Developmental and Comparative Immunology 23 (1999) 329±344

Antimicrobial peptides in insects; structure and function

Phillipe Bulet*, Charles Hetru, Jean-Luc Dimarcq, DanieÂle Ho€mann
Institut de Biologie MoleÂculaire et Cellulaire, Unite Propre de Recherche du CNRS 9022, 15 Rue Rene Descartes, 67084 Strasbourg,
Cedex, France

Accepted 1 January 1999

Abstract

Antimicrobial peptides appear to be ubiquitous and multipotent components of the innate immune defense
arsenal used by both prokaryotic and eukaryotic organisms. During the past 15 years a multitude of these peptides
have been isolated largely from insects. In spite of great di€erences in size, amino acid composition and structure,
most of the antimicrobial peptides from insects can be grouped into one of three categories. The largest category in
number contains peptides with intramolecular disul®de bonds forming hairpin-like b-sheets or a-helical±b-sheet
mixed structures. The second most important group is composed of peptides forming amphipathic a-helices. The
third group comprises peptides with an overrepresentation in proline and/or glycine residues. In general, the insect
antimicrobial peptides have a broad range of activity and are not cytotoxic. Despite a wealth of information on
structural requirements for their antimicrobial activity, the mode of action of these peptides is not yet fully
understood. However, some data suggest the existence of two types of mode of action:

1. through peptide±lipid interaction or

2. through receptor-mediated recognition processes.

This review presents the main results obtained during the last four years in the ®eld of antimicrobial peptides
from insects with a special focus on the proline-rich and cysteine-rich peptides. # 1999 Elsevier Science Ltd. All
rights reserved.

1. Introduction
Insects represent the largest class within the
animal kingdom in terms of species number. The
* Corresponding author: Tel.: +33-88-41-70-62; fax: +33-
88-60-69-22.
E-mail address: bulet@ibmc.u-strasbg.fr (P. Bulet)
resistance of insects to pathogens has certainly
contributed to their extreme proliferation and
diversity. At present, insects are found in most of
the biological niches except for the deep marine
environment and the polar regions. More than
one million of insect species have been described
and it is estimated that an equivalent number of
species remains to be identi®ed.

0145-305X/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
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330 P. Bulet et al. / Developmental and Comparative Immunology 23 (1999) 329±344
The ®rst line of defense of insects against
pathogens and parasites is physical, i.e. the
cuticle. However, once this barrier has been brea-
ched, a complex interaction of innate humoral
and cellular immune reactions is induced in both
tissues and haemocoel, which results in the rapid
elimination of microorganisms. The best charac-
terized aspect of the insect immune response is
the synthesis by the fat body (equivalent of the
mammalian liver) and certain blood cells of anti-
microbial peptides/polypeptides and their rapid
release into the hemolymph. As a rule, a given
insect produces a unique repertoire of antimicro-
bial peptides that have overlapping structural fea-
tures but are often targeted for speci®c
microorganisms. In addition, the simultaneous
presence of several antimicrobial peptides acting
in synergy can provide insects with a more
powerful defense against harmful invaders (bac-
teria, fungi, and protozoa).
There is now evidence that antimicrobial pep-
tides are key elements of the innate immunity
against bacteria and fungi in both the animal
and the plant kingdom (for reviews see [1±3]). Of
the impressive number of antimicrobial structures
reported, at least 50% were identi®ed in invert-
ebrates and predominantly within insects. In
1981, Boman and colleagues isolated and fully
characterized the ®rst inducible antibacterial pep-
tide from an insect, the cecropin from bacteria-
challenged diapausing pupae of the moth
Hyalophora cecropia [4]. Since the original report
of cecropin from Boman, more than 170 antimi-
crobial peptides/polypeptides have been found in
insects. They share common features such as a
low molecular weight (below 5 kDa), a positive
net charge at physiological pH and for most of
them, amphiphilic a-helices or hairpin-like b-
sheets or mixed structures. On the basis of
sequence and structural features, these peptides
can be tentatively classi®ed into three broad
classes:
(i) linear peptides forming a-helices and
deprived of cysteine residues;
(ii) cyclic peptides containing cysteine residues
and
(iii) peptides with an overrepresentation in
proline and/or glycine residues (for review see
[5]).
This review focuses on the insect antimicrobial
peptides described during the last four years and
includes data obtained in other invertebrates. We
will present the structure of:
(i) the cysteine-containing peptides namely
insect defensins, drosomycin and thanatin,
(ii) the proline-rich peptides with a special
emphasis on the O-glycosylated antimicrobial
peptides drosocin, lebocins and formaecins
and
(iii) the gloverins, glycine-rich molecules.
We will discuss the structural features, the bio-
logical activity and the mode of action of these
molecules.
2. Cysteine-rich antimicrobial peptides
2.1. Insect defensins
Insect defensins are members of a widely dis-
tributed family of antibacterial peptides with a
characteristic six cysteine/three disul®de bridge
pattern. All insect defensins have the same
cysteine pairing: Cys1±Cys4, Cys2±Cys5 and
Cys3±Cys6. Insect defensins are 36 to 46 amino
acid long with the exception of the 51-residue bee
and bumblebee defensins (for review see [5],[6]).
The ®rst two insect defensins were independently
isolated from culture cells of the ¯esh¯y
Sarcophaga peregrina by Natori and associates
[7] and by Lambert and collaborators from bac-
teria-challenged larvae of the black blow¯y
Phormia terranovae [8]. Since these initial reports,
some 40 members of insect defensins have been
described in phylogenetically diverse lineages
(Fig. 1). To date, insect defensins have not yet
been reported from Lepidoptera. Insect defensins
are present in endopterygote (Diptera,
Coleoptera, Hymenoptera and Trycoptera) and
exopterygote insects (Hemiptera and Odonata).
In addition, insect defensin-like peptides have
been found in two ancient arthropods, the scor-
 P. Bulet et al. / Developmental and Comparative Immunology 23 (1999) 329±344 331

