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Apical Surface Expression of Aspartic Protease Plasmepsin 4, a Potential Transmission-blocking Target of the Plasmodium Ookinete

2010; Elsevier BV; Volume: 285; Issue: 11 Linguagem: Inglês

10.1074/jbc.m109.063388

1083-351X

Fengwu Li, Kailash P. Patra, Charles A. Yowell, John B. Dame, Karen Chin, Joseph M. Vinetz,

Aquaculture disease management and microbiota

To invade its definitive host, the mosquito, the malaria parasite must cross the midgut peritrophic matrix that is composed of chitin cross-linked by chitin-binding proteins and then develop into an oocyst on the midgut basal lamina. Previous evidence indicates that Plasmodium ookinete-secreted chitinase is important in midgut invasion. The mechanistic role of other ookinete-secreted enzymes in midgut invasion has not been previously examined. De novo mass spectrometry sequencing of a protein obtained by benzamidine affinity column of Plasmodium gallinaceum ookinete axenic culture supernatant demonstrated the presence of an ookinete-secreted plasmepsin, an aspartic protease previously only known to be present in the digestive vacuole of asexual stage malaria parasites. This plasmepsin, the ortholog of Plasmodium falciparum plasmepsin 4, was designated PgPM4. PgPM4 and PgCHT2 (the P. gallinaceum ortholog of P. falciparum chitinase PfCHT1) are both localized on the ookinete apical surface, and both are present in micronemes. Aspartic protease inhibitors (peptidomimetic and natural product), calpain inhibitors, and anti-PgPM4 monoclonal antibodies significantly reduced parasite infectivity for mosquitoes. These results suggest that plasmepsin 4, previously known only to function in the digestive vacuole of asexual blood stage Plasmodium, plays a role in how the ookinete interacts with the mosquito midgut interactions as it becomes an oocyst. These data are the first to delineate a role for an aspartic protease in mediating Plasmodium invasion of the mosquito and demonstrate the potential for plasmepsin 4 as a malaria transmission-blocking vaccine target. To invade its definitive host, the mosquito, the malaria parasite must cross the midgut peritrophic matrix that is composed of chitin cross-linked by chitin-binding proteins and then develop into an oocyst on the midgut basal lamina. Previous evidence indicates that Plasmodium ookinete-secreted chitinase is important in midgut invasion. The mechanistic role of other ookinete-secreted enzymes in midgut invasion has not been previously examined. De novo mass spectrometry sequencing of a protein obtained by benzamidine affinity column of Plasmodium gallinaceum ookinete axenic culture supernatant demonstrated the presence of an ookinete-secreted plasmepsin, an aspartic protease previously only known to be present in the digestive vacuole of asexual stage malaria parasites. This plasmepsin, the ortholog of Plasmodium falciparum plasmepsin 4, was designated PgPM4. PgPM4 and PgCHT2 (the P. gallinaceum ortholog of P. falciparum chitinase PfCHT1) are both localized on the ookinete apical surface, and both are present in micronemes. Aspartic protease inhibitors (peptidomimetic and natural product), calpain inhibitors, and anti-PgPM4 monoclonal antibodies significantly reduced parasite infectivity for mosquitoes. These results suggest that plasmepsin 4, previously known only to function in the digestive vacuole of asexual blood stage Plasmodium, plays a role in how the ookinete interacts with the mosquito midgut interactions as it becomes an oocyst. These data are the first to delineate a role for an aspartic protease in mediating Plasmodium invasion of the mosquito and demonstrate the potential for plasmepsin 4 as a malaria transmission-blocking vaccine target. IntroductionMalaria is the most important parasitic disease of man and a globally dominant cause of morbidity and mortality in humans (1.Sachs J. Malaney P. Nature. 