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 M and differentially inhibited two chromatographically separable P. gallinaceum ookinete-produced chitinase activities with IC 50 values of 7 and 12 M, 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.

Development of optimal passive and active immunization strategies for cryptosporidiosis is dependent on the identification and characterization of functional parasite molecules which can be targeted. Each of a subset of five monoclonal antibodies (mAb) produced against immunoaffinity-isolated C. parvum antigens elicited distinct morphologic changes in sporozoites and merozoites, characterized by progressive formation and eventual release of membranous antigen-mAb precipitates from the sporozoite posterior m g g s MW, Stone AL, Arrowood MJ, Langer RC, Yount PA. Abstract # C46, Thud International Workshop on Pneumocystis, Toxoplarma, Cryptosporidium and Microsporidia (1994) Cleveland, OH; E g g s MW, Stone AL, Yount PA, Arrowood MJ, Langer RC, Bentley DL. Protective MQnQClOnd Antibodies Define a Ckcumspsrozoite-like Glycooproteh Exoantigen of Cryptosporidium parvum Sporozoites and Merozoites, submitted]. These changes were similar to, but more rapid in development and prominent than thoae elicited by therapeutic bovine colostral anti-C. parvum antibody we described previously [8], and resembled the malarial circumsporozoite precipitate (CSP) reaction [3]. Infectivity of sporozoites having undergone the CSP-like reaction was neutralized. Further, mAbs which elicited the CSP-like reaction provided marked passive protection against C. parvum oocyst challenge in neonatal BALB/c mice. The antigen mechanistically involved in the CSP-like reaction was released initially from the apical end of sporozoites. Cytoskeletal inactivation with cytochalasind did not inhibit apical release of the antigen, but did inhibit its posterior translocation. The CSP-like reaction was morphologically and antigenically distinct from P23 trail deposition [2] previously described for motile C. parvum sporozoites. The antigen recognized by mAbs which elicited the CSPlike reaction was localized to sporozoite apical complex organelles, including electrondense granules, by immunoelectron microscopy. In western blots of sporozoites or merozoites, the mAbs recognized multiple 46-230 kDa antigens and a high M, antigen, designated CSL, which variably migrated between 1200-1400 kDa in reducing SDS-

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.

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