The Melanization Reaction Is Not Required for Survival of Anopheles gambiae Mosquitoes after Bacterial Infections

Abstract

The melanization reaction of insects requires activation of pro-phenoloxidase by a proteolytic cascade leading to melanin production. Studies in adult mosquitoes have shown that bacteria are efficiently melanized in the hemocoel, but the contribution of melanization to survival after bacterial infections has not been established. Here we show that the Anopheles gambiae noncatalytic serine protease CLIPA8, an essential factor for Plasmodium ookinete melanization, is also required for melanization of bacteria in adult mosquitoes. CLIPA8 silencing by RNA interference inhibits pro-phenoloxidase activation and melanization of bacteria in the hemolymph following microbial challenge. However, CLIPA8 is not required for wound melanization nor for melanotic pseudotumor formation in serpin2 knockdown mosquitoes, suggesting a specific role for pathogen melanization. Surprisingly, CLIPA8 knockdown mosquitoes are as resistant to bacterial challenge as controls, indicating that melanization is not essential for defense against bacteria and questions its precise role in mosquito immunity. The melanization reaction of insects requires activation of pro-phenoloxidase by a proteolytic cascade leading to melanin production. Studies in adult mosquitoes have shown that bacteria are efficiently melanized in the hemocoel, but the contribution of melanization to survival after bacterial infections has not been established. Here we show that the Anopheles gambiae noncatalytic serine protease CLIPA8, an essential factor for Plasmodium ookinete melanization, is also required for melanization of bacteria in adult mosquitoes. CLIPA8 silencing by RNA interference inhibits pro-phenoloxidase activation and melanization of bacteria in the hemolymph following microbial challenge. However, CLIPA8 is not required for wound melanization nor for melanotic pseudotumor formation in serpin2 knockdown mosquitoes, suggesting a specific role for pathogen melanization. Surprisingly, CLIPA8 knockdown mosquitoes are as resistant to bacterial challenge as controls, indicating that melanization is not essential for defense against bacteria and questions its precise role in mosquito immunity. Melanization is a prominent defense mechanism in arthropods and has received considerable attention in the context of a genetically selected refractory strain of Anopheles gambiae that melanizes several Plasmodium species, including New World strains of Plasmodium falciparum (1Collins F.H. Sakai R.K. Vernick K.D. Paskewitz S. Seeley D.C. Miller L.H. Collins W.E. Campbell C.C. Gwadz R.W. Science. 1986; 234: 607-610Crossref PubMed Scopus (421) Google Scholar). Biochemical studies in large insects such as Manduca sexta, Bombyx mori, and Holotrichia diomphalia (for reviews see Refs. 2Cerenius L. Soderhall K. Immunol. Rev. 2004; 198: 116-126Crossref PubMed Scopus (1280) Google Scholar, 3Jiravanichpaisal P. Lee B.L. Soderhall K. Immunobiology. 2006; 211: 213-236Crossref PubMed Scopus (640) Google Scholar) have led to the current model of the melanization response, in which recognition of nonself-molecular patterns eventually activates a clip-domain serine protease cascade, culminating in limited proteolysis and conversion of inactive pro-phenoloxidase (PPO) 3The abbreviations used are: PPO, pro-phenoloxidase; PO, phenol oxidase; WT, wild type; PBS, phosphate-buffered saline; SPH, serine protease homolog; dsRNA, double-stranded RNA; RHD, Rel homology domain. into active phenol oxidase (PO) by pro-phenoloxidase-activating enzymes. Activated PO catalyzes the rate-limiting step in the melanization reaction, the oxidation of tyrosine to dopaquinone that ultimately leads to production and cross-linking of melanin with proteins on the surfaces of microbes or wounds. Our recent genetic studies in A. gambiae (4Osta M.A. Christophides G.K. Kafatos F.C. Science. 