end of
magainins or the bombinins, and the significance of human ol-globin; this can easily be recovered from the
this finding is still obscure. erythrocytes.

Relationships with antimicrobial peptides from Perspectives

other organisms Studies on antimicrobial peptides from amphibians
Gene-encoded antibiotic peptides have been iso- have, so far, only dealt with a few species, mainly
lated from animals, plants and bacteria. These belonging to the order Anura; little is known about
molecules confer protection against pathogens in the peptides from the order Urodela. Indeed, in most
absence of an immune system. However, the ‘innate cases, only the main constituents of skin secretions
immunity’ provided by antimicrobial peptides is even have been investigated. Therefore, it would be
present in vertebrates, which have an immune system expected that many more skin peptides of this sort
capable of responding to an infinite number of exter- exist in Nature. The variety of different defence pep-
nal stimuli. This nonspecific mechanism may act as a tides present in the skin of frogs from a single species
first line of defence against infections. is surprising, and may be the result of evolutionary
A bewildering variety of antimicrobial peptides has selection to provide the most efficient and broad-
been isolated from different sources; some examples spectrum antibacterial protection.
are the cecropins”” and apidaecins”’ from insect Studies on the isolation of new peptides, their mech-
hemolymph, the defensins”‘, the bactenecins”“, indoli- anism of action and their functional cooperativity are
cidin3j and PK-39 (Ref. 35) from different mam- clearly of great interest. Studies on the correlation
malian cells types, and the tachyplesins from crus- between the variety and/or abundance ofantibacterial
taceans’{). These antimicrobial peptides were discussed peptides in frog-skin secretion and the microorganism
in detail in a recent Ciba Foundation SymposiumJ7. ecosystem in which the animal lives are also import-
Despite a common antimicrobial activity, all these ant. These peptides may have additional functions, as
molecules have very different amino acid sequences has recently been shown for PR-39. This is also found
(Fig. 2); some of them are linear (the cecropins), some in wound fluid, where it induces mammalian cells to
contain one disulphide bridge (the bactenecins), express cell-surface heparan sulfate proteoglycans as
others contain more than one disulphide bridge (the part of the wound-repair processQ. Finally, a compara-
defensins, tachyplesin), and some show an over-rcpre- tive analysis of mature peptide and cDNA precursor
sentation of certain amino acids (Pro and Arg in api- sequences might provide an insight into the evol-
daecin, bactenecin and PR-39, Trp in indolicidin). In utionary relationships between different amphibian
keeping with this structural diversity, it would be species.
expected that the interaction of these peptides with
microbial membranes varies greatly. It has been References
1 Enpamer, V. and Melchlorri, P. (lY73) Pure Aypl. Chern. 35,X+494
demonstrated for some peptides that can form amphi- 2 Daly, J. W. (1995) Proc. hhrl Acad. Sci. USA 92, 9-l 3
pathic helices, that synthetic isomers composed of only 3 Barthalmu~, G. T. (lY94) in Anzylrihinu Biolqy (Vol. 1) (Heatwok, H.,
II-amino acids are as active as the natural L-isomers”“; cd.), pp. 382-410, Surrey Batty & Sons
however, in the case of apidaecin, the synthetic all 4 Enpamer, V. (1994) in Ayhibim Biolqy (Vol. 1) (Heatwok, H., cd.),
II-isomer is inactive”“. pp. 178-330, Surrey Bratty PC Sons

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