Conclusions About Functional Relationships Between the Candida Albicans *Als Proteins Drawn From Analysis of ALS Gene Expression Patterns

196 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2004.The agglutinin-like sequence (ALS) gene family of C. albicans consists of eight genes, some of which encode proteins with adhesive functions. Protein function across the entire family is unknown. The central assumption of this study was that ALS gene expression data could be used to provide information about Als protein function. Family-wide ALS gene expression was surveyed across different models of oral and disseminated candidiasis and across budding and filamentous growth of cultured cells. Methods of detection of gene expression included reverse transcriptase polymerase chain reaction (RT-PCR) analysis of RNA, and flow cytometry and immunohistochemical detection of green fluorescent protein reporter constructs (PALS-GFP). Results demonstrated that differential expression of ALS genes occurred at the level of transcriptional intensity, rather than at the level of "on" or "off". Changes in ALS1 and ALS3 expression were the most readily detectable across culture conditions, infection models, and detection methods. The expression data were used to design experiments to test the functions of selected Als proteins. Microarray analysis was used to profile the genome-wide transcription of a homozygous, als1/ als1 mutant, under conditions where ALS1 expression was determined to be maximal in the PALS1-GFP reporter strain. Microarray data suggested that Als1p plays a role in cell division and that Als4p and Als9p may partially compensate for the lack of Als1p in the als1/als1 mutant. Strains that overproduced the N terminus of Als1p or Als3p, were tested for relative virulence in the murine model of disseminated candidiasis. This approach was suggested by data from the PALS1-GFP and PALS3-GFP reporter strains that revealed high levels of ALS1 and ALS3 expression in lesions within parenchymal organs, and also because the N-terminal domain of Als3p is known to possess adhesive function. Increased survival of mice inoculated with the Als N-terminal overproducing strains suggested the potential for therapeutic usage of Als proteins to reduce C. albicans adhesion and colonization in patients at risk for developing candidiasis. The work presented here is the most thorough analysis of gene expression in a C. albicans gene family to date, and demonstrates the utility of gene expression data in subsequent protein functional analysis.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD