Function of PHR1 in Candida albicans cell wall assembly
Thesis (Ph.D.)--Georgetown University, 2011.; Includes bibliographical references.; Text (Electronic thesis) in PDF format. Candida albicans is usually present as a commensal of the mucosal surfaces of the gastrointestinal and urogenital tracts but it is a major cause of hospital-acquired infection in immunocompromised individuals. Candida spp. are the fourth most common cause of bloodstream infection. Moreover, the mortality rate of systemic candidiasis is very high due to the difficulty in diagnosis and treatment. Candida albicans is a polymorphic fungus that can change its morphology ranging from hyphae to yeast. The ability to switch between these morphologies is believed to be involved in the pathogenesis and virulence of this organism. The Candida albicans PHR1 (pH-regulated) gene is an alkali-expressed gene that encodes a 548 amino acid residue GPI-anchored cell surface glycoprotein. The deletion of PHR1 in C. albicans resulted in pH-conditional defects of cell morphology and virulence. The enzyme encoded by PHR1 is required for proper cross-linking of beta-1,3-glucans and beta-1,6-glucans in the cell wall. Both this enzyme and the cell wall itself are absent in host cells, which makes it an interesting target for new antifungal drug development.; We developed methods to study protein-protein interactions which combined protein cross-linking, Triton℗ʼX-114 extraction, ion exchange chromatography, 2 dimensional SDS-PAGE and mass spectrometry of an epitope tagged Phr1p that proved successful in analyzing interactions of membrane bound Phr1p. Therefore, these procedures might be useful for studying other membrane proteins. The results showed that the Phr1p forms a complex about double the size of Phr1p and mass spectrometry indicated that Phr1p was the only major component of this complex. Together this suggests that Phr1p forms a dimer. The dimer was enriched in the detergent-rich phase after Triton℗ʼX-114 extraction and phase separation, an observation consistent with the dimer retaining a hydrophobic GPI-anchor moiety. Western Blotting results showed about 25-60% of Phr1p was in a dimeric form which may indicate that the function of the dimeric form of Phr1p is distinct from that of the monomeric form.
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