Fig. 1. Alignment of insect defensins with those of the scorpions Androctonus australis and Leiurus quinquestriatus and from two
molluscs Mytilus edulis and Mytilus galloprovinsialis. Defensins or equivalent are listed according to their origin. Gaps are intro-
duced to optimize alignment. The highly conservative cysteine residues are highlighted in grey.

pions Leiurus quinquestriatus [9] and Androctonus
australis [10]. Interestingly, the presence of insect
defensin-like molecules has also been reported
from two species of molluscs, the mussels Mytilus
edulis [11] and M. galloprovincialis [12]. However,
a marked di€erence exists between the two mol-
luscan defensins, since in M. galloprovincialis two
cysteine residues are found in addition to the
conserved consensus disul®de array. A defensin-
like peptide also takes part in the immune
defense of the nematode Ascaris suum [13].
Sequence comparisons between all insect defen-
sins reveal that similarities are ranging from 58
to 95% and 75% similarities are observed
between the defensin isolated from the dragon¯y
Aeschna cyanea which belong to the ancient
order of the Odonata and the scorpion and mus-
sel defensins. Recently, a defensin with an N-
terminal extension of six residues has been iso-
lated from the gut of the dipteran Stomoxys cal-
citrans [14]. The hymenopteran defensins from
Apis mellifera (see in review [5]) and Bombus pas-
cuorum [6], respectively, contrast with all other
defensins by the presence of a 12-amino acid C-
terminal extension. No similar defensin is found
in another hymenopteran insect, the ant Formica
332 P. Bulet et al. / Developmental and Comparative Immunology 23 (1999) 329±344
rufa [15]. This C-terminal extension is supposed
to adopt an a-helix structure. Interestingly,
hymenopteran defensins are so far the only
defensins presenting a C-terminal amidation
[6,16]. This type of post-translational modi®-
cation is shared with the cecropins, insect antimi-
crobial peptides forming a-helices (for reviews,
see [5] and [17]).
A phylogenetic analysis of the invertebrate
defensin family using the six cysteine residues as
landmarks reveals salient features. The dragon¯y
defensin groups closely with the scorpion and
mussel defensins, while all the other defensins ®t
into a second group [11]. Within each group, a
high variability is apparent, for instance, the mol-
ecular distance between the defensin sequence of
the dragon¯y versus the mollusc or the scorpions
defensins (i.e. three organisms belonging to
di€erent phyla) is smaller than that separating
Drosophila melanogaster from Aedes aegypti, two
dipteran species.
3. Biological activity and mode of action of insect
defensins
Insect defensins are active against a wide range
of Gram-positive bacteria. Only few Gram-nega-
tive bacteria, fungi and yeasts are a€ected by
defensins (for review see [5]). Some reports have
implicated insect defensins (Phormia and Aeschna
defensins) in the killing of some mosquito stages
of the Plasmodium gallinaceum parasite [18].
These two defensins have a profound toxic e€ect
on Plasmodium oocystes in A. aegypti as well as
on isolated sporozoites. Defensins a€ect the
Plasmodium oocystes in a time-dependent man-
ner: the oocystes become swollen or show exten-
sive internal vacuolization at the time when the
e€ects of defensins are maximal. In vitro exper-
iments on isolated sporozoites reveal that both
defensins are highly toxic, causing the disruption
of the membrane permeability barrier and a
change in morphology with subsequent loss of
motility. In contrast to the toxicity observed
when cecropin and cecropin derivatives are
injected into mosquitoes, insect defensins showed
no e€ects on the insects when injected at concen-
tration which kill or a€ect the development of
the parasites [18]. Under low salt conditions,
defensins are lytic in vitro at a low micromolar
concentration (0.1 to 10 mM) for most of the sus-
ceptible Gram-positive strains. However, the e-
ciency is strongly reduced when the salt
concentration is increased [19]. This decrease of
activity at high salt concentration is also
observed for mammalian (see review [2]) and
plant (see review [3]) defensins but not for stye-
lins, cysteine-free phenylalanine-rich antimicro-
bial peptides from the solitary tunicate Styela
clava [20]. Numerous studies conducted on var-
ious native insect defensins established that these
peptides have an almost immediate lytic e€ect on
the Gram-positive bacteria. A 1 min exposure to
the peptide is sucient to kill the bacteria. This
is observed for all the insect defensins except for
Aeschna defensin which kills the Gram-positive
bacteria only after several hours of contact [21].
This suggests the existence of a di€erent mode of
action in the latter case. Studies performed on
recombinant Phormia defensin show that the pep-
tide instantly disrupts the permeability barrier of
the cytoplasmic membrane of the Gram-positive
bacterial strain Micrococcus luteus, resulting in a
loss of cytoplasmic potassium, a partial depolar-
ization of the inner membrane, a decrease in
cytoplasmic ATP, and ®nally an inhibition of the
respiration [19]. The addition of divalent cations
and a decrease in the membrane potential below
a threshold of 110 mV results in the inhibition of
the potassium loss. Patch-clamp experiments on
giant liposomes support the hypothesis that
Phormia defensin a€ect the permeabilization bar-
rier through the formation of channels in the
cytoplasmic membrane of M. luteus [19].
4. Drosomycin, an antifungal cysteine-rich peptide
In addition to antibacterial peptides, insects
produce cysteine-rich peptides with antifungal
properties. Drosomycin was the ®rst inducible
antifungal peptide to be isolated from insects. To
date, its presence has only been reported in the
fruit¯y D. melanogaster [22]. Drosomycin is a 44-
residue peptide including eight cysteine residues
 P. Bulet et al. / Developmental and Comparative Immunology 23 (1999) 329±344 333