2002; 415: 680-685Crossref PubMed Scopus (1381) Google Scholar). The lack of an effective vaccine, the emergence of drug-resistant Plasmodium falciparum and Plasmodium vivax, and insecticide-resistant vector mosquitoes all contribute to the increasing human toll of malaria. Malaria is transmitted through the bite of infected Anopheles mosquitoes. Preventing transmission from the human reservoir to the definitive host, the mosquito, is one approach to malaria control (2.Malkin E.M. Durbin A.P. Diemert D.J. Sattabongkot J. Wu Y. Miura K. Long C.A. Lambert L. Miles A.P. Wang J. Stowers A. Miller L.H. Saul A. Vaccine. 2005; 23: 3131-3138Crossref PubMed Scopus (181) Google Scholar). Delineating the mechanisms by which the malaria parasite invades and infects mosquitoes may lead to new strategies to block malaria transmission (3.Carter R. Mendis K.N. Miller L.H. Molineaux L. Saul A. Nat. Med. 2000; 6: 241-244Crossref PubMed Scopus (113) Google Scholar, 4.Vinetz J.M. Curr. Top. Microbiol. Immunol. 2005; 295: 357-382PubMed Google Scholar).After a mosquito ingests infectious Plasmodium gametocytes, male and female gametes emerge in the midgut and rapidly fuse to form diploid zygotes. Parasites must then develop into motile ookinetes, penetrate and traverse the protein- and chitin-containing peritrophic matrix, and then cross the midgut epithelium to form oocysts (4.Vinetz J.M. Curr. Top. Microbiol. Immunol. 2005; 295: 357-382PubMed Google Scholar). Developmentally regulated antigens of these stages are potential targets of antibodies induced by vaccination of the vertebrate host that are co-ingested with parasites as a mosquito takes a blood meal (5.Kaslow D.C. Chem. Immunol. 2002; 80: 287-307Crossref PubMed Scopus (70) Google Scholar). Such antibodies are called transmission-blocking antibodies, which act by interfering with parasite development within the mosquito midgut, thus preventing parasite transmission to the mosquito vector. Importantly, proteins expressed in the mosquito stages are less likely to be mutated in response to human immunological responses (6.Saul A. Curr. Opin. Infect. Dis. 2007; 20: 476-481Crossref PubMed Scopus (69) Google Scholar). Hence, interfering in this part of the life cycle has the potential to reduce both transmission as well as the spread of drug-resistant parasites.Ookinete-expressed proteases have been proposed to play vital roles in ookinete invasion of peritrophic matrix and mosquito midgut (7.Langer R.C. Li F. Vinetz J.M. Infect. Immun. 2002; 70: 102-106Crossref PubMed Scopus (18) Google Scholar, 8.Li F. Templeton T.J. Popov V. Comer J.E. Tsuboi T. Torii M. Vinetz J.M. J. Biol. Chem. 2004; 279: 26635-26644Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 9.Shen Z. Jacobs-Lorena M. J. Biol. Chem. 1997; 272: 28895-28900Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The peritrophic matrix is the first physical barrier faced by the ookinete as it escapes the blood meal. The peritrophic matrix is composed of proteins, glycoproteins, proteoglycans, and chitin (10.Shen Z. Jacobs-Lorena M. J. Biol. Chem. 1998; 273: 17665-17670Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 11.Moskalyk L.A. Oo M.M. Jacobs-Lorena M. Insect Mol. Biol. 1996; 5: 261-268Crossref PubMed Scopus (62) Google Scholar). Proteins, including chitin cross-linking proteins (peritrophins), have been reported to account for 22–55% of the total mass of the peritrophic matrix (10.Shen Z. Jacobs-Lorena M. J. Biol. Chem. 1998; 273: 17665-17670Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 11.Moskalyk L.A. Oo M.M. Jacobs-Lorena M. Insect Mol. Biol. 1996; 5: 261-268Crossref PubMed Scopus (62) Google Scholar). Specific protease inhibitors added to infectious blood meals have been observed to reduce ookinete infectivity for the mosquito (12.Shahabuddin M. Criscio M. Kaslow D.C. Exp. Parasit. 