Characterization of novel Cryptosporidium parvum antigens and host cell cytoskeleton response to infection

Cryptosporidium parvum infection involves attachment to and invasion of enterocytes, with subsequent modifications to the niche occupied by the parasite. My research involves investigations of parasite factors involved in and host cytoskeletal changes induced by the infection process. Specifically, we have generated two hybridoma clones, CMYL3 and CMYL30, by fusing mouse myeloma cells with spleen cells from Balb/c mice immunized with excysted oocysts of Cryptosporidium muris. These clones secreted monoclonal antibodies against an oocyst wall antigen (apparent molecular mass of 250 KDa, called CM250) from both C. muris, and C. parvum. Immunofluorescence and confocal microscopy on purified oocysts and infected mouse tissues revealed that the staining was confined to the oocyst wall of both Cryptosporidium species. In addition, I have cloned a novel C. parvum gene and determined the expression pattern of the gene product. Immunofluorescence microscopy detected CP2 on sporozoites and type I meronts in infected HCT-8 cells. Immunogold electron microscopy revealed that CP2 consistently localized to the parasitophorous vacuole membrane/vacuole membrane (PVM/VM) of trophozoites and meronts. In addition, progression from macrogametocyte to sporulated oocyst revealed CP2 initially at the periphery of amylopectin-like granules, in the cytoplasm and discrete vesicles, the parasitophorous vacuole, on the surface of sporozoites, and finally, on the PVM. The observed expression pattern suggests that CP2 may be involved in the invasion process and/or PVM/VM integrity. I have also characterized the role of the actin-binding protein tropomyosin during infection. CMYL, together with anti-CP2 was used to label infection sites. Infections of the human ileocecal cell line, HCT-8, revealed a consistent accumulation of tropomyosin 5 (TM5) to the site of infection. Infected mouse ileum revealed an accumulation of the actin/tropomyosin network to the apical surface of infected cells. To address a possible function of tropomyosin during the infection process, CHO cell lines overexpressing tropomyosin isoforms were infected. Cell lines overexpressing TM5 consistently revealed a significant increase in the number of mature type I meronts at early time points indicating that regulation of actin dynamics by tropomyosin, specifically TM5, may be involved in the infection process.