Antimicrobial Activity Spectrum, cDNA Cloning, and mRNA Expression of a Newly Isolated Member of the Cecropin Family from the Mosquito Vector Aedes aegypti

Abstract

An antimicrobial peptide belonging to the cecropin family was isolated from the hemolymph of bacteria-challenged adult Aedes aegypti. This new peptide, named cecropin A, was purified to homogeneity and fully characterized after cDNA cloning. The 34-residue A. aegypti cecropin A is different from the majority of reported insect cecropins in that it is devoid of a tryptophan residue and C-terminal amidation. The importance of these two structural features on the activity spectrum was investigated using a chemically synthesized peptide. A comparison of the antimicrobial activity spectrum of A. aegypti and Drosophilacecropin A showed a lower activity for the mosquito molecule. A. aegypti cecropin mRNA expression was not detected by Northern blot or reverse transcription-polymerase chain reaction analysis in any immature stage of the mosquito, nor in naı̈ve adults, but it was observed in challenged adults 6 h after bacteria inoculation, and it continued over 7–10 days. An antimicrobial peptide belonging to the cecropin family was isolated from the hemolymph of bacteria-challenged adult Aedes aegypti. This new peptide, named cecropin A, was purified to homogeneity and fully characterized after cDNA cloning. The 34-residue A. aegypti cecropin A is different from the majority of reported insect cecropins in that it is devoid of a tryptophan residue and C-terminal amidation. The importance of these two structural features on the activity spectrum was investigated using a chemically synthesized peptide. A comparison of the antimicrobial activity spectrum of A. aegypti and Drosophilacecropin A showed a lower activity for the mosquito molecule. A. aegypti cecropin mRNA expression was not detected by Northern blot or reverse transcription-polymerase chain reaction analysis in any immature stage of the mosquito, nor in naı̈ve adults, but it was observed in challenged adults 6 h after bacteria inoculation, and it continued over 7–10 days. One factor contributing to the enormous success that insects have exhibited through evolutionary time is their ability to mount a rapid and effective response against invading pathogens. This response includes the rapid production of potent antimicrobial peptides effective against bacteria and fungi; transcriptional activity may be detected within minutes of a stimulus, resulting in 1–100 μm concentrations of specific peptides in the hemolymph within 24 h (1Hetru C. Hoffmann D. Bulet P. Brey P.T. Hultmark D. Molecular Mechanisms of Immune Responses in Insects. Chapman & Hall, London1998: 40-66Google Scholar). Since the isolation of cecropin fromHyalophora cecropia by Boman and co-workers (2Steiner H. Hultmark D. Engström A. Bennich H. Boman H.G. Nature. 1981; 292: 246-248Crossref PubMed Scopus (1093) Google Scholar), approximately 170 immune peptides have been characterized from insects and other invertebrates (for a recent review, see Ref. 1Hetru C. Hoffmann D. Bulet P. Brey P.T. Hultmark D. Molecular Mechanisms of Immune Responses in Insects. Chapman & Hall, London1998: 40-66Google Scholar). In insects, these molecules are rapidly synthesized, easily stored, and promptly available after infections. These features, together with the low metabolic cost for the insect and a low specificity (see review in Ref.3Shai Y. Brey P.T. Hultmark D. Molecular Mechanisms of Immune Responses in Insects. Chapman & Hall, London1998: 111-134Google Scholar), make antimicrobial peptides a very versatile component of the insect immune response. In mosquitoes, which serve as obligate vectors for serious debilitating human diseases such as malaria, lymphatic filariasis, and numerous arboviruses, the predominant immune peptide is a member of the insect defensin family (4Lowenberger C. Bulet