1995; 80: 212-219Crossref PubMed Scopus (29) Google Scholar). These observations suggest that ookinetes could use proteases to cross the midgut peritrophic matrix.The genome of P. falciparum encodes a large variety of proteases, including a diverse family of 10 aspartic proteases (designated plasmepsins) (13.Coombs G.H. Goldberg D.E. Klemba M. Berry C. Kay J. Mottram J.C. Trends Parasitol. 2001; 17: 532-537Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). Four plasmepsins are known to degrade hemoglobin in the digestive vacuole of asexual stage malaria parasites (14.Banerjee R. Liu J. Beatty W. Pelosof L. Klemba M. Goldberg D.E. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 990-995Crossref PubMed Scopus (373) Google Scholar, 15.Dame J.B. Yowell C.A. Omara-Opyene L. Carlton J.M. Cooper R.A. Li T. Mol. Biochem. Parasitol. 2003; 130: 1-12Crossref PubMed Scopus (67) Google Scholar). Functions of the remaining six plasmepsins have not been determined, although gene expression profiling and comprehensive proteomic analysis have demonstrated the presence of several plasmepsins in the sexual stage forms of P. falciparum, Plasmodium berghei, and Plasmodium yoelii (16.Hall N. Karras M. Raine J.D. Carlton J.M. Kooij T.W. Berriman M. Florens L. Janssen C.S. Pain A. Christophides G.K. James K. Rutherford K. Harris B. Harris D. Churcher C. Quail M.A. Ormond D. Doggett J. Trueman H.E. Mendoza J. Bidwell S.L. Rajandream M.A. Carucci D.J. Yates 3rd, J.R. Kafatos F.C. Janse C.J. Barrell B. Turner C.M. Waters A.P. Sinden R.E. Science. 2005; 307: 82-86Crossref PubMed Scopus (657) Google Scholar, 17.Young J.A. Fivelman Q.L. Blair P.L. de la Vega P. Le Roch K.G. Zhou Y. Carucci D.J. Baker D.A. Winzeler E.A. Mol. Biochem. Parasitol. 2005; 143: 67-79Crossref PubMed Scopus (253) Google Scholar). No role for any plasmepsin has been demonstrated in Plasmodium-mosquito interactions.In this study, we demonstrate that the P. gallinaceum plasmepsin 4 (PgPM4) 2The abbreviations used are: PgPM4P. gallinaceum plasmepsin 4IFAindirect immunofluorescence assaymAbmonoclonal antibodyALLNcalpain inhibitor I (sequence N-acetyl-Leu-Leu-norleucinal (N-acetyl-Leu-Leu-Nle-CHO)ALLMcalpain inhibitor II (sequence N-acetyl-Leu-Leu-Met-CHO)PBSphosphate-buffered salineHPLChigh pressure liquid chromatographyCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidrPgPM4recombinant PgPM4. synergizes with the P. gallinaceum chitinase PgCHT2 (the ortholog of the P. falciparum chitinase (18.Li F. Patra K.P. Vinetz J.M. J. Infect. Dis. 2005; 192: 878-887Crossref PubMed Scopus (52) Google Scholar)) to facilitate malaria parasite invasion of the mosquito midgut and/or may be involved in the development of ookinete to oocyst. These data are the first to indicate a specific mechanistic function for a plasmepsin in any stage of the malaria parasite other than the asexual blood stage. This aspartic protease plays an important role in ookinete invasion of the mosquito midgut and/or parasite development, and thus may be a novel target of blocking malaria transmission.DISCUSSIONHere, we demonstrate that the Plasmodium ookinete-expressed plasmepsin, PgPM4, plays a role in parasite invasion of the mosquito midgut and is involved somewhere along the pathway along which ookinetes develop into oocysts. These observations are the first to demonstrate, at the protein level, the role of a plasmepsin expressed by the mosquito midgut stages of the malaria parasite, although gene expression and proteomic profiling have shown the presence of plasmepsin gene products in the sexual stages (16.Hall N. Karras M. Raine J.D. Carlton J.M. Kooij T.W. Berriman M. Florens L. Janssen C.S. Pain A. Christophides G.K. James K. Rutherford K. Harris B. Harris D. Churcher C. Quail M.A. Ormond D. Doggett J. Trueman H.E. Mendoza J. Bidwell S.L. Rajandream M.A. Carucci D.J. Yates 3rd, J.R. Kafatos F.C. Janse C.J. Barrell B. Turner C.M. Waters A.P. Sinden R.E. Science. 