2004; 303: 2030-2032Crossref PubMed Scopus (350) Google Scholar, 5Michel K. Budd A. Pinto S. Gibson T.J. Kafatos F.C. EMBO Rep. 2005; 6: 891-897Crossref PubMed Scopus (135) Google Scholar, 6Volz J. Osta M.A. Kafatos F.C. Muller H.M. J. Biol. Chem. 2005; 280: 40161-40168Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 7Volz J. Muller H.M. Zdanowicz A. Kafatos F.C. Osta M.A. Cell Microbiol. 2006; 8: 1392-1405Crossref PubMed Scopus (135) Google Scholar) identified several proteins that regulate Plasmodium berghei ookinete melanization and highlighted the underlying genetic complexity of this immune module. However, field-caught A. gambiae (8Niare O. Markianos K. Volz J. Oduol F. Toure A. Bagayoko M. Sangare D. Traore S.F. Wang R. Blass C. Dolo G. Bouare M. Kafatos F.C. Kruglyak L. Toure Y.T. Vernick K.D. Science. 2002; 298: 213-216Crossref PubMed Scopus (111) Google Scholar) and most laboratory strains rarely utilize melanization to kill Plasmodium parasites, although certain mutants do so (4Osta M.A. Christophides G.K. Kafatos F.C. Science. 2004; 303: 2030-2032Crossref PubMed Scopus (350) Google Scholar, 7Volz J. Muller H.M. Zdanowicz A. Kafatos F.C. Osta M.A. Cell Microbiol. 2006; 8: 1392-1405Crossref PubMed Scopus (135) Google Scholar). Although melanization does not seem to be a major parasite-killing mechanism, it remains unclear whether it is important for elimination of other pathogens such as bacteria. In this study we address specifically this question. To date the role of melanization in defense against bacteria in mosquitoes remains controversial. Although melanized bacteria have been detected in the hemocoel of mosquitoes, including Aedes aegypti (9Hillyer J.F. Schmidt S.L. Christensen B.M. J. Parasitol. 2003; 89: 62-69Crossref PubMed Scopus (137) Google Scholar, 10Hillyer J.F. Schmidt S.L. Christensen B.M. Microbes Infect. 2004; 6: 448-459Crossref PubMed Scopus (69) Google Scholar) and Anopheles albimanus (11Hernandez-Martinez S. Lanz H. Rodriguez M.H. Gonzalez-Ceron L. Tsutsumi V. J. Med. Entomol. 2002; 39: 61-69Crossref PubMed Scopus (57) Google Scholar), no genetic studies have investigated whether this defense mechanism promotes survival of mosquitoes to bacterial infections. Using RNA interference, we have previously identified a noncatalytic clip-domain serine protease (serine protease homolog), CLIPA8, which is essential for ookinete melanization in a refractory A. gambiae strain as well as in certain reverse genetic mutant backgrounds (4Osta M.A. Christophides G.K. Kafatos F.C. Science. 2004; 303: 2030-2032Crossref PubMed Scopus (350) Google Scholar, 5Michel K. Budd A. Pinto S. Gibson T.J. Kafatos F.C. EMBO Rep. 2005; 6: 891-897Crossref PubMed Scopus (135) Google Scholar, 6Volz J. Osta M.A. Kafatos F.C. Muller H.M. J. Biol. Chem. 2005; 280: 40161-40168Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 7Volz J. Muller H.M. Zdanowicz A. Kafatos F.C. Osta M.A. Cell Microbiol. 2006; 8: 1392-1405Crossref PubMed Scopus (135) Google Scholar). Here we show that CLIPA8 is also essential for melanization of both Gram-positive and Gram-negative bacteria in A. gambiae; however, melanization was not required for mosquito survival after bacterial infections. Furthermore, CLIPA8 is not required for melanization of wound surfaces nor for the formation of circulating melanotic pseudotumors. Anopheles gambiae Strain and Gene Silencing by RNA Interference—All experiments were performed with the A. gambiae G3 strain. In vivo gene silencing by RNA interference was performed as reported (12Blandin S. Moita L.F. Kocher T. Wilm M. Kafatos F.C. Levashina E.A. EMBO Rep. 2002; 3: 852-856Crossref PubMed Scopus (296) Google Scholar). Double-stranded RNAs (dsRNA) for lacZ (control), CLIPA8, and the Rel homology domain (RHD) of Rel2 were synthesized as described in Refs. 7Volz J. Muller H.M. Zdanowicz A. Kafatos F.C. Osta M.A. Cell Microbiol. 