REVIEW Sci. USA 88, 4328-4332 62 Okamoto, T., Ogiwara, H., Kawabe, T. et al. (1992) Int. Immunol. 7, 811-820 63 Fujii, S., Nanbu, Y., Nonogaki, H. etal. (1991) Cancer 68, 1583-1591

64 Schreck, R., Rieber, P. and Baeuerle, P.A. (1991) EMBO J. 10, 2247-2258 65 Buhl, R., Holyroyd, K.J., Mastrangeli, A. et al. (1989) Lancet ii, 1294-1297 66 Mitsui, A. Biochem. Biophys. Res. Commun. (in press)

Insect defensins: inducible antibacterial peptides* Jules A. Hoffmann and Charles Hetru In response to bacterial challenge or trauma, insects produce a battery of bactericidal or bacteriostatic molecules with a broad spectrum of activity against Gram-positive and~or Gram-negative bacteria; most are smallsized cationic peptides. This review focuses on insect defensins, a large group of inducible antibacterial peptides that are present both in ancient and recent insect orders. This immune response of insects shares many of the characteristics of the mammalian acute phase response. Insects have long been known to be particularly resistant to bacterial infection. Approximately a century ago, Kowalevsky ~ in St Petersburg, and Cu~not 2 in Nancy, provided the first detailed reports on antibacterial &fence reactions in insects, and highlighted the role of phagocytosis and capsule formation by blood cells. After the first World War, Glaser -~ in Munich, Paillot 4 in Lyon and Metalnikow s at the Pasteur Institute of Paris, conclusively showed that insect host defence was not limited to cellular reactions, but that injections of bacterial cultures induced the appearance in the blood of bacteriolytic substances. Surprisingly, these pioneering studies were followed by a long period of stagnation in the field, and it was not until 1980 that any of the induced antibacterial substances were characterized, apart from the ubiquitous lysozyme 6. Boman and his associates in Stockholm, in 1981, succeeded in identifying the major inducible bactericidal factor from the blood of bacteria-challenged diapausing pupae of a silkworm, Hyalophora cecropia, and named this novel peptide cecropin -'~. In the decade that followed, a small number of laboratories isolated seven antibacterial peptides or peptide families in insect species that belonged to the phylogenetically recent orders of the Lepidoptera, Diptera, Hymenoptera and Coleoptera. The structural characterization of these molecules revealed that they were novel, mostly cationic, small-sized peptides with a broad spectrum of activity against Gram-positive and/or Gram-negative bacteria. For convenience, these peptides or polypeptides can be categorized into four groups: (1) the cecropins are 4 kDa cationic peptides which form two amphipathic (z-helices; they are active against Gram-positive and

Gram-negative bacteria and are believed to form channels in the bacterial membranes~; (2) three as yet incompletely characterized, small-sized (2-4 kDa) cationic proline-rich peptides - the apidaecins 9, abaecin ~° and drosocin (P. Bulet, J.L. Dimarcq et al., unpublished) - which are primarily bactericidal against Gram-negative bacteria; (3) several distinct polypeptides ranging in size from 8 to 27 kDa, mostly cationic and frequently rich in glycine residues: the attacins ~, sarcotoxins II ~-', diptericins t~ and coleoptericin ~4. These polypeptides affect Gram-negative bacteria; they are either bactericidal or bacteriostatic. All members of these three groups of molecules are devoid of cysteines. (4) In sharp contrast, the fourth group, the insect defensins ~5,~ have six cysteines engaged in three intramolecular disulfide bridges. These molecules prb marily affect Gram-positive bacteria and appear to be the most widespread group of inducible antibacterial peptides so far characterized in insects: they are present in Diptera, Coleoptera, Hymenoptera, Hemiptera and Odonata. This review will focus on these molecules. Insect defensins: structure

The term 'insect defensins' was initially coined by this laboratory for two 4 kDa peptides isolated from blood of larvae of a large dipteran species, the fleshfly Phormia terranovae, in which they appear a few hours after a bacterial challenge ~s. As these two molecules (isoforms A and B, which differ by a single amino acid substitution) show sequence similarities with a group "This article is dedicated to the late Dr Jean-Pierre Lecocq, Director of Transg~ne, who enthusiastically supported studies on insect defcnsins.