2005; 307: 82-86Crossref PubMed Scopus (657) Google Scholar, 17.Young J.A. Fivelman Q.L. Blair P.L. de la Vega P. Le Roch K.G. Zhou Y. Carucci D.J. Baker D.A. Winzeler E.A. Mol. Biochem. Parasitol. 2005; 143: 67-79Crossref PubMed Scopus (253) Google Scholar). In the avian-infecting malaria parasite P. gallinaceum, both PgPM4 and the chitinase PgCHT2 are present in micronemes, consistent with their roles as secreted proteins, and both proteins are present on the apical surface of the ookinete. Collectively, these observations suggest the possibility that both chitinase and PgPM4 are involved in penetrating the chitin- and peritrophin-containing peritrophic matrix. Alternatively, PgPM4 might have additional protease functions involved in parasite-mosquito midgut interactions other than at the level of the peritrophic matrix, such as crossing the epithelial surface or cleaving other parasite or mosquito protein targets. Further experiments are needed to distinguish among these possibilities.The biological importance of plasmepsin 4 was demonstrated in experimental mosquito infections using membrane feeding experiments that incorporated plasmepsin inhibitors and anti-plasmepsin 4 monoclonal antibodies into the infectious blood meal. The data from these experiments provided further evidence of the importance of plasmepsin 4 in parasite-mosquito interactions and strongly suggested its role as a potential target of new transmission blocking strategies.In the process of midgut invasion, the ookinete must cross a series of physical barriers, the first of which is the chitin- and peritrophin-containing peritrophic matrix, followed by the epithelial surface. A series of ookinete-expressed proteins have been reported to be critical for host cell recognition, binding, and motility and have been involved in passage of the ookinete through the mosquito midgut (8.Li F. Templeton T.J. Popov V. Comer J.E. Tsuboi T. Torii M. Vinetz J.M. J. Biol. Chem. 2004; 279: 26635-26644Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 26.Yuda M. Yano K. Tsuboi T. Torii M. Chinzei Y. Mol. Biochem. Parasitol. 2001; 116: 65-72Crossref PubMed Scopus (85) Google Scholar, 29.Tsai Y.L. Hayward R.E. Langer R.C. Fidock D.A. Vinetz J.M. Infect. Immun. 2001; 69: 4048-4054Crossref PubMed Scopus (89) Google Scholar, 30.Limviroj W. Yano K. Yuda M. Ando K. Chinzei Y. J. Parasitol. 2002; 88: 664-672Crossref PubMed Scopus (19) Google Scholar, 33.Winger L.A. Tirawanchai N. Nicholas J. Carter H.E. Smith J.E. Sinden R.E. Parasite Immunol. 1988; 10: 193-207Crossref PubMed Scopus (99) Google Scholar, 34.Tomas A.M. Margos G. Dimopoulos G. van Lin L.H. de Koning-Ward T.F. Sinha R. Lupetti P. Beetsma A.L. Rodriguez M.C. Karras M. Hager A. Mendoza J. Butcher G.A. Kafatos F. Janse C.J. Waters A.P. Sinden R.E. EMBO J. 2001; 20: 3975-3983Crossref PubMed Scopus (164) Google Scholar), none of which have been demonstrated to be proteases. Plasmepsins (PM1, PM2, PM4, and HAP in P. falciparum but only PM4 in other Plasmodium spp. (15.Dame J.B. Yowell C.A. Omara-Opyene L. Carlton J.M. Cooper R.A. Li T. Mol. Biochem. Parasitol. 2003; 130: 1-12Crossref PubMed Scopus (67) Google Scholar)) have previously only been reported to be present in blood stage parasites. These proteases, localized to the digestive vacuole of the asexual blood stage parasites, participate in the metabolism of hemoglobin and perform other lysosomal functions (14.Banerjee R. Liu J. Beatty W. Pelosof L. Klemba M. Goldberg D.E. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 990-995Crossref PubMed Scopus (373) Google Scholar, 35.Wyatt D.M. Berry C. FEBS Lett. 2002; 513: 159-162Crossref PubMed Scopus (67) Google Scholar, 36.Bonilla J.A. Bonilla T.D. Yowell C.A. Fujioka H. Dame J.B. Mol. Microbiol. 