2006; 8: 1392-1405Crossref PubMed Scopus (135) Google Scholar, 13Blandin S. Shiao S.H. Moita L.F. Janse C.J. Waters A.P. Kafatos F.C. Levashina E.A. Cell. 2004; 116: 661-670Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar, 14Meister S. Kanzok S.M. Zheng X.L. Luna C. Li T.R. Hoa N.T. Clayton J.R. White K.P. Kafatos F.C. Christophides G.K. Zheng L. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 11420-11425Crossref PubMed Scopus (195) Google Scholar, respectively. RNA Isolation and Quantitative Real Time PCR—Mosquitoes were challenged with Escherichia coli or Staphylococcus aureus either by injecting bacterial suspensions at A600 = 0.4 or by pricking with overnight bacterial cultures. Mosquitoes injected or pricked with sterile PBS were used as controls. Total RNA was isolated from whole mosquitoes at the indicated time points using TRIzol reagent (Invitrogen) according to the supplier's instructions, and contaminant genomic DNA was removed by DNase I treatment. First strand cDNA synthesis and CLIPA8 primers used for quantitative real time PCR are as described previously (7Volz J. Muller H.M. Zdanowicz A. Kafatos F.C. Osta M.A. Cell Microbiol. 2006; 8: 1392-1405Crossref PubMed Scopus (135) Google Scholar). Generation of Monoclonal Antibodies against CLIPA8—A 719-bp fragment corresponding to the protease domain of CLIPA8 was amplified using primers CLIPA8expF, 5′-CCATGGAATTTCCTTGGGTTGTGGCGATTCT-3′, and CLIPA8expR, 5′-GCGGCCGCTCACAGTATTCCCTCGATTGTAGCAT-3′, containing the restriction sites NcoI and NotI, respectively. The amplicon was first subcloned in pGEM®-T Easy vector (Promega) and then cloned between the NcoI and NotI sites of the pETM11 expression vector. Fusion protein containing an N-terminal His tag was expressed in E. coli strain BL21 (DE3) and extracted from inclusion bodies using 8 m urea, pH 8, as described (15Schrodel A. Volz J. de Marco A. J. Biotechnol. 2005; 120: 2-10Crossref PubMed Scopus (21) Google Scholar). Monoclonal antibodies against CLIPA8 were produced at the EMBL Monoclonal Antibody Core Facility as described (16De Masi F. Chiarella P. Wilhelm H. Massimi M. Bullard B. Ansorge W. Sawyer A. Proteomics. 2005; 5: 4070-4081Crossref PubMed Scopus (69) Google Scholar). Western Blot Analysis—Bacteria from overnight cultures were washed and resuspended in PBS; suspensions of 69 nl (E. coli, Enterobacter cloacae, Enterococcus faecalis, and S. aureus, each at A600 = 0.8) were injected into mosquitoes using a Nanoject II injector (Drummond). For immunoblotting, hemolymph proteins were collected in a reducing SDS buffer by proboscis clipping from 10 mosquitoes 6 h post-bacterial challenge, separated using 12% SDS-PAGE, and transferred as described (17Danielli A. Loukeris T.G. Lagueux M. Muller H.M. Richman A. Kafatos F.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7136-7141Crossref PubMed Scopus (67) Google Scholar). Blots were incubated with mouse anti-CLIPA8 monoclonal antibody (1/20), rat anti-PPO (1/1000; a kind gift from H. M. Müller), and rabbit anti-SRPN3 (1/1000) polyclonal antibodies (18Michel K. Suwanchaichinda C. Morlais I. Lambrechts L. Cohuet A. Awono-Ambene P.H. Simard F. Fontenille D. Kanost M.R. Kafatos F.C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 16858-16863Crossref PubMed Scopus (84) Google Scholar). Anti-mouse, anti-rabbit, and anti-rat IgG horseradish peroxidase-conjugated secondary antibodies (Promega) were used at 1/15,000, 1/30,000, and 1/15,000 dilutions, respectively. Relative protein band quantitation was done using the Phoretix 1D software. Measurements of PO Activity—PO activity was assayed 6 h post-challenge with a mixture of S. aureus