MOLECULAR BASIS OF VIRULENCE FACTORS OF ORAL FUNGAL SP.

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DESCRIPTION (provided by applicant): This is a Revision of a K22 Scholar Development and Faculty Transition Award application, submitted on behalf of Mary Ann Jabra-Rizk who is currently a Post-Doctoral Fellow in the Department of Diagnostic Sciences and Pathology at the U of MD Dental School. A 2 year Scholar Development phase and three years of Faculty Transition win be centered at U of MD involving co-mentors and consultants at Johns Hopkins, the U of VA and the U of Wurzburg and will establish Dr. Jabra-Rizk as an independent expert in oral fungal diseases. An Advisory Committee at the U of MD will evaluate her progress and aid in the Faculty Transition. The hypothesis to be tested during the Scholar Development is that C. dubliniensis is a species that exhibits constant cell surface hydrophobicity, possessing a homologue to the C. albicans CSHl gene which encodes a hydrophobic cell wall protein involved in cell adherence but differs in the expression of the MNN mannosylation family of genes and other putative virulence genes. Two aims are planned: 1. (a) Determine the presence of the C. albicans CSHl and CaMNN9 genes or homologues of the genes in C. dubliniensis using PCR primers designed based on the C albicans CSIII and CaMNN9 gene sequences. These will be subsequently amplified and cloned using C. dubliniensis CDS6 genomic DNA and probe hybridization. A C. dubliniensis knockout of the CSH 1 homologue gene will be generated using the latest technique (ura3 amxotrophic C. dubliniensis mutants in the URA-blaster) in gene disruption technique (b) Determine the effect of the disruption of the CdCShl gene on adherence by assessing the C. dubliniensis knockout clone for cell surface hydrophobicity7 adhesion to fibronectin, adherence to Fusobacterium nucleatum and pooled human buccal epithelial cells, in comparison to the wild type (c) Determine differences in the ability of macrophages to phagocytize 37C-grown C. albicans, 37C grown CdCSH1 mutant and wild type C. dubliniensis and the C. albicans mnn9 mutant in order to determine the effect of structural changes in the side chains of cell wall mannan on the ability of C. dubliniensis and C. albicans to evade host cell phagocytosis. 2. Based on cDNA microarray sequences of genes in the C. albicans genome, we will determine levels of differential expression of the CSHl and CaMNN9 genes between hydrophilic (37C-grown C. albicans) and hydrophobic (250C-grown C. albicans, 25 and 37C-grown C. dubliniensis and C. albicans mutants, A9V10 and Camnn9) yeast cells, as well as the C. dubliniensis CdCSHl mutant generated in Aim 1. Dr. Jabra-Rizic will subsequently utilize the information and mutants generated to investigate whole genome differences between C. dubliniensis and C. albicans in the Faculty Transition phase using DNA microarrays. She will also analyze differences in the levels of expression of hsp90 gene and other glycosylation and heat shock proteins genes in C. dubliniensis. In addition, a hsp90 knock out mutant of C. albicans will be generated and assessed for thermotolerance and phagcytosis by macrophages. Long range plans include using the mutants generated to study Candida mannoprotein-specific host immunomodulation, such as stimulation of cytokines and chemokines.