Fig. 2. (A) Alignment of the inducible antifungal drosomycin from Drosophila melanogaster and two plant defensins AFP1 and
AFP2 from the radish Raphanus sativus and a g-thionin from wheat endosperm (g-1P thionin). Gaps are introduced for a better
alignment. The highly conservative residues are highlighted in grey and the connectivity of the cysteine residues indicated by broken
lines. (B) 3-D structure of drosomycin and of defensin A from Phormia terranovae. Based on the coordinates from the Brookhaven
Protein Data Bank and draw with Swiss PDBviewer program.

engaged in the formation of four internal disul-
®de bridges: Cys1±Cys8, Cys2±Cys5, Cys3±Cys6
and Cys4±Cys7 [23] (Fig. 2). In comparison to
insect defensins which contain three disul®de
bonds, drosomycin exhibits an additional disul-
®de bond between the ®rst and the C-terminal
cysteine residues conferring a highly compact
structure to the molecule. This certainly accounts
for the remarkable protease resistance of droso-
mycin as observed in vitro [22,23] as well as in
vivo [24]. Drosomycin has striking similarities
with cysteine-rich antifungal peptides from
plants, i.e. plant defensins and g-thionins [25].
These two groups of plant antifungal peptides
are either constitutively present or induced upon
an infection, and are part of the plant defense
mechanisms (for review see [26]). Drosomycin,
plant defensins and g-thionins share a consensus
sequence including [1] eight cysteine residues that
form a conserved array of four disul®de bonds
(see above for the cysteine sca€old and in the
next section) and [2] ®ve additional residues con-
served at approximately identical positions,
namely two glycine residues, a serine residue, an
aromatic amino acid and a glutamine residue
[Fig. 2(A)]. The structural similarities between
drosomycin and the plant defense peptides are
even more striking than those observed between
drosomycin and insect defensins. The degree of
sequence similarity is around 40% between dro-
334 P. Bulet et al. / Developmental and Comparative Immunology 23 (1999) 329±344
somycin, the plant defensins extracted from the
radish Raphanus sativus [25] and some g-thionins
[27]. In contrast, the similarity between drosomy-
cin and the insect defensins is almost restricted to
the six invariant cysteine residues [Fig. 2(A)].
5. Biological activity and mode of action of
drosomycin
Drosomycin has a potent antifungal activity
while it is ine€ective against yeasts and bacteria
and has no hemolytic e€ect on bovine erythro-
cytes. The concentration of drosomycin in the he-
molymph of bacteria-challenged adult Drosophila
(100 mM) is much higher than the concentration
required to a€ect most of the fungal strains
tested [22]. Drosomycin is active against human
and phytopathogenic fungi at concentrations
often below 5 mM. The relevance of drosomycin
in the innate immunity in Drosophila ¯ies is
underscored by the observation that mutant ¯ies
impaired in the induction of the drosomycin gene
show a lower survival rate than wild-type ¯ies
when challenged with an infectious fungus [28].
Drosomycin inhibits spore germination at high
concentrations and delays growth of hyphae at
lower concentrations. This delay in growth causes
reduced hyphal elongation with a concomitant
increase in hyphal branching. Drosomycin is fun-
gicidal against the model fungus Neurospora
crassa, and induces partial lysis of the hyphae of
growing Botrytis cinerea [22]. However this par-
tial lysis, which results in extrusion of cyto-
plasmic material from the hyphae, is only
observed in the presence of divalent cations
(Ca++). A comparable e€ect is observed with
plant defensins (for review, see [26]). So far, no
structural±activity relationships have been
reported for drosomycin. In addition, the mode
of action of this inducible antifungal peptide
from an insect remains to be elucidated. Recent
data obtained by Broekaert and collaborators
[29] on a drosomycin related plant defensin tend
to prove that, in contrast to insect defensins,
these peptides do not cause ion channel for-
mation in either arti®cial planar lipid bilayers or
arti®cial liposomes. However, when added to
fungi, the plant defensins cause calcium in¯ux
and potassium e‚ux, an alkalinization of the in-
cubation medium and changes in membrane po-
tential. These modi®cations are the consequences
of the interaction of the antifungal peptide with a
target receptor acting as an anchor point allow-
ing the peptide to slip into the membrane [29].
This target receptor remains to be identi®ed.
6. Three-dimensional structure of drosomycin
compared to insect defensins
The structure of drosomycin in solution has
been determined from two-dimensional 1H±
NMR data [27], as was the case for Phormia
defensin [30,31] and Sarcophaga sapecin [32]. A
comparable architecture exists for both insect
defensins while marked di€erences exist between
drosomycin and the insect defensins. The two
insect defensins adopt a compact globular fold
comprising an amino-terminal loop, a central a-
helix and a double stranded C-terminal b-sheet
(for review see [5]). The amino-terminal loop is
connected to the ®rst strand of the b-sheet by the
disul®de bond Cys1±Cys4. The second strand of
the b-sheet is connected to the central a-helix by
the two remaining disul®de bridges (Cys2±Cys5
and Cys3±Cys6). The three-dimensional structure
of drosomycin compared to that of insect defen-
sins [Fig. 2(B)] di€ers by the presence of an ad-
ditional strand of b-sheet in the N-terminal
extension of drosomycin [27]. In insect defensins
the sequence between Cys1 and Cys2 is longer
(12 amino acids) than the segment between Cys2
and Cys3 of drosomycin (7 amino acids), which
both form the loops in the corresponding mol-
ecules. The fourth disul®de bridge Cys1±Cys8 of
drosomycin connects this N-terminal short strand
of b-sheet to the C-terminal cysteine residue. The
drosomycin sca€old (babb) is identical to the
sca€old observed for plant defensins [25]. A
striking feature that emerges from the compari-
son of the three-dimensional structures of insect
defensins, drosomycin and defense molecules
from plants (defensins and g-thionins) is a motif
named `Cysteine Stabilized a-Helix' (CSH). This
sca€old refers to an invariant Cys±Xaa±Cys
 P. Bulet et al. / Developmental and Comparative Immunology 23 (1999) 329±344
sequence found in a strand of a b-sheet in which
two cysteine residues are attached to two cysteine