P. Charlet M. Hetru C. Hodgeman B. Christensen B.M. Hoffmann J.A. Insect Biochem. Mol. Biol. 1995; 25: 867-873Crossref PubMed Scopus (127) Google Scholar, 5Chalk R. Albuquerque C.M.R. Ham P.J. Townson H. Proc. R. Soc. Lond. B. Biol. Sci. 1995; 261: 217-221Crossref PubMed Scopus (36) Google Scholar, 6Cho W.L. Fu Y.C. Chen C.C. Ho C.M. Insect Biochem. Mol. Biol. 1996; 26: 395-402Crossref PubMed Scopus (47) Google Scholar, 7Richman A.M. Bulet P. Hetru C. Barillas-Mury C. Hoffmann J.A. Kafatos F.C. Insect Mol. Biol. 1996; 5: 203-210Crossref PubMed Scopus (106) Google Scholar) Although it has been suggested that inducible antimicrobial peptides have no impact on eukaryotic parasites (8Cociancich S. Bulet P. Hetru C. Hoffmann J.A. Parasitol. Today. 1994; 10: 132-139Abstract Full Text PDF PubMed Scopus (181) Google Scholar), several studies suggest otherwise. Reports have implicated cecropins or synthetic cecropin derivatives (9Gwadz R.W. Kaslow D. Lee J.Y. Maloy W.L. Zasloff M. Miller L.H. Infect. Immun. 1989; 57: 2628-2633Crossref PubMed Google Scholar, 10Chalk R. Townson H. Ham P.J. Exp. Parasitol. 1995; 80: 401-406Crossref PubMed Scopus (50) Google Scholar, 11Rodriguez M.C. Zamudio F. Torres J.A. Gonzalez-Ceron L. Possani L.D. Rodriguez M.H. Exp. Parasitol. 1995; 80: 596-604Crossref PubMed Scopus (71) Google Scholar, 12Jaynes J.M. Burton C.A. Barr S.B. Jeffers G.W. Julian G.R. White K.L. Enright F.M. Klei T.R. Laine R.A. FASEB J. 1988; 2: 2878-2883Crossref PubMed Scopus (88) Google Scholar), insect defensins (13Shahabuddin M. Fields I. Bulet P. Hoffmann J.A. Miller L.H. Exp. Parasitol. 1998; 89: 103-112Crossref PubMed Scopus (134) Google Scholar), or a general immune activation process (14Lowenberger C. Ferdig M.T. Bulet P. Khalili S. Hoffmann J.A. Christensen B.M. Exp. Parasitol. 1996; 83: 191-201Crossref PubMed Scopus (55) Google Scholar, 15Lowenberger C.A. Kamal S. Chiles J. Paskewitz S. Bulet P. Hoffmann J.A. Christensen B.M. Exp. Parasitol. 1999; 91: 59-69Crossref PubMed Scopus (115) Google Scholar) in reducing the development of parasites in mosquitoes. Because of the potential role cecropins might play in the anti-parasite responses cited above and the recent reports of synthesized cecropin-like molecules that kill Plasmodium sp. (16Boisbouvier J. Prochnicka-Chalufour A. Nieto A.R. Torres J.A. Nanard N. Rodriguez M.H. Possani L.D. Delepierre M. Eur. J. Biochem. 1998; 257: 263-273Crossref PubMed Scopus (23) Google Scholar, 17Possani L.D. Zurita M. Delepierre M. Hernandez F.H. Rodriguez M.H. Toxicon. 1998; 36: 1683-1692Crossref PubMed Scopus (23) Google Scholar), we have attempted to isolate and characterize cecropins from mosquitoes. Cecropins are a family of inducible antibacterial peptides of 4 kDa, devoid of cysteine residues, constituted by two α-helices linked by a short hinge, with a strongly basic N-terminal region and a long hydrophobic domain in the C-terminal half. Cecropins integrate into the bacterial cell membrane and cause lysis and cell death by a loss of cations (18Durell S.R. Raghunathan G. Guy H.R. Biophys. J. 1992; 63: 1623-1631Abstract Full Text PDF PubMed Scopus (83) Google Scholar). Cecropins have been characterized from Lepidoptera and Diptera (1Hetru C. Hoffmann D. Bulet P. Brey P.T. Hultmark D. Molecular Mechanisms of Immune Responses in Insects. Chapman & Hall, London1998: 40-66Google Scholar, 19Boman H.G. Hoffmann J.A. Janeway Jr., C.A. Natori S. Phylogenetic Perpectives in Immunity: The Insects Host Defense. Landes Company, Austin, TX1994: 3-17Google Scholar), but in mosquitoes, there has been only limited information (20Knapp T. Crampton J. Trans. R. Soc. Trop. Med. Hyg. 1990; 84: 459Google Scholar, 21Hernandez V.P. Gerenday A. Fallon A.M. Am. J. Trop. Med. Hyg. 1994; 50: 440-447Crossref PubMed Scopus (18) Google Scholar) until recently, when Sun et al.(22Sun D. Eccleston E.D. Fallon A.M. Biochem. Biophys. Res. Commun. 