O 1992, Ftscvier Science" Publisl~ers l.~d, UK.

lmmunology Today

411

V,,t ,3

lO i9 e

REVIEW

1 (a) Phormia

A ]Phormia B ~Sarcophaga

(b)lZophobas Z B

( c ) ~

I0

20

I A T C D L L I . . . . ISGTGIN"SAC IATCDLLI

-

"CL

"

C"I

-ISGTGINHSACAA~CLLRI-IGNRGGYCNI

R

~A T C D L LJ-

-IS G T G I N H S A C A A H C L L RI-|G N R G G Y C ~ G

IAT C ~ L L I -

-IsI-#--L"-'fi--~N

=l

c F~C~VlLIG F G ~ P - ~

H~A

C AA H C L S~-IGmR

Fq

"

40

30

RI-I

G G Y C~G

=1 L -IGI L

s

r E I AIG TIK L ~ S A~CIG~H C~A L -IGIR TIG G Y C ~ S . . . . . . . D Q M Q ~ H R H L ~ Q T I T ~ R S l G G Y C ~ G

- V I G i v e v c R NI -IKIGI v C v C R NI - I K I A I V C V C R NI

-Iq

lv

-IKISl v c

vc

R/

-,K,s,v c v c R, LW , R

Fig. 1. Amino acid sequence of insect defensins. To date, seven defensins have been fully sequenced in species belonging to three insect orders: (a) Diptera, (b) Coleoptera and (c) Odonata. They are also present in Hymenoptera and Hemiptera (incomplete data, not presented). Bars indicate gaps to optimize sequence alignments. Identicalamino acids are boxed. Not surprisingly, the sequence homology among the four dipteran defensins presented in Fig. 1 is very high, whereas the coleopteran and dipteran defensins show only 60% homology. The defensin of Aeschna, a member of the order of the Paleoptera, which appeared 100 million years before the recent insect groups, has a sequence homology with dipteran defensins of less than 30%, but the conserved amino acid residues reside in key positions. Detailed nuclear magnetic resonance (NMR) studies of Phormia defensin in water suggest that this antibacterial peptide consists of three distinct domainsn, as illustrated in the model of Fig. 3: first an amino-terminal loop formed by residues 1 to 13; secondly, an 0thelix consisting of residues 14 to 24; and third, an antiparallel [5-sheet, comprising residues 27 to 40, with a turn involving residues 32 to 34. The amino-terminal loop is linked by one of the disulfide bridges to the first strand of the 13-sheet, whereas the s-helix is stabilized via the two other disulfide bridges to the second strand of the 13-sheet. As far as can be judged from the studies published on sapecin 23, the global folding of this molecule in methanol does not differ significantly from that of defensin A in water. The three-dimensional structure of the other insect defensins has not yet been investigated. Assuming that they are similar to those of Phormia and Sarcophaga, the analysis of the primary sequences presented in Fig. 1 would suggest that the strongest variability between the dipteran, coleopteran and paleopteran homologues resides within the amino-terminal loop, which is longest in Zophobas (14 residues) and shortest in Aeschna (4 residues). In contrast, the sizes of the putative s-helices and antiparallel ]3-sheets appear relatively

of bactericidal peptides from mammalian phagocytes, named defensins by Lehrer and associates 17'18, they were given the name of insect defensins. Mammalian defensins are variably cationic, relatively arginine rich, non-glycosylated peptides of 29-34 amino acid residues. To date, the primary amino acid sequences of 15 mammalian defensins have been established. They all contain a characteristic cysteine motif consisting of six cysteines engaged in three intramolecular disulfide bridges. Mammalian defensins are predominantly present in phagocytes and account for -30% of the total protein in azurophil granules of human neutrophil leucocytes. They participate in the non-oxidative microbicidal mechanisms through membrane permeabilization of ingested microorganisms. In contrast to insect defensins, however, they are not secreted into the blood and are not considered to be acute phase reactants. Independently of the studies on inducible antibacterial peptides of the fleshfly Phormia, the group of Natori in Tokyo showed that an embryonic cell line, NIH-Sape-4, derived from another large dipteran species Sarcophaga peregrina, secreted an insect defensin isoform, differing by one single substitution; they named this isoform sapecin 16. In addition to being constitutively expressed in this cell line, the synthesis of this defensin isoform is induced in larvae of Sarcophaga by injection of bacteria. To date, seven members of the insect defensin family have been characterized (Fig. 1). They are moderately cationic (pI 8-8.5), non-glycosylated peptides, composed of 38 to 43 amino acid residues, and all contain a characteristic motif of six cysteines engaged in three intramolecular disulfide bridges 19-21(Fig. 2).

C1

Phormia

A

Rabbit

NP-I

02

VV

C

0 3

R

04

R

F

R

4

I' 01

C2

05

I R

06

I H P L C C R R 24

I

'

C3

C4

I 0 5 06

Fig. 2. Sequence similarities between insect defensin from Phormiaand the mammalian defensin, rabbit neutrophilic peptide 1 (NP-1). Identical residues are boxed; a one residue gap is introduced for optimal alignment (bar). Both molecules contain six cysteines (C~ to C6) linked via three disulfide bridges.