residues of the stretch Cys±Xaa±Xaa±Xaa±Cys
present in an a-helix. This motif was ®rst ident-
i®ed by Kobayashi and collaborators [33], and it
is observed in a series of small cysteine-rich neu-
rotoxins from venom of scorpions, spiders, and
honeybees. Cornet and collaborators [31], follow-
ing the resolution of the three-dimensional struc-
ture of Phormia defensin, proposed the term
`Cysteine Stabilized ab' motif (Csab) for the par-
ticular sca€old of the insect defensins. This motif
containing at least the a-helix±two-stranded b-
sheet (abb) type structure found in the insect
defensins can be extended in the N-terminal part
of drosomycin to a babb type structure. From
these observations, the Csab motif which con-
tains two disul®de bridges with invariant pos-
itions of cysteine residues in many small defense
molecules from di€erent origins (insects, scor-
pions, spiders and plants) and exhibiting various
biological activities (antibacterial, antifungal and
toxins) may be derived from a common ancestor
gene. However, this structural Csab sca€old
adopted by these invertebrate and plant defense
molecules is not present in mammalian defensins.
The latter have a conformation consisting of a
triple-stranded b-sheet structure and a connecting
loop as a base from which a b-hairpin hydrophi-
lic ®nger protrudes nearly orthogonal [34]. This
di€erence in the three-dimensional structures
between mammalian and insect defensins is not in
favor of a common ancestor, as proposed earlier.
7. Thanatin, an antimicrobial peptide from a bug
Recently, a second inducible peptide with anti-
fungal properties, thanatin, has been isolated
from an immune challenged insect belonging to
the order of Hemiptera, the bug Podisus maculi-
ventris [35]. Thanatin is a 21-residue peptide
including two cysteine residues engaged in the
formation of an internal disul®de bridge (Fig. 3).
Thanatin has no sequence similarity with any
other insect defense peptides. However, it has
noticeable similarities with frog skin secretion
antimicrobial peptides, and more speci®cally
335
(40%) with the brevinin family [36,37]. Both
types of peptides include a C-terminal cationic
loop, 6 amino acids for thanatin and 5 amino
acids for brevinins, delineated by the two cysteine
residues which form a disul®de bridge [Fig.
3(A)]. The loop has a strong positive net charge
and in both types of molecules, a central threo-
nine residue separates two subgroups of basic
residues. A similar motif, a central threonine or
serine ¯anked by groups of basic residues clus-
tered between two cysteines, is present in a num-
ber of antimicrobial peptides (esculentins,
gaegurins and ranalexin) from frog-skin se-
cretions. This motif named `Rana box' [38] is
also present in thanatin. However, the `Insect
box' [35] as opposed to the `Rana box' contains
an additional asparagine residue within the loop.
In both thanatin and brevinin 1 a cluster of six
hydrophobic residues extends N-terminally to a
common lysine residue. In contrast, thanatin is
C-terminally extended by a three amino acid
sequence [Fig. 3(A)].
8. Biological activity and mode of action
Thanatin is the ®rst reported insect peptide to
have a broad spectrum of activity against both
Gram-positive and Gram-negative bacterial
strains as well as against ®lamentous fungi.
Thanatin is fungicidal and bactericidal and exhi-
bits its activity at a minimal inhibitory concen-
tration often below 2.5 mM [35]. Thanatin has no
hemolytic e€ect on porcine red blood cells. A
structural±activity relationship investigation on
thanatin and several truncated forms point out
four regions within thanatin that are crucial for
its full activity:
1. the C-terminal loop,
2. the C-terminal three-residue extension,
3. a stretch of seven N-terminal mostly hydro-
phobic residues, and
4. three additional N-terminal residues that are
necessary for full antifungal activity but dis-
pensable for antibacterial activity.
Although the mode of action of thanatin on
336 P. Bulet et al. / Developmental and Comparative Immunology 23 (1999) 329±344
Fig. 3. (A) Comparison of the amino acid sequence of thanatin
the amphibian Rana brevipoda. Gaps are introduced to optimize
grey and the connectivity of the two cysteine residues indicated
thanatin and the `Rana box' in brevinin 1. (B) 3-D structure of
Data Bank and draw with Swiss PDBviewer program.
bacteria and fungi is not yet fully understood,
several data point to a mode of action which dif-
fers from that of insect defensins and cecropins.
An all-D-enantiomer of thanatin has nearly the
same antifungal activity as natural all-L-thanatin,
while all-D-thanatin is nearly inactive against
Gram-negative and some Gram-positive bacteria
[35]. This suggests more than one mechanism of
action for thanatin, depending on the target
microorganism. This is in contrast to brevinins
which exert their antibiotic e€ect through disrup-
tion of the permeability of the bacterial mem-
brane [38].
9. Three-dimensional structure of thanatin
The three-dimensional structure of thanatin
obtained by two-dimensional 1H±NMR in sol-
ution, and molecular modeling, show that thana-
tin adopts a structure di€erent from that of
drosomycin and insect defensins. The core of tha-
natin is a well-de®ned two-stranded (®ve residues
from Podisus maculiventris and the antimicrobial brevinin 1 from
the alignment. The highly conservative residues are highlighted in
by a broken line. The bold characters outline the `Insect box' in
thanatin. Based on the coordinates from the Brookhaven Protein
each) b-sheet structure slightly twisted and main-
tained by the single disul®de bridge, while the N-
terminus (the seven ®rst N-terminal amino acids)
corresponds to a long extended and poorly
de®ned arm [39] [Fig. 3(B)]. This antiparallel
two-stranded b-sheet structure assimilated to a
hairpin-like b-sheet structure is also found in bre-
vinins [37], protegrins antimicrobial peptides iso-
lated from porcine leukocytes [40,41] and
tachyplesins isolated from the hemocytes of
horseshoes crabs [42].
10. Proline and glycine-rich peptides
10.1. Structural features of the proline-rich
peptides
Several proline-rich peptides have been isolated
from Hymenoptera (apidaecins and abaecins
from Apoidea, Sphecoidea, Vespoidea and
Ichneumonoidea), Diptera (drosocin from D.
melanogaster ) and Hemiptera (pyrrhocoricin
 P. Bulet et al. / Developmental and Comparative Immunology 23 (1999) 329±344 337