1998; 249: 410-415Crossref PubMed Scopus (35) Google Scholar) reported the purification of a 35-amino acid cecropin from anAedes albopictus cell line. We report herein the identification and full characterization of a new member of the cecropin family isolated from the hemolymph of immune-activatedAedes aegypti. A. aegypti (Liverpool strain) mosquitoes were reared as described previously (23Beerntsen B.T. Christensen B.M. Exp. Parasitol. 1990; 71: 406-414Crossref PubMed Scopus (38) Google Scholar). Adult female mosquitoes were immunized (4Lowenberger C. Bulet P. Charlet M. Hetru C. Hodgeman B. Christensen B.M. Hoffmann J.A. Insect Biochem. Mol. Biol. 1995; 25: 867-873Crossref PubMed Scopus (127) Google Scholar), and 24 h later, the hemolymph was collected from individual mosquitoes by perfusing the hemocoel with Aedes saline as described previously (23Beerntsen B.T. Christensen B.M. Exp. Parasitol. 1990; 71: 406-414Crossref PubMed Scopus (38) Google Scholar). The hemolymph from about 2600 bacteria-challenged mosquitoes or 2000 naı̈ve mosquitoes was centrifuged at 5000 × g for 5 min at 4 °C, and the supernatant was frozen until use. Antibacterial peptides were purified from acidified cell-free hemolymph. Acidification was performed with ammonium acetate buffer at pH 3.5 (final concentration, 25 mm), supplemented with aprotinin (Sigma) as a protease inhibitor (final concentration, 1.5 μm) and phenylthiourea (Prolabo) as a melanization inhibitor (final concentration, 20 μm). After extraction under gentle stirring for 30 min in an ice-cold water bath, the sample was centrifuged at 10,000 × g for 20 min at 4 °C. The supernatant then was loaded onto a 12-cc Sep-Pak Vac C18 cartridge (WatersTM) equilibrated with acidified water (0.05% trifluoroacetic acid). Stepwise elution was performed with 2, 40, and 80% acetonitrile in acidified water. The 40% Sep-Pak fraction was analyzed by reversed-phase HPLC 1The abbreviations HPLChigh performance liquid chromatographyRTreverse transcriptionPCRpolymerase chain reactionMICminimal inhibitory concentrationMALDI-TOF MSmatrix-assisted laser desorption time-of-flight mass spectrometry on an Aquapore RP 300 C8 column (250 × 7 mm; BrownleeTM) using a linear gradient of 2–60% acetonitrile in acidified water for 120 min (flow rate 1.3 ml/min). The antibacterial activity of the collected fractions was detected by liquid growth inhibition assay againstMicrococcus luteus A270 and Escherichia coliSBS363, as described previously (4Lowenberger C. Bulet P. Charlet M. Hetru C. Hodgeman B. Christensen B.M. Hoffmann J.A. Insect Biochem. Mol. Biol. 1995; 25: 867-873Crossref PubMed Scopus (127) Google Scholar). The active fractions were further purified on a Delta-Pak HPIC18 column (150 × 2 mm; WatersTM) with a linear biphasic gradient of acetonitrile in acidified water from 2 to 22% over 10 min and from 22 to 32% over 50 min at a flow rate of 0.2 ml/min at 35 °C. The first step of HPLC purification was performed with a Beckman Gold HPLC system equipped with a photodiode-array detector Beckman 168. The subsequent steps were carried out using a Waters HPLC system (Waters, model 626) attached to a tunable absorbance detector (Waters, model 486) and equipped with an internal oven. For all HPLC purification steps, the effluent was monitored by its UV absorption at 225 nm. Fractions were hand collected, concentrated under vacuum, and reconstituted in MilliQ water (MilliporeTM) before antimicrobial assays. high performance liquid chromatography reverse transcription polymerase chain reaction minimal inhibitory concentration matrix-assisted laser desorption time-of-flight mass spectrometry The purity of the isolated peptides was ascertained by capillary zone electrophoresis as described previously (4Lowenberger C. Bulet P. Charlet M. Hetru C. Hodgeman B. Christensen B.M. Hoffmann J.A. Insect Biochem. Mol. Biol. 1995; 25: 867-873Crossref PubMed Scopus (127) Google Scholar). Analyses were conducted on a model 270A-HT capillary electrophoresis system (PEApplied Biosystems) equipped with a fused silica capillary. Measurements were carried out on a Bruker (BIFLEXTM) mass spectrometer equipped with a delayed extraction ion source. Mass spectrometry analysis was performed as described previously (24Uttenweiler-Joseph S. Moniatte M. Lagueux M. Van Dorsselaer A. Hoffmann J.A. Bulet P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11342-11347Crossref PubMed Scopus (138) Google Scholar), using α-cyano-4-hydroxycinnamic acid (Sigma) as matrix. The isolated peptides were subjected to Edman degradation on a pulse liquid automatic sequenator (PEApplied Biosystems, model 473A). Total RNA was extracted from whole bodies of immune-activated mosquitoes 1–24 h after inoculation and reverse-transcribed as described previously (25Lowenberger C.A. Smartt C.T. Bulet P. Ferdig M.T. Severson D.W. Hoffmann J.A. Christensen B.M. Insect Mol. Biol. 1999; 8: 107-118Crossref PubMed Scopus (84) Google Scholar), using the primer 5′-CGGGCAGTGAGCGCAACGT14-3′ for the RT reaction. PCR amplification of cecropin cDNA was performed using a reverse primer, consisting of the initial of the RT primer, and two and to the cecropin by Edman PCR were and 30 of 50 and by at °C. PCR were and as previously described (25Lowenberger C.A. Smartt C.T. Bulet P. Ferdig M.T. Severson D.W. Hoffmann J.A. Christensen B.M. Insect Mol. Biol. 1999; 8: 107-118Crossref PubMed Scopus (84) Google Scholar). were using the cDNA using specific to the cDNA and the PCR amplification and cDNA were as described Phylogenetic analysis and of A. A with other insect cecropins were performed with the with Northern analysis was performed as described previously (25Lowenberger C.A. Smartt C.T. Bulet P. Ferdig M.T. Severson D.W. Hoffmann J.A. Christensen B.M. Insect Mol. Biol. 1999; 8: 107-118Crossref PubMed Scopus (84) Google Scholar) using 5 of RNA extracted from whole bodies of immature of A. aegypti and naı̈ve and immune-activated were using specific to 50 of a cecropin cDNA in a PCR described previously (25Lowenberger C.A. Smartt C.T. Bulet P. Ferdig M.T. Severson D.W. Hoffmann J.A. Christensen B.M. Insect Mol. Biol. 1999; 8: 107-118Crossref PubMed Scopus (84) Google Scholar). The was as a RNA from naı̈ve and and naı̈ve and immune-activated adults was and as described PCR with the cecropin specific in were on a using an system were using specific was performed by as described previously P. Bulet P. S. L. Hetru C. Hoffmann J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). The synthetic cecropin was by extraction on Sep-Pak C18 and by two reversed-phase and purity of the peptide was by Edman mass spectrometry and capillary zone were performed as described previously C. Bulet P. in Molecular Scholar). the antibacterial activity spectrum we in to the bacterial reported in a (4Lowenberger C. Bulet P. Charlet M. Hetru C. Hodgeman B. Christensen B.M. Hoffmann J.A. Insect Biochem. Mol. Biol. 1995; 25: 867-873Crossref PubMed Scopus (127) Google Scholar), the The and were activity was in the for antibacterial assays. The peptides and were from M. A. Zasloff to on (4Lowenberger C. Bulet P. Charlet M. Hetru C. Hodgeman B. Christensen B.M. Hoffmann J.A. Insect Biochem. Mol. Biol. 1995; 25: 867-873Crossref PubMed Scopus (127) Google Scholar), we the analysis on the with 40% that the of antibacterial This fraction was subjected to reversed-phase HPLC analysis A antibacterial activity against M. luteus and was observed in fractions from 25 to individual active fraction was further subjected to a reversed-phase The collected fractions were against M. luteus and as The are only reported for the fractions After the antibacterial individual active were purified from fractions A, and C. 2, and 2 in at and were active luteus and at high a lower concentration to the absorbance at 225 an