and E. coli (each at A600 = 0.4) or PBS (control). Hemolymph was collected in ice-cold phosphate-buffered saline (PBS) containing protease inhibitors, and protein concentration was determined using the protein assay kit from Bio-Rad. Five μg of total hemolymph proteins in 40 μl of PBS containing protease inhibitors were mixed with 120 μl of saturated l-3,4-dihydroxiphenylalanine solution; absorbance at 492 nm was measured after incubation at room temperature for 30 min. Survival Experiments—E. coli and S. aureus were cultured to A600 = 0.7, pelleted, washed, and resuspended in phosphate-buffered saline to the indicated concentrations. Bacterial infections and survival assays were done as described (12Blandin S. Moita L.F. Kocher T. Wilm M. Kafatos F.C. Levashina E.A. EMBO Rep. 2002; 3: 852-856Crossref PubMed Scopus (296) Google Scholar). CLIPA8, a Hemolymph Protein, Is Cleaved by Wounding and Bacterial Infections—Immunoblotting of hemolymph from naive mosquitoes (Fig. 1A, c lane) revealed a full-length CLIPA8 protein (CLIPA8-F) with an apparent molecular mass of 47 kDa. However, soon after S. aureus injection into mosquitoes, a cleaved form of CLIPA8 (CLIPA8-C) corresponding to the noncatalytic protease domain appeared at 38 kDa in immunoblotted hemolymph extracted in reducing SDS buffer (Fig. 1A). Quantitative analysis of band intensity (Fig. 1A, bar graph) revealed that the CLIPA8-C levels increased after S. aureus infection, peaked at 6 h, and returned to the basal level by 24 h post-infection. Furthermore, CLIPA8 mRNA expression peaked at 12 h post-infection with S. aureus and E. coli (2.5- and 1.7-fold, respectively, relative to PBS-injected controls) and remained relatively high at 24 h post-infection (Fig. 2). In contrast, CLIPA8-F did not increase in response to infection (data not shown), suggesting that the observed transcriptional induction during this immune response may serve protein replenishment rather than accumulation. Both CLIPA8-F and CLIPA8-C are absent from hemolymph of mosquitoes treated with double-stranded (ds) CLIPA8 RNA (Fig. 1B, lane 6). Further analysis revealed that CLIPA8 cleavage in the hemolymph is induced robustly by additional bacterial species, including E. coli, E. cloacae, and E. faecalis, but is relatively minor after sterile PBS injection (Fig. 1B). Quantitation of CLIPA8-C band intensities revealed a 17-, 43-, 26-, 40-, and 5-fold increase in the abundance of CLIPA8-C in hemolymph in response to the injection of E. coli, E. cloacae, E. faecalis, S. aureus, or sterile PBS alone, respectively, as compared with control noninjected mosquitoes. Hence, CLIPA8 cleavage is induced much more effectively by infection rather than by injury per se.FIGURE 2Transcript levels of CLIPA8 in whole mosquitoes challenged with E. coli (gray), S. aureus (black), or PBS (white). The ordinate represents relative expression values. Error bars represent standard deviations in the pooled data set from two independent biological experiments (one performed with mosquitoes injected with bacterial suspensions at A600 = 0.4, and the second mosquitoes pricked with overnight bacterial cultures).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cleavage of serine protease homologs (SPHs) involved in PPO activation has been reported in other insect species, including M. sexta (19Yu X.Q. Jiang H. Wang Y. Kanost M.R. Insect Biochem. Mol. Biol. 2003; 33: 197-208Crossref PubMed Scopus (207) Google Scholar) and H. diomphalia (20Kim M.S. Baek M.J. Lee M.H. Park J.W. Lee S.Y. Soderhall K. Lee B.L. J. Biol. Chem. 2002; 277: 39999-40004Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), where it occurs at a specific site within the clip-domain; however, the clip and protease domains remain linked by a disulfide bond. Immunoblotting of mosquito hemolymph, extracted under nonreducing conditions, revealed that both CLIPA8-C and CLIPA8-F migrated as a single band (Fig. 1B, lane 7), indeed indicating the presence of a disulfide bond that bridges both domains. CLIPA8 Is Essential for Melanization of Bacteria—The facts that CLIPA8 is essential for P. berghei ookinete melanization (7Volz J. Muller H.M. Zdanowicz A. Kafatos F.C. Osta M.A. Cell Microbiol. 