Fig. 4. Comparison of proline-rich antimicrobial peptides from insects. (A) The short-chain proline-rich peptides apidaecins are
compared to formaecins, drosocin, pyrrhocoricin and metalnikowins. (B) The long-chain proline-rich lebocins from Bombyx mori
are compared to Apis mellifera abaecin and to Drosophila melanogaster metchnikowins. Conserved residues are highlighted in grey.
Gaps are introduced to optimize alignments. The O-glycosylated threonine residues are boxed (substitution can be the monosac-
charide N-acetylgalactosamine or the disaccharide galactose 4 N-acetylgalactosamine).

from Pyrrhocoris apterus ), (for review see [5]).
Since 1995, antimicrobial peptides containing
proline residues have been found in the lepidop-
teran Bombyx mori (lebocins, [43]) and in two
hymenopterans, namely B. parcuorum (apidaecin
and abaecin, [6]) and Myrmecia gulosa (formae-
cins, [44]). In addition, metchnikowin, a proline-
rich peptide, was found in D. melanogaster [45]
and a group of molecules (metalnikowins) similar
to the hemipteran pyrrhocoricin was character-
ized from two bugs, Palomena prasina [46] and
Podisus maculiventris [35] (Fig. 4).
The size of the proline-rich peptides varies
from 15 (metalnikowins) to 39 residues for the
abaecins from B. pascuorum. The proline residues
(over 25% of the amino acid composition) are
often associated in doublets and triplets with
basic residues (arginine and lysine). This class of
insect antibiotics is subdivided into two groups:
1. the unsubstituted and
2. the O-glycosylated peptides.
10.1.1. The unsubstituted short-chain peptides
To this subgroup belong apidaecins, abaecin
and metalnikowins. Apidaecins are a group clo-
sely related peptides that were found only in
Hymenoptera [47], including B. parcuorum [6].
338 P. Bulet et al. / Developmental and Comparative Immunology 23 (1999) 329±344
Seventeen isoforms have been isolated [Fig.
4(A)]. Not surprisingly, the recently described B.
pascuorum apidaecin shows maximum homology
with the apidaecin from B. terrestris which is
belonging to the same genus. The molecules di€er
by a single amino acid replacement, a glycine
residue at position 1, common to 9 of the 17 api-
daecin isoforms, in B. pascuorum versus an ala-
nine residue in B. terrestris. This substitution to
an alanine appears to be unique to B. terrestris.
From the proposed alignment, an evolutionary
conserved sequence can be deduced with the
remarkable C-terminal octapeptide Pro±Arg±
Pro±Pro±His±Pro±Arg±Ile/Leu, an additional
Arg/Lys±Pro dipeptide within the N-terminus
and a proline residue in the central part of the
apidaecins [Fig. 4(A)]. The variations observed
between the conserved amino acids are rather
limited between apidaecins and the closely related
hymenopteran peptides and more apparent
between distantly related superfamilies.
Recently, several isoforms of a group of api-
daecin-like peptides have been isolated from two
di€erent hemipteran species, the bugs P. prasina
[46] and P. maculiventris [35]. These molecules (8
forms), named metalnikowins, contain between
15 to 17 amino acids including ®ve proline resi-
dues if we except the four proline residues of
metalnikowin II from P. prasina [Fig. 4(A)]. The
di€erence in the primary structures between
metalnikowins is found mainly at the C-terminus.
In fact, a part of the conserved consensus
sequence found in the apidaecins, such as the
dipeptide Arg/Lys±Pro and the two ®rst residues
of the remarkable C-terminal conserved octapep-
tide are also present in metalnikowins.
10.1.2. The unsubstituted long-chain peptides
Soon after the discovery of apidaecins,
Casteels and collaborators isolated an additional,
34-amino acid long, member of the proline-rich
family from the honeybee Apis mellifera, which
they named abaecin [48]. Recently, an abaecin
was also reported from the bumblebee B. pas-
cuorum [6]. The B. pascuorum abaecin with 39
amino acids is the largest proline-rich antibacter-
ial peptide from insects isolated so far. It shares
approximately 54% (21/39 residues) primary
sequence homology with its counterpart in the
honeybee and has a Pro±Arg±Pro motif found in
apidaecins and metalnikowins but not in honey-
bee abaecin. In addition, the abaecins from
Hymenoptera have structural similarities with
two classes of antimicrobial peptides, metchniko-
wins and lebocins isolated from the fruit¯y D.
melanogaster [45] and from the silkworm B. mori
[43], respectively [Fig. 4(B)].
Two forms of metchnikowins di€ering by only
one amino acid (histidine/arginine at position 3)
have been isolated. They are composed of 26
amino acids including seven proline residues
(27%) with a triplet Pro±Arg±Pro, a common
feature of all the proline-rich peptides described
above with the exception of the abaecin from A.
mellifera (Fig. 4). The similarity between metch-
nikowins and the abaecins, the largest members