activity was detected only against M. In 2, was active only against Mass of fractions and 2 by the of with mass to of the A. aegypti defensins A, and (4Lowenberger C. Bulet P. Charlet M. Hetru C. Hodgeman B. Christensen B.M. Hoffmann J.A. Insect Biochem. Mol. Biol. 1995; 25: 867-873Crossref PubMed Scopus (127) Google Scholar) not N-terminal by Edman degradation that was Aedes defensin and that 2 the A and as by an capillary zone electrophoresis analysis not structural information on the in was further purified to homogeneity by an chromatography step using the reversed-phase column as in step 2 not After the antibacterial assay against only a active fraction was The purity was ascertained by capillary zone electrophoresis and a mass at was the purity of the molecule. The peptide then was subjected to Edman degradation and the acid was The mass for peptide was lower the mass by approximately that the was not We the cDNA (see and to were Edman we that the N-terminal residue of the peptide was a This residue was at 24 in the cDNA Molecular mass of the peptide that it at the residue at in the cDNA The mass of the was in with the mass by at the active of to a acid peptide with the the acid by Edman degradation A in the showed high with antibacterial peptides from Lepidoptera and We the new isolated peptide as A. aegypti cecropin A. The A. aegypti cecropin cDNA a of The with an and a of a to the acid previously by Edman The region a a region of the and before the The a peptide the first and the cecropin by a The peptide region is hydrophobic and is to with an residue This is on the that the cecropin peptide in the hemolymph of mosquitoes and not any of the Northern we not cecropin mRNA in any immature stage of A. nor in naı̈ve adults was in immune-activated transcription was detected 6 h and continued for 7–10 days. by we not cecropin in isolated from or naı̈ve adults of A. aegypti a for cecropin was detected in immune-activated mosquitoes. After HPLC and mass spectrometry to the of the of cecropin were The activity spectrum of A. aegypti cecropin A was with Drosophilacecropin A, which the features of cecropins of a tryptophan residue and a C-terminal The activity of these two peptides are in with two and In the antibacterial aegypti and cecropins were effective against against the only bacteria was not by A. cecropins at the concentration μm). The insect cecropins were active the two against the bacteria and the cecropin was several effective the A. aegypti The were against and and S. against P. and against bacteria were by the two A. aegypti cecropin A was against of the B. and B. as as against L. to a concentration of B. and S. the to the cecropin the growth of B. was not by μm of A. aegypti cecropin A but was to the μm). In the growth was not by Drosophilacecropin A but was by the A. aegypti peptide μm). The two cecropins were active against bacteria and A. aegypti cecropins were active against the F. F. and N. and A. and B. were to In aegypti cecropin showed a activity C. and S. spectrum of Aedes and cecropins with two and P. C. F. P. J. Biol. Full Text PDF PubMed Google Scholar) is as concentration not detected at the concentration μm). in a new The P. C. F. P. J. Biol. Full Text PDF PubMed Google Scholar) is as concentration not detected at the concentration μm). The that the cell-free hemolymph of bacteria-challenged adults of A. aegypti several antibacterial the previously described A. aegypti defensins (4Lowenberger C. Bulet P. Charlet M. Hetru C. Hodgeman B. Christensen B.M. Hoffmann J.A. Insect Biochem. Mol. Biol. 1995; 25: 867-873Crossref PubMed Scopus (127) Google Scholar, 5Chalk R. Albuquerque C.M.R. Ham P.J. Townson H. Proc. R. Soc. Lond. B. Biol. Sci. 1995; 261: 217-221Crossref PubMed Scopus (36) Google Scholar). a by reversed-phase HPLC of the defensin we have isolated and characterized a antibacterial peptide belonging to the cecropin This is the first report the full of a mosquito cecropin isolated from the insect hemolymph. the only report in mosquitoes was the cecropin isolated from an A. albopictus cell D. Eccleston E.D. Fallon A.M. Biochem. Biophys. Res. Commun. 