Biochemical Characterization of Cytochrome b5 Reductase in Candida albicans

University Of South Florida, Tampa FL

Investigators

Abstract

DESCRIPTION (provided by applicant): Candida albicans is a commensal member of the human microflora and is the most common causative agent of fungal-related disease with particular significance in immuno-compromised individuals with HIV disease/AIDS, ranging from mild clinical manifestations such as oral thrush to a more serious disseminated candidiasis. Emerging drug resistance is a major problem in Candida, contributed by enzymes involved in the detoxification of xenobiotics and pharmacological agents. One such enzyme, cytochrome b5 reductase (cb5r), has a high pharmacological significance owing to its role in fatty acid elongation, ergosterol (or cholesterol in mammals) biosynthesis, and cytochrome P450-mediated detoxification of xenobiotics. The objective of this project is to characterize Candida cb5r and its electron acceptor, cytochrome b5 (cb5), as potential novel drug targets. Hence we have cloned cb5r and cb5 from C. albicans and have optimized recombinant protein expression in E. coli. SPECIFIC AIM 1 will characterize the biochemical, kinetic, and pharmacological properties of Candida cb5r in order to test the hypothesis that the fungal enzyme differs from its mammalian counterpart. SPECIFIC AIM 2 will assess the knockout phenotype of the two cb5r isoforms, CBR1 and MCR1, on cell growth and viability in response to pH, heat shock, and oxidative stress in Candida albicans and the yeast model system, Saccharomyces cerevisiae. This will allow me to test the hypothesis that the cb5r knockouts affect susceptibility to various environmental stresses. SPECIFIC AIM 3 will utilize protein pull-down assays and proteomic analysis to identify cytoplasmic and/or membrane-bound proteins that interact with C. albicans cb5r and cb5. Hence, in the final specific aim I will test the hypothesis that Candida cb5r interacts with other cellular proteins in addition to cb5. Phylogenetic analysis revealed that both cb5r and cb5 from Candida have highest homology to their plant counterparts, but lower homology to human cb5r and cb5. Interestingly, plant cb5r has been identified as a novel herbicidal target, making these plant inhibitors promising candidates as a new class of antifungals against Candida cb5r. This proposal will characterize a novel drug target in Candida, an opportunistic fungus that affects immuno-compromised individuals such as AIDS patients. Hence, identification of new drug targets has particular significance to combat multiple drug resistance in opportunistic fungal pathogens. PUBLIC HEALTH RELEVANCE: My overall long-term goal is to pursue a career in molecular microbiology, infectious disease, and biomedical research. I have had previous experience in these fields and know it is my passion and desire to remain in research. I find it particularly exciting that this project merges infectious disease with biochemistry and proteomics, with the overall goal of identifying novel drug targets for an important human pathogen. The research training plan in this proposal allows me to learn new cutting edge methods in biochemistry and proteomics, while also enhancing my training as a molecular microbiologist. To accomplish these goals, this project will advance my knowledge of experimental design and accompanying literature review. It will furthermore improve my skills in molecular methods, such as targeted gene disruption (gene knockout) and recombinant protein expression. I will also learn new techniques used in biochemistry and proteomics, such as mass spectrometry, protein pull-down assays, and other methods involved in the characterization of proteins.