2007; 65: 64-75Crossref PubMed Scopus (84) Google Scholar). Our data, based upon experiments of an avian model of malaria transmission, P. gallinaceum, suggest a novel role of plasmepsin 4 in the malaria parasite life cycle.Remarkably, little is known about transport and trafficking of secretory proteins in Plasmodium ookinete stages. Apicomplexan parasites, although morphologically defined based on the apical structure, characteristically have three types of secretory organelles found in Plasmodium in merozoites and sporozoites but not ookinetes (which only have micronemes) as follows: rhoptries (involved in invasion and formation of parasitophorous vacuole); dense granules (thought to be involved in maintenance of the parasitophorous vacuole); and micronemes (involved in the secretion of both soluble and transmembrane proteins). Transmission-blocking targets such as chitinase, CTRP, and WARP are found in micronemes and are discharged onto the ookinete surface to facilitate invasion of the mosquito midgut (8.Li F. Templeton T.J. Popov V. Comer J.E. Tsuboi T. Torii M. Vinetz J.M. J. Biol. Chem. 2004; 279: 26635-26644Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 25.Dessens J.T. Beetsma A.L. Dimopoulos G. Wengelnik K. Crisanti A. Kafatos F.C. Sinden R.E. EMBO J. 1999; 18: 6221-6227Crossref PubMed Scopus (230) Google Scholar, 37.Kaneko O. Templeton T.J. Iriko H. Tachibana M. Otsuki H. Takeo S. Sattabongkot J. Torii M. Tsuboi T. Parasitol. Int. 2006; 55: 227-231Crossref PubMed Scopus (14) Google Scholar). The micronemal localization and prominent apical staining pattern for PgPM4 and co-localization with chitinase, as demonstrated by deconvolution immunofluorescence microscopy, imply that these enzymes synergize in mosquito invasion at the level of the peritrophic matrix. This interpretation is further supported by P. gallinaceum membrane feeding experiments in which the combination of mAbs raised against PgCHT2 and PgPM4 reduces the oocyst number significantly compared with using either one of them individually. Again, the mechanism by which plasmepsin 4 is involved in ookinete infection of the mosquito has not been definitely established but must be experimentally distinguished from other potential protein and structural targets with which the ookinete comes into contact in the mosquito midgut.One potential concern about the presence of plasmepsin 4 in the ookinete culture supernatant is that perhaps it arises either from contaminating asexual stage parasites or spontaneous lysis of ookinetes during in vitro culture. In the P. gallinaceum cultures we performed, significant numbers of asexual forms are not observed because the gradient centrifugation step eliminates this stage of parasite. It is formally possible that ookinetes lysed under suboptimal culture conditions to a degree sufficient to yield detectable plasmepsin 4 in the culture supernatant. The results presented indicate that plasmepsin 4 is present within micronemes and on the parasite surface, and therefore its presence in ookinete culture supernatant is not parsimoniously explained by parasite lysis.Further evidence of the role of plasmepsin activity in parasite invasion of the mosquito midgut was suggested by the transmission blocking activity of aspartic protease-specific inhibitor pepstatin A, other peptidomimetic aspartic protease inhibitors, and calpain inhibitors.A number of studies have revealed an effect of pepstatin and other aspartic protease inhibitors on asexual blood stage parasite, suggesting one or more of the plasmepsins are essential for the survival of the parasites (38.Francis S.E. Gluzman I.Y. Oksman A. Knickerbocker A. Mueller R. Bryant M.L. Sherman D.R. Russell D.G. Goldberg D.E. EMBO J. 1994; 13: 306-317Crossref PubMed Scopus (250) Google Scholar, 39.Haque T.S. Skillman A.G. Lee C.E. Habashita H. Gluzman I.Y. Ewing T.J. Goldberg D.E. Kuntz I.D. Ellman J.A. J. Med. Chem. 1999; 42: 1428-1440Crossref PubMed Scopus (172) Google Scholar, 40.Nezami A. Luque I. Kimura T. Kiso Y. Freire E. Biochemistry. 2002; 41: 2273-2280Crossref PubMed Scopus (87) Google Scholar, 41.Rosenthal P.J. Exp. Parasitol. 