of the proline-rich peptides, is particularly strong
in the C-terminal part. In fact, a consensus
sequence of Pro±Phe±Asn±Pro appears close to
this region. Such a conserved consensus sequence
is also observed in formaecins, the proline-rich
peptides from the ant M. gulosa (see Section
10.1.3, O-glycopeptides, below). This evolution-
ary conserved sequence may well represent a
functionally important domain as observed for
the apidaecin type motif (see above). In addition,
some sequence similarities are also observed in
the N-terminal segment of apidaecins and metal-
nikowins, however to a lower extent, as it is
restricted to few dispersed residues.
Attention has recently been focused on the
abaecins as a prototype member of the proline-
rich family [45]. Abaecin potentially links the lar-
ger members of this class (metchnikowins and
formaecins) with the shorter members of this
group (apidaecins, metalnikowins and the O-gly-
cosylated peptides).
10.1.3. The O-glycosylated antibacterial peptides
In contrast to the group of unmodi®ed com-
pounds, an attractive structural feature is the pre-
sence of an O-glycosidic substitution in some of
the proline-rich peptides (Fig. 4, sequences with
boxed threonine residues). The prototype of this
subgroup is drosocin, a 19-residue peptide con-
taining six proline and four arginine residues in-
 P. Bulet et al. / Developmental and Comparative Immunology 23 (1999) 329±344
corporated into three repeated triplet sequences
Pro±Arg±Pro. This peptide, isolated from D. mel-
anogaster, carries a post-translational modi®-
cation on the threonine residue at position 11.
This threonine residue is O-glycosylated by a dis-
accharide comprised of N-acetylgalactosamine
and galactose moieties [49]. The terminal galac-
tose is linked to N-acetylgalactosamine which is
directly linked through its anomeric carbon to
the hydroxyl group of threonine. Since this ®rst
report, additional O-glycosylated antibacterial
peptides have been described from three other
insect orders, (i) Hemiptera (pyrrhocoricin from
P. apterus, [50]), (ii) more recently from
Lepidoptera (lebocins from B. mori, [43]) and (iii)
very recently from Hymenoptera (formaecins
from M. gulosa, [44]). Brie¯y, pyrrhocoricin is a
20-residue peptide with sequence similarities to
drosocin and metalnikowins and a threonine resi-
due carrying a disaccharide motif identical to
that observed in drosocin. It is interesting to note
that pyrrhocoricin and drosocin are closer to
metalnikowins than to the hymenopteran apidae-
cins and abaecins. The major di€erence between
these molecules is the absence of the O-substi-
tuted threonine residue in metalnikowins (Fig. 4).
During the last three years, the existence of
proline-rich antimicrobial peptides from insects
was reported in Lepidoptera and Hymenoptera.
In the Lepidoptera B. mori, Hara and collabor-
ators mentioned proline-rich O-glycopeptides,
lebocins 1, 2 and 3 [43]. Lebocins are 32-residue
peptides (25% proline residues) which are O-gly-
cosylated on their threonine residue. Lebocins 1
and 2 di€er in their sugar moiety, the disacchar-
ide galactose 4 N-acetylgalactosamine versus N-
acetylgalactosamine, respectively. Lebocin 3 har-
bors the same substituting group than lebocin 2
but it has a leucine residue instead of a proline
residue at position 16 [Fig. 4(B)]. The variation
in the glycan structure in lebocins, one sugar or
two sugars, has been proposed to re¯ect a di€er-
ent stage in the glycan maturity (elongation or
degradation) [43]. Such a glycoheterogeneity was
also reported for drosocin [49], pyrrhocoricin [50]
and for diptericin, a glycine/proline-rich antibac-
terial O-glycosylated peptide from the black
blow¯y P. terranovae [51]. A study of the origin
339
of the glycoheterogeneity observed on Phormia
diptericin suggests the presence of glycosidases in
the hemolymph of P. terranovae [52]. A recent
report on the di€erential display of peptides
involved in the immune response of D. melanoga-
ster supports this hypothesis through the in vivo
study of drosocin stability in the hemolymph of
D. melanogaster [24]. Lebocins show the highest
(41%) similarity with abaecins and metchniko-
wins. However, in contrast to the other proline-
rich peptides, lebocins do not contain any Pro±
Arg±Pro triplet.
In 1998, Mackintosh and colleagues [44]
reported the presence in the bulldog ant M.
gulosa (Hymenoptera) of two O-glycosylated pro-
line-rich peptides (®ve proline residues out of 16
amino acids) which they named formaecins. The
primary structure of formaecins di€ered at two
sites: an asparagine residue versus a threonine
residue at position 8 and an histidine residue ver-
sus a tyrosine residue at position 13 for formae-
cin 1 and 2, respectively. Formaecins 1 and 2
have respectively 50 and 44% sequence identities
with drosocin. In both cases the O-glycosylation