Immunology Today

412

rot. 13 5,70. 10 1992

REVIEW 57

well conserved. In all defensins, the (x-helix has an amphipathic character with a hydrophobic surface (note, for example, the three Ala and two Leu residues in Phormia) and a charged surface (for example, Hisl 9 and Arg23 in Phormia) although the latter is less marked in Aeschna. As indicated above, in Phormia defensin, the c~-helix is linked to the ~-sheet by two disulfide bridges, the positions of which are illustrated in Fig. 1 and Fig. 3. The cysteine pairs involved are separated by three residues on the ~-helix (one turn) and one residue on the ~-sheet. This arrangement, CXXXC/CXC (where C stands for Cys and X for any residue) is known as a cysteine-stabilized (x-helix motif and is present in all seven defensins. It was originally described in endothelins24 and is present in toxins from various venoms (see below).

i

L

D4

\

jl~

°=

H,3

I

N,,

\o,,,

\

38"~

Structural homologies and nonhomologies

These structural data indicate that insect defensins differ markedly from mammalian defensins. The latter are dominated by a three-stranded antiparallel ]3-sheet stabilized by three disulfide bridges, but contain no ~zhelix2S"L The initial idea ~ that mammalian and insect defensins are homologous peptides derived from an Fig. 3. Spatial arrangement of the backbone (Ca atoms) of Phormia ancestral bactericidal molecule was based on the sub- defensm A as deduced from NMR studies. The disulfide bridges are stantial sequence similarity between residue fragment symbolized by dotted lines. 15-34 in insect defensin from Phormia and residue fragment 4-24 in a rabbit defensin: 10 of 21 residues are identical and several replacements are conservative and Phormia defensin show many additional simi(Fig. 2). However, the structural information which has larities, while Aeschna defensin and scorpion charybdobeen gained on these molecules over the last three years toxin also share certain additional features. The does not substantiate the initial proposal. For one, recent elucidation of the tertiary structure of charybdomost of the residues in rabbit defensin which are ident- toxin 2'~ has revealed significant structural similarities ical to those of insect defensin from Phormia are not between this scorpion toxin and defensins: in particuconserved among the 15 mammalian defensins that lar, both types of molecule have a ]3-sheet linked, via have now been reported ~, and the same remark is valid two disulfide bridges, to a well-defined c~-helix and, by for the seven insect defensins which have since been the third disulfide, to the amino-terminal fragment. In sequenced. More importantly, the connectivities of the light of these data, it seems plausible that insect three intramolecular disulfide bonds are totally differ- defensins that act on prokaryotic membranes and ent in the two families of molecules ~s'lg, as are their charybdotoxin (a K+ channel blocker that acts on eukaryotic membranes), derive from a common ancesrespective three-dimensional structures 22'25'2~. The name of insect defensins has now entered gen- tral gene (see also Refs 22 and 28) which is different eral literature and we believe that it should continue to from the ancestor of mammalian defensins. be used. However, it should be appreciated that, while mammalian and insect defensins participate in the Insect defensins: synthesis The synthesis of insect defensins, like that of most of antibacterial defence, sharing similarities in size, charge and predominant activity against Gram-positive bac- the other immune effector molecules of insects, takes place in the fat body and in some blood cells. In teria, they are not structural homologues. Interestingly, insect defensins share structural simi- Phormia, an insect which has been studied in detail, all larities with two types of peptides that do not partici- the cells of the fat body participate in this response. pate in the inducible antibacterial response. These are This tissue is a functional equivalent of the vertebrate royalisin 2-, a 53 residue antibacterial peptide consti- liver and extends throughout all parts of the body. In tutively present in the royal jelly of honey bees, and addition, one blood cell type, the thrombocytoids, also charybdotoxin 's, a 37 residue K+ channel blocker syn- produce defensin. These cells are, to a certain degree, thesized by scorpion venom glands. Figure 4 compares morphologically and functionally analogous to mamthe primary sequences of these two peptides with malian megakaryocytes and are actively involved in Phormia and Aeschna defensins. The four molecules agglutinating bacteria and sealing off wounds of the share a superimposable cysteine array (allowing for an integument 2~. The response of the insect to a bacterial challenge is amino-terminal gap in charybdotoxin and Aeschna defensin) and two conserved residues (Gly28 and rapid: within 15 rain following an injection of bacteria, Lys33; numbering for Phormia defensin) which are also defensin transcripts are apparent in the cells of the fat present in the other defensins. Interestingly, royalisin body. In Phormia, the response reaches its maximum at

Immunology Today

413

v,,/13 No. 10 1~)92

REVIEW

Phormia defensin

Royalisln Charybdotoxin

z

•

Aeschna defensin

v

+ +'

P L ....... .

.

.

.

.

.

.

.

,+

-

o + m