1995; 80: 272-281Crossref PubMed Scopus (125) Google Scholar). However, these studies did not address whether plasmepsins or aspartic proteases may also be required for the parasite development outside the asexual stage. To determine the role of plasmepsins in midgut invasion and oocyst development, studies with pepstatin A and a variety of peptidomimetic and natural product aspartic protease inhibitors were performed using membrane feeding assays. As shown in Table 1, nontoxic concentrations of pepstatin A, SQ 3000, and Po/vPM led to inhibited oocyst formation and reduced Plasmodium infectivity to the mosquito in a dose-dependent manner. The effective concentration started as low as 0.1 μm. Consistent with the results of pepstatin A on asexual stages parasites (28.Banerjee R. Francis S.E. Goldberg D.E. Mol. Biochem. Parasitol. 2003; 129: 157-165Crossref PubMed Scopus (52) Google Scholar), immunoblot experiments on in vitro culture of ookinetes incubated with pepstatin A demonstrated that pepstatin A did not interfere with the processing of pro-plasmepsin 4, suggesting blocking of pro-plasmepsin 4 processing requires not only aspartic protease inhibitors but also other inhibitors, such as cysteine protease inhibitors, like what happened at asexual blood stages (28.Banerjee R. Francis S.E. Goldberg D.E. Mol. Biochem. Parasitol. 2003; 129: 157-165Crossref PubMed Scopus (52) Google Scholar). Furthermore, ookinete formation assay revealed that pepstatin A had no effect on the development of zygote to ookinetes in ookinete culture in vitro. These results suggest that PgPM4 was involved in penetration of peritrophic matrix/epithelial membrane and/or development of ookinete to oocyst.ALLN (calpain inhibitor I) and ALLM (calpain inhibitor II), inhibitors of the Ca2+-dependent neutral cysteine protease calpain I and calpain II, are also thought to be plasmepsin convertase inhibitors (28.Banerjee R. Francis S.E. Goldberg D.E. Mol. Biochem. Parasitol. 2003; 129: 157-165Crossref PubMed Scopus (52) Google Scholar). Previous studies have shown that at asexual stages calpain inhibitors I and II prevent activation of Plasmodium aspartic proteases (28.Banerjee R. Francis S.E. Goldberg D.E. Mol. Biochem. Parasitol. 2003; 129: 157-165Crossref PubMed Scopus (52) Google Scholar) through inhibition of falcipain (31.Drew M.E. Banerjee R. Uffman E.W. Gilbertson S. Rosenthal P.J. Goldberg D.E. J. Biol. Chem. 2008; 283: 12870-12876Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). However, our data indicate that although activation of pro-plasmepsin is unaffected by the addition of ALLN or ALLM to in vitro P. gallinaceum zygote cultures, membrane feeding experiments demonstrated that ALLM and high dose (20 μm) ALLN reduced oocyst numbers significantly. Calpain inhibitors did not interfere with the activation of the pro-plasmepsin 4 during the development of zygote to ookinete (data not shown), but calpain II and high concentrations of calpain I remarkably reduced ookinete formation and even block the ookinete development. It was obvious that the processing of pro-plasmepsin 4 occurred previous to the zygote stage, and some calpain-like proteins (other than PgPM4), which were critical for the development of zygote to ookinete, were inhibited by calpain inhibitors. In vitro, the activation of the rPgPM4 was pH-dependent and autocatalytically activated at pH 4.5. It is possible that in the ookinete plasmepsins traffic through the secretory organelle, microneme, to the plasma membrane of the parasite. However, it is not clear where the processing of pro-plasmepsin 4 takes place and whether an acidic pH is required inside the mosquito midgut. Western immunoblot experiments showed that PgPM4 found in the ookinete-conditioned medium is a catalytically active enzyme identical to that in the lysate of ookinete and zygote. There was no difference of PgPM4 expression patterns between conditioned medium/lysate under reduced and unreduced conditions (Fig. 2). The immunoelectron microscopy results demonstrated that PgPM4 was present in the microneme of ookinete, indicating it may be secreted via the microneme. The pH of the microneme remains unknown; however, it is possible that the pro-plasmepsin 4 is activated within the microneme prior to secretion. Alternatively, the activation/processing of pro-plasmepsin 4 may occur in acidic vacuoles in the zygote before secretion; EM micrographs have revealed many previously unreported cellular organelles in P. falciparum 3V. Bounkeua and F. Li, unpublished data. or mediated by other proteases at neutral pH after secretion. This interpretation is supported by evidence that P. falciparum plasmepsin PfPM2 not only digests hemoglobin in the food vacuole during its asexual proliferation in the erythrocytes but is also involved in remodeling the erythrocyte cytoskeleton by cleaving spectrin at neutral pH (32.Le Bonniec S. Deregnaucourt C. Redeker V. Banerjee R. Grellier P. Goldberg D.E. Schrével J. J. Biol. Chem. 1999; 274: 14218-14223Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar).Because it is possible that these inhibitors are affecting mosquito-secreted as well as ookinete-secreted proteases, we repeated these studies using monoclonal antibodies that specifically recognize ookinete-secreted plasmepsins. Comparison of both the mAbs and polyclonal antibody raised against PgPM4 with negative controls using isotype control or normal mouse serum, we demonstrate that anti-PM4 antibodies markedly reduced oocyst number and infection rate in a reproducible, dose-dependent manner. The concentration of mAb 2G4 that started to affect the transmission was as low as 50 μg/ml, consistent with the Western immunoblot result that showed that 2G4 had a stronger affinity for the mature enzyme, suggesting the efficiency and specificity of the mAb. Compared with negative control, the mAbs had no effect on ookinete formation and the processing of pro-plasmepsin 4 in the ookinete culture in vitro. These results suggested a possible role of PgPM4 involved in the mosquito midgut invasion and/or development of oocyst from ookinete. The experiments here indicate an important role of plasmepsin 4 in the molecular interaction of the malaria parasite with the mosquito midgut and/or the development of ookinete to oocyst. Further delineation of the mechanisms by which Plasmodium ookinete-secreted PM4 facilitates parasite invasion of the mosquito vector and/or development in mosquito midgut may suggest novel approaches to malaria control. IntroductionMalaria is the most important parasitic disease of man and a globally dominant cause of morbidity and mortality in humans (1.Sachs J. Malaney P. Nature. 2002; 415: 680-685Crossref PubMed Scopus (1381) Google Scholar). The lack of an effective vaccine, the emergence of drug-resistant Plasmodium falciparum and Plasmodium vivax, and insecticide-resistant vector mosquitoes all contribute to the increasing human toll of malaria. Malaria is transmitted through the bite of infected Anopheles mosquitoes. Preventing transmission from the human reservoir to the definitive host, the mosquito, is one approach to malaria control (2.Malkin E.M. Durbin A.P. Diemert D.J. Sattabongkot J. Wu Y. Miura K. Long C.A. Lambert L. Miles A.P. Wang J. Stowers A. Miller L.H. Saul A. Vaccine. 2005; 23: 3131-3138Crossref PubMed Scopus (181) Google Scholar). Delineating the mechanisms by which the malaria parasite invades and infects mosquitoes may lead to new strategies to block malaria transmission (3.Carter R. Mendis K.N. Miller L.H. Molineaux L. Saul A. Nat. Med. 2000; 6: 241-244Crossref PubMed Scopus (113) Google Scholar, 4.Vinetz J.M. Curr. Top. Microbiol. Immunol. 