A novel insect defensin from the ant Formica rufa

Biochbnie (1998) 80, 343-346 © Soci6t6 fi'angaise de biochimie et biologic molOculah-e / Flscvier, Paris

Short cmmnunication

A hove| insect defensin from the ant Formica rufa S Taguch] ~, P B u l e t b, J A H o f f m a n n b aDcpartment t~f Biological Science amt Technolog3; Science University of Tokyo, 2641 Yamazaki. Noda-sld. Chiba 278. Japan: binstitut de Biologic Moleculaire et Ceihdaire (IBMC), UPR 9022, CNRS, 'Response hmmmitaire et Developpement chez les lnsectes" 15, rue Rene-Descartes, 67(184 Strasbourg cedex, France Received 13 November 1997: accepted 31 January ! 998) Summary - - By combination of size exclusion and reversed-phase chronmlogral~hy+ we have isolated a novel member of insect defensin+type antimicrobiai peptides from the entire bodies of bacteria-challenged Formica tufa ¢hymenoptera, tormicidae). The molecular mass of the purified peptide was estimated to be 4120.42 by matrix-assisted laser desorption/ionization+time of flight/mass spectromctry. Sequence analysis revealed that this peptide consisted of 40 amino acid residues with six cysteines engaged in the formation of three intramolecular disulfide bridges. This peptide is unique among the arthropod defensins in terms of the presence of asparatic acid and alanine at posflion 33 and as C-terminal residue, respectively. In addition, this novel defensin from Formwa rtqa has the particularity to have no C+tennmal extension in contrast to those reported for other hymenoptera defensins. (© Soci6t6 franqaise de biochimie et biologic mol6culaire / Elsevier. ParisL antimicrobial peptide I Formica tufa I insect defensin I sequence a|ignmem

Introduction Inducible production of antimicrobiai peptides has been rec° ognized as an ancient ettkaryotic defense strategy ('innate immunity': reviewed in II 1), which inhibits the cell growth of microbes in bacteriostatistical or bactericidal nmnners. To date, several types of antinficrobial peptides have been iso!ated, and their structures have been fully or pataiaily characterized 111, In particttlar, 21) members of the insect defensin superfimfily have been characterized. They are known It bc small (4~6 kDa) and cationic antimicrobia! peptides with a conserved framework of six disnlphide°linked cysteines 121. Insect del'ensins have been shown to disrupt tire permeability of the cytoplasmic membrane of the Gram+positive bacterium, Micn+coccus luteus, resulting from the formation of voltagedependent ion channels in the cytoplasmic membrane 131. We are interested in the phylogenetic aspects of the immune response in insects and me widespread family of insect defensin would be a good biochemical criterion for such a study. In the present communication, we describe the isolation and characterization from hymenoptera Formica n
Materials and methods Ants, Formica rufa, were collected by S Chemysh, St Petersburg State University, in the field of St Petersburg. Insects were individually pricked with a needle previously dipped into a combined pellet of Micrococcus iuteus as Gram-positive bacteria and Escherichia colt 1106 as Gram-negative bacteria by the procedure established for Dn~-

sophih+ 141.4.7 g of bacteria+challenged ants were kept at r+~+mten+pemtum for 24 h and frozen in liquid nitrogen. The procedm+eused for the extraction of the amimicrobial substances wits derived fl'om the methodology established for the characterization of antimicrobial pep+ tides from small-sized insects 15]. Briefly. the entire ants +verc ground in liquid nitrogen, quickly supplied to acidification witl! 0.t)5++ trifluoroacetic acid containing aprotinm as protease inhibitor and phe~ nylthiomca as inhibitor of melanization for 30 rain in an ige++coldwater bath, centrililged at 4+C dnrmg 30 rain ill 10 ()(}{)rpm, and the .~ttl~'l'+ natant was prepurified by solid+phase extraction on Scp-Pak C I 8 cat'tritlge~ (Waters). Stcpwise elutitm wa~ carried ~mt with Ih|+cc proportion+s of acetonitrilc t5%, 40%, mid 80')L) i|+ +t¢idificd ++~,'alcr

