Investigating the role of CNAG_05113 in the carnitine biosynthesis pathway in \u3ci\u3eCryptococcus neoformans.\u3c/i\u3e

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 Current treatments for severe cryptococcal infections are extensive and outdated.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

Elucidating Acetate Metabolism: Identification of Transporters and Enzymes Required for Acetate Utilization in the Fungal Pathogen Cryptococcus Neoformans

Cryptococcus neoformans is the leading cause of fungal meningitis world-wide. While exposure to this environmental sporophyte is common during childhood, those who are immune compromised are at risk of infection. Following inhalation, this basidiomycetous fungus subsequently colonizes other organs though hematogenous dissemination, eventually crossing the blood brain barrier and colonizing the brain where it causes as cryptococcal meningitis. Changes in the availability of carbon sources stemming from the movement from soil to the lungs induce changes in fungal metabolism. Specifically, alveolar macrophages, which present a first line of defense against infection, provide a glucose-/amino acid-poor environment. As such, the use of alternate carbon sources such as acetate and lactate may be needed to establish pulmonary infection. The focus of this study is the utilization of the non-preferred carbon source acetate. Following the generation of acetyl-CoA in the cytosol, Cryptococcus possesses two parallel pathways for import of acetyl-CoA into the mitochondria. The first is the carnitine shuttle which imports acetyl groups via acetyl-carnitine. We have identified three genes which could encode for carnitine acetyltransferases, CNAG_00537, CNAG_006551 and CNAG_05042, and a gene for the acyl-carnitine translocase ACUH (CNAG_00499). The carnitine shuttle has not been characterized in C. neoformans and its role in virulence is unreported. The second pathway, the glyoxylate shunt, imports glyoxylate cycle intermediates into the mitochondria in exchange for metabolites required for gluconeogenesis. We have identified two potential transporters, AcuL (CNAG_02288) and Dic (CNAG_04712). Our characterization of the C. neoformans deletion mutants of the genes encoding for components of the acetate utilization pathway reveal differences to the pathways previously described from Saccharomyces cerevisiae and Candida albicans. This suggests that mechanisms of alternative carbon source utilization are not conserved across fungi. Based on the requirement of ACUL, CNAG_00537, and CNAG_006551 for virulence, and their divergence from or complete lack of human orthologs, we propose these enzymes and transporters could be targets for improved antifungal drugs