2005; 295: 357-382PubMed Google Scholar).After a mosquito ingests infectious Plasmodium gametocytes, male and female gametes emerge in the midgut and rapidly fuse to form diploid zygotes. Parasites must then develop into motile ookinetes, penetrate and traverse the protein- and chitin-containing peritrophic matrix, and then cross the midgut epithelium to form oocysts (4.Vinetz J.M. Curr. Top. Microbiol. Immunol. 2005; 295: 357-382PubMed Google Scholar). Developmentally regulated antigens of these stages are potential targets of antibodies induced by vaccination of the vertebrate host that are co-ingested with parasites as a mosquito takes a blood meal (5.Kaslow D.C. Chem. Immunol. 2002; 80: 287-307Crossref PubMed Scopus (70) Google Scholar). Such antibodies are called transmission-blocking antibodies, which act by interfering with parasite development within the mosquito midgut, thus preventing parasite transmission to the mosquito vector. Importantly, proteins expressed in the mosquito stages are less likely to be mutated in response to human immunological responses (6.Saul A. Curr. Opin. Infect. Dis. 2007; 20: 476-481Crossref PubMed Scopus (69) Google Scholar). Hence, interfering in this part of the life cycle has the potential to reduce both transmission as well as the spread of drug-resistant parasites.Ookinete-expressed proteases have been proposed to play vital roles in ookinete invasion of peritrophic matrix and mosquito midgut (7.Langer R.C. Li F. Vinetz J.M. Infect. Immun. 2002; 70: 102-106Crossref PubMed Scopus (18) Google Scholar, 8.Li F. Templeton T.J. Popov V. Comer J.E. Tsuboi T. Torii M. Vinetz J.M. J. Biol. Chem. 2004; 279: 26635-26644Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 9.Shen Z. Jacobs-Lorena M. J. Biol. Chem. 1997; 272: 28895-28900Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The peritrophic matrix is the first physical barrier faced by the ookinete as it escapes the blood meal. The peritrophic matrix is composed of proteins, glycoproteins, proteoglycans, and chitin (10.Shen Z. Jacobs-Lorena M. J. Biol. Chem. 1998; 273: 17665-17670Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 11.Moskalyk L.A. Oo M.M. Jacobs-Lorena M. Insect Mol. Biol. 1996; 5: 261-268Crossref PubMed Scopus (62) Google Scholar). Proteins, including chitin cross-linking proteins (peritrophins), have been reported to account for 22–55% of the total mass of the peritrophic matrix (10.Shen Z. Jacobs-Lorena M. J. Biol. Chem. 1998; 273: 17665-17670Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 11.Moskalyk L.A. Oo M.M. Jacobs-Lorena M. Insect Mol. Biol. 1996; 5: 261-268Crossref PubMed Scopus (62) Google Scholar). 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Parasitol. 2005; 143: 67-79Crossref PubMed Scopus (253) Google Scholar). No role for any plasmepsin has been demonstrated in Plasmodium-mosquito interactions.In this study, we demonstrate that the P. gallinaceum plasmepsin 4 (PgPM4) 2The abbreviations used are: PgPM4P. gallinaceum plasmepsin 4IFAindirect immunofluorescence assaymAbmonoclonal antibodyALLNcalpain inhibitor I (sequence N-acetyl-Leu-Leu-norleucinal (N-acetyl-Leu-Leu-Nle-CHO)ALLMcalpain inhibitor II (sequence N-acetyl-Leu-Leu-Met-CHO)PBSphosphate-buffered salineHPLChigh pressure liquid chromatographyCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidrPgPM4recombinant PgPM4. synergizes with the P. gallinaceum chitinase PgCHT2 (the ortholog of the P. falciparum chitinase (18.Li F. Patra K.P. Vinetz J.M. J. Infect. Dis. 2005; 192: 878-887Crossref PubMed Scopus (52) Google Scholar)) to facilitate malaria parasite invasion of the mosquito midgut and/or may be involved in the development of ookinete to oocyst. These data are the first to indicate a specific mechanistic function for a plasmepsin in any stage of the malaria parasite other than the asexual blood stage. This aspartic protease plays an important role in ookinete invasion of the mosquito midgut and/or parasite development, and thus may be a novel target of blocking malaria transmission.