(0.05% trifluoroaceti¢ acid, TFA). Antimicmbiai activity was moni+ tored during the purification process using a liquid+growthinhibition assay using Gram+positivebacteria (M htteus LGrmn°negativebacteria (E colt 363). and a fungus (N crassa) as described previously 141. Marked antimicrobial activities against M luteus were recovered in the 40% acetonitrile fraction. This fraction was subjected to size+exclusion chromatography columns tandemly connected (Ultraspherogel SEC 20(X) and 3000 columns, 7.5 × 300 mm; Beckmmt) and eluted under isocratic conditions with 30% acetonitrile in 0.05% TFA solution at a flow rate ofO.5 mL hnin. Anti M lutetts activity was found to be present in over ten fractions under our conditions. The final purification of the IYaction containing the activity against M luteus included three suc+ cessive reversed-phase chrommographies on an Aquapore RP300 C8 column (250 × 4.6 mm; Brownlee) using adaptive linear gradients of acetonitrile in acidified water: i) 2-4"+0%over 90 rain at 0.8 mlJmin; it) a diphasic gradient 2-24% over !0 min and 24-3(1% over 5(1 rain at 0.5 nfl.,/min; iii) tbr the final step a lineal"diphasic gradient 2+23% in 10 rain followed by a 60-rain gradient of 23+28% at 0.5 mL/min. Peptide purity was ascertained by capillary zone electrophorcsis using a model 270A-HT system (Applied Biosystems, division of Perkin Elmer) as described previously 15, 61. For reduction and

344 alkylation, an aliquot of the purified peptide was dissolved in 40 pL of 0.5 M Tris-HCI, 2 mM EDTA (pH 7.5). containing 6 M guanidine hydrochloride to which 2 laLof 2.2 M dithiothreitol were added. The sample was flushed with nitrogen and incubated for i h at 45~C in the dark. 2 pL of freshly distilled 4-vinylpyridine was and the .~unple was incubated for an additional tO min at 45°C in the dark and under nitrogen atmosphere. The S-pyridylet h y l ~ peptide was purified: by reversed-phase HPLC prior to sequencing by Edman degradation. Automated Edman degradation of the native and the S-pyridylathylated peptide and detection of phenylthiohydantoin derivatives were performed on a pulse liquid automatic sequenator (model 473A. Applied Biosystems, division of Perkin Elmer), Matrix-assisted laser desorptiontionization-time of flighl/mass spectrometry (MALDI-TOF-MS)was performed on a Broker biflex (Bruker. Bremen. Germany) laser desorption time of flight mass spectrometer operating in a positive linear mode. Briefly. the purified peptide samples (0.5 laL for each one) were deposited on a thin layer of ct-cyano-4-hydroxycinnamicacid matrix (7% in 50% acetonitrile containing 0.05% CF~COzH)made by fast evaporation of a saturated solution in acetone, i laL of this peptide matrix solution was transferred onto the stainless steel target. The droplet was allowed to dry under gentle vacuum before introduction into the mass spectrometer. Mass spectra were obtained by averaging 80 shots and externally calibrated with a mixture of three standard peptides (angiotensin Ii, ACTH 18-39. and bovine insulin with [M'HI* ion at m/z of 1047.2, 2466.1. and 5734.6. respectively).

