Date of Award

8-2007

Document Type

Thesis

Degree Name

Master of Science (MS)

Legacy Department

Microbiology

Advisor

Temesvari, Lesly

Committee Member

Scott , Thomas

Committee Member

Paul , Kimberly

Abstract

Entamoeba histolytica is a parasitic human protozoan that infects 500,000,000 people worldwide annually. In the course of the parasite's life cycle, motile trophozoites breach the colonic mucosa, invade through the epithelial layer and extracellular matrix (ECM) and occasionally disseminate through portal blood vessels to distant organs. Membrane rafts are small heterogeneous, highly dynamic, sterol- and sphingolipid- enriched domains whose functional significance entails compartmentalization of cellular processes and regulation of cellular signaling. Recent studies reveal the physiological role of membrane rafts in adhesion to host epithelium in E. histolytica. In the current study we examined the role of lipid rafts in adhesion of trophozoites to host ECM components, collagen and fibronectin. A high throughput fluorescence based assay was developed to assess parasitic adhesion to commercial collagen type I- and fibronectin-coated microtiter plates. Disruption of membrane rafts by treatment with a cholesterol extracting agent, methyl-beta-cyclodextrin (MβCD), resulted in inhibition of adhesion to ECM. Replenishment of cholesterol by treatment with a lipoprotein-cholesterol concentrate (LCC) restored adhesion. Confocal microscopy, using fluorescent lipid analogs, revealed enrichment of lipids at the parasite-ECM interface. The galactose inhibitable Gal/GalNAc lectin is a glycoprotein on E. histolytica that is a known resident of lipid rafts and mediates adhesion to host cells. Adhesion to collagen was observed to decline in the presence of galactose, suggesting a role for the Gal/GalNAc lectin as a putative receptor mediating adhesion to collagen. On the other hand, adhesion to fibronectin was not impaired by galactose, suggesting that the Gal/GalNAc lectin is not involved in adhesion of E. histolytica to fibronectin. This study has offered new insight into the molecular mechanisms of adhesion, which is important to the pathogenesis of amoebiasis. Such insight may lead to the development of innovative therapeutic modalities and vaccines.

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Microbiology Commons

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