Cryptosporidium Parvum Microbial Adhesion: Interactions Between Sporozoites and Host Cells

147 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2000.Cryptosporidium parvum is a significant protozoal pathogen of both humans and animals worldwide. Since little is known about the mechanisms of attachment and invasion of C. parvum sporozoites to host cells, our goal was to elucidate the mechanism of sporozoite recognition of receptors on the host cell membrane. Knowledge of these mechanisms may allow for novel therapeutic strategies for cryptosporidiosis. An in vitro cell suspension binding assay was developed and used to investigate binding between C. parvum sporozoites and host cells. Morphologic features of binding events visualized with this assay were identical to those described in natural infections. A variety of glycoconjugates, as well as plasma membrane vesicles, were screened for their ability to block C. parvum sporozoite binding to MDBK cells. Of the naturally occurring and synthetic glycoconjugates tested, only mucins inhibited binding between sporozoites and host cells. Plasma membrane vesicles, which represent intact portions of plasma membrane, exhibited dose-dependent inhibition of binding. Solubilization of inhibitory activity from aqueous extracts of MDBK cells, followed by purification with ion exchange chromatography, was attempted. Complete solubility of inhibitory activity in aqueous buffers was not achieved, and inhibitory activity was polydispersed following ion exchange chromatography. The inhibitory activity of MDBK cell extracts was resistant to heat, protease digestion, and glycosidase treatment, suggesting it may be lipid or a lipid-like component. Organic solvent extraction of MDBK cells and bovine mucosa resulted in recovery of inhibitory activity. Purification of these extracts by preparative thin-layer chromatography yielded two major inhibitory fractions, a polar lipid fraction and a nonpolar fraction, with the nonpolar fraction containing the major inhibitory activity. This lipid was purified from bovine small intestinal mucosa by semipreparative HPLC followed by preparative HPTLC. Biochemical analyses, TLC staining techniques, mass spectrometry, and elemental analysis were used to partially characterize this lipid. The results of these analyses indicate the isolation of a small molecular weight, nonsaponifiable lipid lacking carbohydrate moieties, nitrogen, and phosphorus. Recognition of this novel intestinal membrane lipid by C. parvum sporozoites likely represents an early event in the overall adhesion and invasion process.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD