Plasmodium ookinete-secreted chitinase and parasite penetration of the mosquito peritrophic matrix.

Malaria transmission-blocking strategies aimed at disrupting parasite-mosquito interactions have the potential to make important contributions to global malaria control. It has been suggested that Plasmodium-secreted chitinase plays a crucial role in allowing the ookinete to initiate its invasion of the mosquito midgut, which suggests that this enzyme is a candidate target for blocking malaria transmission. In this review, the authors discuss Plasmodium chitinases from the molecular, biochemical and cell biology viewpoints. Future directions of study could involve developing strategies for interrupting the function of Plasmodium chitinases within the mosquito midgut, including transmission-blocking drugs or vaccines, or the development of chitinase-inhibitor-producing transgenic mosquitoes.

Disruption of Plasmodium falciparum chitinase markedly impairs parasite invasion of mosquito midgut.

To initiate invasion of the mosquito midgut, Plasmodium ookinetes secrete chitinolytic activity to penetrate the peritrophic matrix surrounding the blood meal. While ookinetes of the avian malaria parasite Plasmodium gallinaceum appear to secrete products of two chitinase genes, to date only one chitinase gene, PfCHT1, has been identified in the nearly completed Plasmodium falciparum strain 3D7 genome database. To test the hypothesis that the single identified chitinase of P. falciparum is necessary for ookinete invasion, the PfCHT1 gene was disrupted 39 bp upstream of the stop codon. PfCHT1-disrupted parasites had normal gametocytogenesis, exflagellation, and ookinete formation but were markedly impaired in their ability to form oocysts in Anopheles freeborni midguts. Confocal microscopy demonstrated that the truncated PfCHT1 protein was present in mutant ookinetes but that the concentration of mutant PfCHT1 within the apical end of the ookinetes was substantially reduced. These data suggest that full-length PfCHT1 is essential for intracellular trafficking and secretion and that the PfCHT1 gene product is necessary for ookinetes to invade the mosquito midgut.

Characterization of an intestinal epithelial cell receptor recognized by the Cryptosporidium parvum sporozoite ligand CSL.

The protozoan parasite Cryptosporidium parvum is a leading cause of diarrhea in humans and neonatal calves. The absence of approved parasite-specific drugs, vaccines, and immunotherapies for cryptosporidiosis relates in part to limited knowledge on the pathogenesis of zoite attachment and invasion. We recently reported that the C. parvum apical complex glycoprotein CSL contains a zoite ligand for intestinal epithelial cells which is defined by monoclonal antibody (MAb) 3E2. In the present study, the host cell receptor for CSL was characterized. For these studies, a panel of epithelial and mesenchymal cell lines was examined for permissiveness to C. parvum and the ability to bind CSL. Cells of epithelial origin were significantly more permissive and bound significantly greater quantities of CSL than cells of mesenchymal origin. Caco-2 intestinal cells were selected from the epithelial panel for further characterization of the CSL receptor. Immunoelectron microscopy demonstrated that CSL bound initially to the surface of Caco-2 cells and was rapidly internalized. The molecule bound by CSL was identified as an 85-kDa Caco-2 cell surface protein by radioimmunoprecipitation and CSL affinity chromatography. Sporozoite incubation with the isolated 85-kDa protein reduced binding of MAb 3E2. Further, attachment and invasion were significantly inhibited when sporozoites were incubated with the 85-kDa protein prior to inoculation onto Caco-2 cells. These observations indicate that the 85-kDa protein functions as a Caco-2 cell receptor for CSL. CSL also bound specifically to intestinal epithelium from calves, indicating receptor expression in a second important host species. Molecular characterization of the CSL receptor may lead to novel avenues for disrupting ligand-receptor interactions in the pathogenesis of C. parvum infection.

Micronemal transport of Plasmodium ookinete chitinases to the electron-dense area of the apical complex for extracellular secretion.

Plasmodium ookinetes secrete chitinases to penetrate the acellular, chitin-containing peritrophic matrix of the mosquito midgut en route to invasion of the epithelium. Chitinases are potentially targets that can be used to block malaria transmission. We demonstrate here that chitinases of Plasmodium falciparum and P. gallinaceum are concentrated at the apical end of ookinetes. The chitinase PgCHT1 of P. gallinaceum is present within ookinete micronemes and subsequently becomes localized in the electron-dense area of the apical complex. These observations suggest a pathway by which ookinetes secrete proteins extracellularly.

Chitinases of the avian malaria parasite Plasmodium gallinaceum, a class of enzymes necessary for parasite invasion of the mosquito midgut.