2006; 8: 1392-1405Crossref PubMed Scopus (135) Google Scholar) and is cleaved in the hemolymph following bacterial challenge prompted us to investigate the potential involvement of CLIPA8 in bacterial melanization. We injected E. coli or S. aureus in wild type, dslacZ, or dsCLIPA8-treated adult A. gam-biae and utilized light microscopy to score for the presence of melanized bacterial clumps in the mosquito abdomens, after dissection 3 days post-infection. Such clumps were detected in wild type mosquitoes injected with E. coli (Fig. 3B, arrows) or S. aureus (Fig. 3C, arrows) but not in noninjected (Fig. 3A) or PBS-injected mosquitoes (data not shown). These clumps appear as easily identifiable black bodies clustering mainly on both sides of the dorsal blood vessel. Studies in another mosquito species established that the large melanotic clumps appearing after bacterial infections are formed by aggregation of singly melanized bacteria in the hemocoel (11Hernandez-Martinez S. Lanz H. Rodriguez M.H. Gonzalez-Ceron L. Tsutsumi V. J. Med. Entomol. 2002; 39: 61-69Crossref PubMed Scopus (57) Google Scholar). Interestingly, after challenge with E. coli or S. aureus, melanized clumps were present in dslacZ-treated controls (Table 1) but absent from dsCLIPA8-treated mosquitoes (Fig. 3, D and The was observed (data not with two additional E. and E. indicating that CLIPA8 is indeed required for melanization of bacteria in A. silencing CLIPA8 also hemolymph PPO activation by bacteria. We measured PO activity in the hemolymph of and mosquitoes 6 h after infection with a mixture of E. coli and S. The controls induction of PO the mosquitoes basal as did wild type noninjected or PBS-injected mosquitoes (Fig. The that sterile PBS injection did not PPO in the hemolymph that PPO activation induced by is in to the activation that occurs after bacterial challenge. that CLIPA8 is essential for bacterial melanization in A. Interestingly, mosquitoes melanized wound surfaces as efficiently as did controls (Fig. 3, and suggesting that CLIPA8 is not essential for that CLIPA8 is also not required for the formation of large melanotic (Fig. 3, and which in the of injected bacteria in mosquitoes for the major melanization K. Budd A. Pinto S. Gibson T.J. Kafatos F.C. EMBO Rep. 2005; 6: 891-897Crossref PubMed Scopus (135) Google bacterial clumps in and A. gambiae mosquitoes Mosquitoes were challenged with E. coli or S. aureus (each at A600 = were 3 days post-challenge and for the presence of melanized bacterial of with melanized with melanized in a dsCLIPA8-treated Mosquitoes to mosquitoes were to PPO in the hemolymph following bacterial us to whether the to is for to bacterial To this we compared the survival of and dsCLIPA8-treated mosquitoes days post-infection with E. coli or S. To that CLIPA8 was efficiently this we performed analysis on hemolymph extracted from mosquitoes at time points after The that CLIPA8 is absent from the hemolymph of mosquitoes at days after injection (Fig. mosquitoes E. coli and S. aureus infections as efficiently as did controls (Fig. and of bacteria as controls (Fig. were much than in mosquitoes, which to E. coli and S. aureus infections S. Kanzok S.M. Zheng X.L. Luna C. Li T.R. Hoa N.T. Clayton J.R. White K.P. Kafatos F.C. Christophides G.K. Zheng L. