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Cryptococcus neoformans, the leading cause of fungal meningitis, is a fungal pathogen that causes severe infection of the central nervous system in patients with compromised immune systems, typically caused by HIV/AIDS. C. neoformans infections are present in developed countries including the United States, but most fatalities occur in sub-Saharan Africa where antiretroviral therapy, the treatment for HIV/AIDS, is less accessible. Current treatments for severe cryptococcal infections are extensive and outdated. There is a critical need for an improved understanding of the fungus and new targeted therapies. Our goal is to identify metabolic pathways important to the survival of C. neoformans in the human host that can then be targeted for the development of new antifungal reagents. Lung alveolar macrophages, which present a first line of host defense against C. neoformans infection, provide a glucose- and amino acid-poor environment, and nonpreferred carbon sources such as lactate and acetate are likely important early in establishment of a pulmonary infection. Utilizing a genetic screen performed by a graduated PhD student in my lab to identify genes necessary for growth on acetate, we have discovered that the last step of carnitine biosynthesis is required. Our goal was to identify other steps of the carnitine biosynthetic pathway. Using the amino acid sequence of the fungal Candida albicans 4-trimethylaminobutyraldehyde dehydrogenase (TMABADH), the third enzyme of the carnitine biosynthesis pathway which converts 4-trimethylaminobutyraldehyde (TMABA) to gamma-butyrobetaine (γBB), we identified CNAG_05113 as the encoding gene in C. neoformans. Using a strain in which the CNAG_05113 gene was deleted, the mutant was tested for growth and virulence deficiencies. CNAG_05113 cells have inhibited growth in conditions with acetate as the sole carbon source. When reintroduced to carnitine and carnitine pathway intermediates, growth of mutant cells was restored. These results indicate that CNAG_05113 encodes the third step in carnitine biosynthesis. Future research is to identify the genes encoding other steps of the carnitine biosynthesis pathway and to biochemically characterize the encoded enzymes.