is on threonine residue 11 like in drosocin.
Formaecins are glycosylated with the monosac-
charide N-acetylgalactosamine while drosocin is
glycosylated with the disaccharide galactose 4 N-
acetylgalactosamine.
10.2. Gloverins, a family of glycine-rich
polypeptides
Several antibacterial glycine-rich polypeptides
have been isolated from various insect species
(for review see [5]): Diptera (diptericins, attacins
and sarcotoxins II), Lepidoptera (attacins),
Hymenoptera (hymenoptaecins), Coleoptera
(coleoptericin, holotricin II and III, tenecin III)
and Hemiptera (hemiptericin). Holotricin II and
III and tenecin III are constitutive glycine-rich
peptides active against Candida spp. The size of
the glycine-rich polypeptides varies from 8 kDa
(holotricin) to 30 kDa (sarcotoxin II) and they
can be O-glycosylated [e.g. diptericin from the
black blow¯y P. terranovae [51]]. Recently, glo-
verins, glycine-rich polypeptides, were isolated
from immune hemolymph of two lepidopteran
340 P. Bulet et al. / Developmental and Comparative Immunology 23 (1999) 329±344
Fig. 5. Amino acid sequence of gloverins from Hyalophora gloveri and Helicoverpa armigera. Glycine residues are in bold. The
sequence of gloverin from H. armigera is only partial.
species, the giant silk moth Hyalophora gloveri
[60] and the old world bollworm Helicoverpa
armigera [61]. Gloverins have a mass of 14 kDa
and contain 18% of glycine residues (Fig. 5).
Gloverins have amino acid sequences that reveal
no strong degree of similarity with any known
antimicrobial peptide. They are e€ective against
Gram-negative bacteria and are inactive against
Gram-positive bacteria, yeasts, mammalian cell
lines and pestivirus [61]. Experimental data
obtained on their mode of action indicate that
gloverin may act by inhibiting the synthesis of
vital outer membrane proteins resulting in an
increase in permeability of the outer membrane
of the bacteria [60]. This mode of action is re-
lated to that of attacin (for review see [5]).
11. Biological activity and mode of action of the
proline-rich peptides
As a general feature, most of the short-chain
proline-rich peptides are active against Gram-
negative bacteria while Gram-positives remain
mostly una€ected. The higher resistance of
Gram-positive bacteria is suggested to result
from degradation by extracellular proteases. In
general, distinct functional variability, in terms of
antibacterial spectra and speci®c activity exists
among the proline-rich peptides. In a careful
inspection of apidaecin-type sequences and of
antibacterial spectra, Casteels and collaborators
have demonstrated that di€erences in antibacter-
ial spectra/speci®city can be correlated, in several
cases, with very subtle sequence modi®cations
[47]. This was also observed for metalnikowins.
In fact, metalnikowins, closely related to each
other [70% to 90% amino acid sequence simi-
larities, see Fig. 4(A)], have a speci®c activity
against Gram-negative bacteria ranging from 50
to 200 mM according to the isoform. The low
speci®city observed for metalnikowins (average
125 mM) is compensated by the high concen-
tration of metalnikowins in the hemolymph of
the bugs [up to 100 mM per isoform form
[35,46]]. Interestingly, abaecins, the longest pro-
line-rich peptides are active against both Gram-
negative and Gram-positive bacteria while metch-
nikowins have no activity against Gram-negative
bacteria but are a€ecting the growth of M. luteus
and the ®lamentous fungus Neurospora crassa
[45].
Unlike active peptides such as insect defensins
and cecropins which kill the microorganisms
within minutes through lytic/ionophoric, non
stereospeci®c mechanisms (for review see [53]),
the killing of bacteria by apiadaecin and drosocin
is found to take several hours. In addition, all-D-
enantiomers of both peptides are completely
inactive [54,55] suggesting a reaction mechanism
involving a stereospeci®c interaction of the pep-
tide with a bacterial target.
Apidaecin has an immediate e€ect on protein
synthesis, as the incorporation of a labeled pre-
cursor for protein synthesis is immediately
stopped upon addition of the peptide, which in
turn blocks the replication and transcription
within a few hours (for review see [56]). In ad-
dition, further evidence for a speci®c interaction
of apidaecin with sensitive microorganisms comes
from detailed analysis of activities in relation to
variability in the primary structures of di€erent
apidaecins [47]. An alignment of the primary
amino acid sequences of 17 isolated apidecins
 P. Bulet et al. / Developmental and Comparative Immunology 23 (1999) 329±344
results in the delineation of the strictly conserved
core sequence described above (the C-terminal
octapeptide: Pro±Arg±Pro±Pro±His±Pro±Arg±
Ile/Leu). Studies of the biological activities of (i)
the natural variants, (ii) derivatives obtained by
proteolytic cleavage, and (iii) truncated analogues