Results and discussion Figure I A shows the final HPLC profile of active fraction which strongly exlfibits antimicrobial activity against M luo teas, The present study focuses on the most anti-M hat,tqs frac+ tion (cross-hatching zone in fig I A), From the profile of capillmy zone electrophoresis (fig I B), an active fraction was ibund to be pure enough ibr ix~ptide sequencing and mass spectrometry analysis, The final yield of an active peptide was spt~t~met~¢ally estl[tlatcd to b¢ 0,88 ~g, The purthed ant0oM haeus pept,de was subjected to chemical ¢hm'acterization. Mass speetrometo, measurement by MALDI-TOF yielded a mass of4119,4 Da (fig IC), The fact thin only one mass was ob~rved provides an additional demonstration of the purity of the molecule, For facilitated ~'queneing and assignment of cysteine residues prior to sequencing by automatic Edman degradation, the native peptide (starting material, 182 pmol) was submitt~ to reduction and alkylation with 4-vinyipyridine, After isolation by teversed,pha~ HPLC, the S-pyridylethylated peptide was subjected first to mass spectrometry measuren~nt and s~ond to Edman degradation, The following 40 amino acid ~luence was obtained: FTCDLLSGAGVDHSACAAHCILRGKTG* GRCNSDRVCVCRA, The mass measured on the S-pyridylethylated peptide (4753,3 Do, fig I D) in excess of634 Da coml~lred to the mass measured on the native peptide (4119,4 Do) and of 640 Da compared to the calculated mass from the amino acid ~xluence

obtained by Edman degradation (4125,4 Da), clearly indicated that this anti-M luteus peptide contains the six cysteines engaged in three intramolecular disulfide bridges ,and has no additional modification such as glycosylation as observed |br other antimicrobial peptides from insects [1, 71. The sequence of 40 residues obtained shows a high degree of similarity with arthropod defensins, a large family ofcysteine-rich antimicrobial cationic peptides. The positions of the cysteines in this superfamily are highly conserved, and their array is identical with that of the mlt peptide (fig 2) including the CS a ~ motif [81. Over 20 defensins have now been isolated and fully or partially characterized from various arthropod sources Ill. Among them, a refined three-dimensional solution structure was first established for insect defensin A secreted by Phor. mia terranovae larvae [81. Insect defensin A consists of three distinct domains: a N-terminal flexible loop (residues 4-14), a central amphipathic a-helix (residues 15-23), and a C-terminal double-stranded antiparallel 13-sheet (residues 27-31 and 35-39) with an unusual a-turn consisting of three residues (residues 32-34). Figure 2 shows the sequence alignment of the insect defensin family and regions for secondary structures established in the defensin isoform A from P terranovae. A good alignment of the Formica defensin with Phonnia defensin A strongly suggests that Formica def~nsin would form almost the same tertiary structural framework as Phenicia defensin A. The presence of asparatic acid residue at the 33th position which corresponds to an unusual ~-turn is very unique among del~nsins. It is of interest to investigate the effect of the negatively ch~wged residue in the turn structure on kinetics of action, the level of activity and antimicrobial specificity. A defensin possessing an alanine as C4emdnal t~sidue is also unique among the atthropt~ds. Variations in a C4emdnal e.~tolsion ranging from 2 to 4 t~sidue p~cetled by the last cysleiue i~nlahled to I~ den~onslratcd with resider to SlnlCtUl'eoac+ tivity relationship of this lype of anlimicrohial peptide. Front the sequence sinlilarity observed, I.~mnica dcfensin is closest to the royalisin from Apis mell(l~'ra 19!, and to the defensins from Apis mel!ih'ra I I0l and Bombus I~+scuormn I I il, two other hymenopteran insects. However, Fornffca defensin is not C-terminally amidated and in addition the Cterminal is much smaller with two residues following the last cysteine compared to 11 residues for the Apis and Bombus defensins, This is in gotxl agreement with most species' phylogenetie relations on the order level already constructed 1121. in plate growth-inhibition assay 141, no colony forming unit was observed when plating M luteus (OD~,~,~= 0.001 in poor broth medium) alter an overnight incubation with 0,69 pM of native purified peptide, indicating that the molecule has a bactericidal effect against M haeus, as well as the other members of insect defensins isolated so far. We have also lk~und potent anti Gram-negative bacteria activities in the crude extract of Formica tufa. Thus, in terms of phylogeny, it is of interest to investigate the relationship between the corresponding activity in Formica nq'a and proline-rich peptides, apidaecins Jl31 and hymenoptaecin 1141, which were isolated from Apis mell~h,ra as the anti-