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 11420-11425Crossref PubMed Scopus (195) Google Scholar). the of an response, other than melanization, of The phenol oxidase cascade is an of insect and immune including and melanization of Melanization requires the limited proteolysis of inactive PPO to PO by pro-phenoloxidase-activating and including the serine protease studies in mosquito species revealed that bacteria are efficiently melanized in the hemocoel (9Hillyer J.F. Schmidt S.L. Christensen B.M. J. Parasitol. 2003; 89: 62-69Crossref PubMed Scopus (137) Google Scholar, 10Hillyer J.F. Schmidt S.L. Christensen B.M. Microbes Infect. 2004; 6: 448-459Crossref PubMed Scopus (69) Google Scholar, S. Lanz H. Rodriguez M.H. Gonzalez-Ceron L. Tsutsumi V. J. Med. Entomol. 2002; 39: 61-69Crossref PubMed Scopus (57) Google however, no genetic analysis has been to the contribution of melanization to mosquito survival after bacterial infections. Here we have this using in vivo genetic analysis on RNA We show that CLIPA8, an essential for the melanization of P. berghei in the mosquito is required for PPO activation and the melanization of bacteria in the mosquito The that CLIPA8 is essential for the melanization of both Plasmodium and bacteria the of at a in the genetic that regulate the melanization response of A. gambiae to both We also by that CLIPA8 is cleaved following bacterial however, both the protease and clip domains remain by a disulfide bond as has been shown for other insect (19Yu X.Q. Jiang H. Wang Y. Kanost M.R. Insect Biochem. Mol. Biol. 2003; 33: 197-208Crossref PubMed Scopus (207) Google Scholar, M.S. Lee B.L. J. Biochem. 2000; PubMed Scopus Google Scholar). Cleaved CLIPA8 is most the form of the and recent analysis of the H. diomphalia pro-phenoloxidase-activating factor which to the revealed that is a cleaved is a large that to PO S. Park S.Y. Park J.W. Lee B.L. EMBO J. 2005; PubMed Scopus Google Scholar). The CLIPA8-C band detected in is compared with and a has been reported for M. sexta and (19Yu X.Q. Jiang H. Wang Y. Kanost M.R. Insect Biochem. Mol. Biol. 2003; 33: 197-208Crossref PubMed Scopus (207) Google Scholar). be either to of the cleaved from the hemolymph to the melanization response or to with PO and other proteins in the melanin that Interestingly, CLIPA8 cleavage in the hemolymph was also detected after injection of sterile PBS and 3 days of the major melanization (data not shown), CLIPA8 was not required for melanization of wound nor for the formation of melanotic in mosquitoes. These the of for PPO activation during wound and melanotic pseudotumor shown that the PPO more in A. gambiae than in G.K. E. C. E. S. Blass C. Collins F.H. A. G. C. Hoa N.T. Kanzok S.M. I. Levashina E.A. Loukeris T.G. G. S. K. Moita L.F. Muller H.M. Osta M.A. Paskewitz S.M. A. L. Vernick K.D. D. Volz J. C. J. Zheng L. P. Kafatos F.C. Science. 2002; 298: PubMed Scopus Google no of the essential CLIPA8 melanization factor have been identified in or other insects to date G.K. E. C. E. S. Blass C. Collins F.H. A. G. C. Hoa N.T. Kanzok S.M. I. Levashina E.A. Loukeris T.G. G. S. K. Moita L.F. Muller H.M. Osta M.A. Paskewitz S.M. A. L. Vernick K.D. D. Volz J. C. J. Zheng L. P. Kafatos F.C. Science. 2002; 298: PubMed Scopus Google and data not shown). These the that hemolymph phenol oxidase activity may be essential for defense in although it is in V. L. C. P. EMBO Rep. 2006; PubMed Scopus Google Scholar, H. B. C. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). data do not this dsCLIPA8-treated mosquitoes were as resistant to bacterial infections as controls, although to hemolymph However, do not a potential role for melanization in the of immune as in H. B. C. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). of the of the well melanization immune response genetic studies in We for the PPO K. for antibodies and dsRNA, and G. K. Christophides for We also the Monoclonal Antibody Facility for production of CLIPA8 monoclonal We are to for mosquito