generated by chemical synthesis, indicate that the
major activity of apidaecin-type peptides resides
within the COOH-terminal core. Di€erences in
the antibacterial spectra of the natural variants
are due to minor amino acid replacements in the
variable N-terminal domain. A functional map-
ping of amino acid residues responsible for the
antibacterial activity of apidaecin reveals the im-
portance of the proline and arginine residues
including the proline residue at position 9, a con-
served residue in all apidaecins [Fig. 4(A)] for the
biological activity [57]. A synthetic apidaecin
lacking this proline residue has a 10-fold re-
duction in its activity towards certain bacterial
strains (i.e. Escherichia coli ). Interestingly, this
proline residue is absent in metalnikowins which
are approximately 10 times less ecient than api-
daecins from Hymenoptera.
Concerning the O-glycopeptides, they are
active mostly against Gram-negative bacteria if
we do not consider the activity of drosocin and
pyrrhocoricin against a few Gram-positive bac-
teria. The antibacterial activity of the glycopep-
tides is closely associated with the presence of the
glycan moiety as their synthetic non-glycosylated
counterparts have a markedly reduced activity.
In most cases, the N-acetylgalactosamine (the
most proximal monosaccharide) is sucient to
restore the activity to the level of the natural
compound. An elongation of the glycan chain
with a galactose tends to reduce the antibacterial
activity of lebocins [43] while such an elongated
glycan is naturally present on drosocin and pyr-
rhocoricin.
The mechanism by which the sugar moiety
a€ect biological activity is not yet understood.
The attractive features of glycopeptides, in gen-
eral, when compared to their non-glycosylated
parent analogues, include increased solubility,
serum stability and broader biological activity
spectrum. Sugars can modify the conformation
of the peptide backbone resulting in a change in
341
the anity with stereospeci®c bacterial targets or,
alternatively, this can change the physicochemical
properties of the peptide. Conformational studies
have been restricted to the analysis of drosocin
and its non-glycosylated derivative by circular
dichroism in aqueous and membrane-mimicking
solvents which conclude to the existence of few
di€erences between the two peptide forms forms
[55]. A recent study by NMR spectroscopy has
revealed that glycosylated and non-glycosylated
drosocins exist as random coil conformations in
aqueous solution and that the addition of 50%
tri¯uoroethanol (a membrane-mimicking solvent)
causes the development of small populations of
folded conformations, mainly in the form of
turns [58]. This result demonstrates that glycosy-
lation results in a local straightening of the pep-
tide backbone while it is often considered that
glycoylation is a turn-inducer [59].
12. Concluding remarks
In the last 20 years, it has become clear that
endogenous antimicrobial cationic peptides are
part of the innate immune response of plants, in-
vertebrates and vertebrates. The antimicrobial
arsenal used by insects to ®ght o€ invading
microorganisms is essentially based on short cat-
ionic antibacterial and antifungal peptides/poly-
peptides. According to the species, the fat body
can respond to bacterial infection by the syn-
thesis of either a large panel of antimicrobial sub-
stances (e.g. Drosophila adults produce more
than ten di€erent molecules including drosocin,
drosomycin, defensin, metchnikowins, diptericin,
cecropins) or by producing relatively few mol-
ecules (e.g. a single peptide defensin in the dra-
gon¯y Aeschna cyanea ). One of the most
interesting features of the insect antimicrobial
peptides is their structural similarity to antimi-
crobial substances from the plant and animal
kingdoms. Cecropins, not discussed in this
review, have similarities with the amphibian a-
helical antimicrobial peptides magainins found in
frog skin secretions while, as discussed above,
drosomycin from Drosophila and thanatin from
the bug Podisus maculiventris have similarities
342 P. Bulet et al. / Developmental and Comparative Immunology 23 (1999) 329±344
with plant defensins and frog skin secretion anti-
microbial peptides, respectively. Although insect
antimicrobial peptides manifest great structural
diversity, they can be considered as good candi-
dates to overcome the alarming problem of
acquired bacterial drug-resistance and of emer-
gence of opportunistic pathogens, especially in
immunosuppressed hosts, since they present sev-
eral advantages:
(i) they rapidly kill target microorganisms,
(ii) they have broad activity spectra,
(iii) they are ine€ective against mammalian
cells and
(iv) they have a mode of action that should
restrict the selection of resistant strains.
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