1998; 249: 410-415Crossref PubMed Scopus (35) Google Scholar). In with all isolated from aegypti with an residue In of the cecropins in the region of the peptide has a the from the showed at the and other within the the In to the from Lepidoptera all in a by an or a acid residue the of the cecropin S. Y. M. Biochem. 1994; Scholar) and the cecropin A D. G. H. H. Insect Biochem. Mol. Biol. 1996; 26: PubMed Scopus Google Scholar), the with the that the cecropin molecules in the insects before the of the Diptera and cecropins have been isolated from invertebrates belonging to the C. L. Lee PubMed Scopus Google Scholar), which of the in the region of the of these peptides other comparison of A. aegypti cecropin A with other cecropins showed of and with the A. 2 and of A. aegypti cecropin with previously characterized and cecropins several specific is the of a tryptophan residue in the A. aegypti a tryptophan residue has been at acid or 2, in all characterized insect for the other mosquito cecropins D. Eccleston E.D. Fallon A.M. Biochem. Biophys. Res. Commun. 1998; 249: 410-415Crossref PubMed Scopus (35) Google Scholar) P. M. C. A. C. G. J. F. C. and A. for and cecropin S. Y. M. Biochem. 1994; Scholar). A. aegypti cecropin A is not at the C-terminal as from the characterization of the full cDNA This is in to the of a by an in all insect cecropins the A. albopictus cecropin A D. Eccleston E.D. Fallon A.M. Biochem. Biophys. Res. Commun. 1998; 249: 410-415Crossref PubMed Scopus (35) Google Scholar) The A. aegypti cecropin A has in with the other cecropins over the first N-terminal This high of was not observed in the C-terminal is that only two and two are the 10 of mosquito cecropins and the C-terminal region of other cecropins A analysis of the insect cecropin family was performed at the acid The of the peptides that mosquito cecropins in and all other and cecropins are in This an the cecropins isolated from mosquito and the other Diptera and and from only the cecropins are in two This of is when only the of these cecropins are not the analysis is on a acid to the hydrophobic C-terminal of the is mosquito cecropins are to to cecropins and are to B. cecropin not for A. aegypti cecropin was to immune-activated In with the A. aegypti defensin (25Lowenberger C.A. Smartt C.T. Bulet P. Ferdig M.T. Severson D.W. Hoffmann J.A. Christensen B.M. Insect Mol. 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A. 80: PubMed Scopus Google Scholar, L.D. Boman H.G. PubMed Scopus Google Scholar). In and J. B. M. J. G. PubMed Scopus Google Scholar) showed that only the with an an activity to the In B. S. Y. M. Biochem. 1994; Scholar), a cecropin showed a against after a C-terminal amidation. In A. aegypti cecropin exhibited a activity against and such and have been reported for cecropins Insect Biochem. Mol. Biol. PubMed Scopus Google Scholar, J.M. C. PubMed Google Scholar). The activity of A. aegypti cecropin is to the growth of bacteria that are in mosquito A.M. E.D. M. J. Med. 1998; PubMed Scopus Google Scholar). the concentration of the cecropin in A. aegypti 5 μm) is to the growth of such it is to the role of peptide in mosquitoes from eukaryotic pathogens. studies have implicated cecropins or synthetic in the development of parasites (see the In a and other in an to be The characterization of the cecropin reported and the identification of inducible immune peptides that is in of mosquito This to further into the role these peptides may play insect development and of mosquito vectors from pathogens. We are to S. for mass spectrometry and to J. P. aegypti cecropin We J. Chiles and L. Christensen for mosquito and We J. A. Hoffmann for continued in