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Fill ,2. Sequence alignment of insect defensins including the novel dcfensin from b);nnicu ruJ& Abbreviations of dcfcnsin producers are as follows, t~om top to bottom: P. t. A. P tcrranovae (isoform A); P. t. B. P terranovue (isoform B); S. p. A. S peregrim~ (sapecin A ); S. p. C, S petvgrimt (sapecin C): B. t.. g tenax; D. m.. D mehmog,ster: L. s.. L stigma; A. m.. A mell(h,r, (royalisin); F. r., Fro'mica rulit; Z. a. A, Z atmtus (isolbrm A); Z. a. B, Z ammos (isofom~ B l: T. m.. Tmoliu; (Tenecin 1); P. a.. P,pten¢s; A. e., A cyanea; L. q.. L quhtquestriatus; S. p, B, S peregrina. Residue numbering is that for i~ffonn A of defensin fronl R terranovae. Tenus. loop.structure, c~-helix, and ~-strantl, are secondary structures established in the defensin A from P terrmmvae. Gaps (~) are introduced to maximize the alignnaent.

microbial peptides exhibiting bacteriostatistical activity ag,ainsl Gram-negalive bacteria. These members of insect defensins can be recognized as 'natural mutants', For isolation of evolved defensins with increased antimicrobial activity, uatura! screening would he powcrflll, In contrast, an in vivo monitoring assay system established by Taguchi et al 115, 161 is an 'artificial ev()!~ alien' approach to perform structure~activity reh~tionship analysis and to gain ilupl'oved antiulicrobial peptide. Both app~'oaches are complementary to each other.

Acknowledgments W e ar~ Vela 3 indebt~l to DI: S Chenlysh for his contribution to tile challenging of bacteria to ants and C Hetru tbr valuable advice on phylogenyof insect dcfensins. We also thank Drs M Charlet. SUtten-. wailer and M Scl~neiderfor thdr excdlent analytical exl~rimenls.

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stn~ctur~s. J Bhd Chem 269, 261 ()7-2(~ ] 15 I I Rees J, Monialte M, Bulet P 11997) Novel antibacterial tx~'plidesisoo luted l'rom a European bumblebee, Ilombux I)aScuorum f t*i~~vepop.

lera, al~fidea), bz.s'ect Iliochem Mol Biol 27, 413-422 12 Chcrnysh S, Cociancich S, Briand ,I-E Hetru C, Bulet P (,,)t}(~) The inducible antibacterial peptidc,,, of the hcmiptcrml insect l)~,':mrvr, I,'asina: identification of a unique linnily of prtdiue-rich pept!des and ol'a novel insect del'cn.sin. J b~sect Phvsio142, 81-89 13 Castecls P, Ampe C, Jacobs E Vaeck t <, Tcmpst !)11989) Apitlaeci~ts: antibacterial i~ptide,,, from houeybces. E M R O d 8, 2387.-2391 14 ('astecls i~ Anll~- l~ Jacobx I~ Tcnlpst P [ 19931 Fuuctiunal aad chemical characteri~alion of hymenoptaecin, an antibacterial polyi~,pti(le thai is ilffection-inducible in tile Iioncyhee (Ai,ls mellili'ra). J Biol ('hem 268, 7044-7054 15 Taguchi S, Nakagawa K, Maenu M, MoluOSe H (1994) !11vivo monitoring system for strudurc-function relationship analysis of the antibacterial peptide Apidaecin, Appl Era.iron Micmbhd 60. 356(v-3572 16 'Paguchi S, Ozaki A, Nakagawa K, Momose H (1996) Functional mapping of amino acid residnes repunsible for antibacterial action of apidaecin, Appl Environ Micmbio162, 4652-4()55