The Plasmodium ookinete produces chitinolytic activity that allows the parasite to penetrate the chitin-containing peritrophic matrix surrounding the blood meal in the mosquito midgut. Since the peritrophic matrix is a physical barrier that the parasite must cross to invade the mosquito, and the presence of allosamidin, a chitinase inhibitor, in a blood meal prevents the parasite from invading the midgut epithelium, chitinases (3.2.1.14) are potential targets of malaria parasite transmission-blocking interventions. We have purified a chitinase of the avian malaria parasite Plasmodium gallinaceum and cloned the gene, PgCHT1, encoding it. PgCHT1 encodes catalytic and substrate-binding sites characteristic of family 18 glycohydrolases. Expressed in Escherichia coli strain AD494 (DE3), recombinant PgCHT1 was found to hydrolyze polymeric chitin, native chitin oligosaccharides, and 4-methylumbelliferone derivatives of chitin oligosaccharides. Allosamidin inhibited recombinant PgCHT1 with an IC(50) of 7 microM and differentially inhibited two chromatographically separable P. gallinaceum ookinete-produced chitinase activities with IC(50) values of 7 and 12 microM, respectively. These two chitinase activities also had different pH activity profiles. These data suggest that the P. gallinaceum ookinete uses products of more than one chitinase gene to initiate mosquito midgut invasion.

Cryptosporidium parvum apical complex glycoprotein CSL contains a sporozoite ligand for intestinal epithelial cells.

Cryptosporidiosis, caused by the apicomplexan parasite Cryptosporidium parvum, has become a well-recognized diarrheal disease of humans and other mammals throughout the world. No approved parasite-specific drugs, vaccines, or immunotherapies for control of the disease are currently available, although passive immunization with C. parvum-specific antibodies has some efficacy in immunocompromised and neonatal hosts. We previously reported that CSL, an approximately 1,300-kDa conserved apical glycoprotein of C. parvum sporozoites and merozoites, is the antigenic species mechanistically bound by neutralizing monoclonal antibody 3E2 which elicits the circumsporozoite precipitate (CSP)-like reaction and passively protects against C. parvum infection in vivo. These findings indicated that CSL has a functional role in sporozoite infectivity. Here we report that CSL has properties consistent with being a sporozoite ligand for intestinal epithelial cells. For these studies, native CSL was isolated from whole sporozoites by isoelectric focusing (IEF) following observations that the approximately 1,300-kDa region containing CSL as seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was comprised of approximately 15 molecular species (pI 3 to 10) when examined by two-dimensional (2-D) electrophoresis and silver staining. A subset of six approximately 1,300-kDa species (pI 4.0 to 6.5) was specifically recognized by 3E2 in 2-D Western immunoblots of IEF-isolated CSL. Isolated native CSL bound specifically and with high affinity to permissive human intestinal epithelial Caco-2 cells in a dose-dependent, saturable, and self-displaceable manner. Further, CSL specifically bound to the surface of live Caco-2 cells inhibited sporozoite attachment and invasion. In addition, sporozoites having released CSL after incubation with 3E2 and occurrence of the CSP-like reaction did not attach to and invade Caco-2 cells. These findings indicate that CSL contains a sporozoite ligand which facilitates attachment to and invasion of Caco-2 cells and, further, that ligand function may be disrupted by CSL-reactive monoclonal antibody. We conclude that CSL is a rational target for passive or active immunization against cryptosporidiosis.

Protective monoclonal antibody defines a circumsporozoite-like glycoprotein exoantigen of Cryptosporidium parvum sporozoites and merozoites.

The apicomplexan protozoan parasite Cryptosporidium parvum causes a diarrheal disease in humans and other mammals for which specific therapy and immunoprophylaxis are unavailable. Passive immunization with Abs against whole C. parvum organisms has variable efficacy in immunocompromised or neonatal hosts. Because apical and surface-exposed zoite Ags of the Apicomplexa are critical to infectivity and targets of protective immunity, we examined the ability of mAbs generated against such Ags in C. parvum sporozoites to passively protect against infection and identify biologically relevant parasite molecules. A panel of mAbs was produced against affinity-purified native Ags using sporozoite apical- and surface-reactive mAb C4A1 as binding ligand. One resulting mAb, designated 3E2, elicited prominent morphologic changes in sporozoites and merozoites characterized by rapid and progressive formation, posterior movement, and release of membranous Ag-mAb precipitates. These changes had a striking resemblance to the malarial circumsporozoite precipitate (CSP) reaction. Sporozoite infectivity was completely neutralized after in vitro exposure to 3E2 and the CSP-like reaction. Furthermore, orally administered 3E2 completely prevented or markedly reduced infection in neonatal BALB/c mice. 3E2 bound to apical complex and surface molecules of zoites and was demonstrated in membranous precipitates by immunoelectron microscopy. In Western blots, 3E2 recognized multiple 46 to approximately 770 kDa sporozoite Ags and an approximately 1300-kDa Ag designated CSL, also expressed by merozoites. CSL was characterized as a soluble glycoprotein exoantigen released by infectious sporozoites. Further, CSL was determined to be the molecular species mechanistically involved in the CSP-like reaction by its identification in SDS-PAGE gels and Western blots of purified membranous precipitates. These findings indicate that CSL has a functional role in sporozoite infectivity and is a candidate molecular target for passive or active immunization against cryptosporidiosis.