Investigation on Replicative Aging in Cryptococcus Neoformans Populations

This application is a competitive renewal of a research proposal that investigated replicative aging in Cryptococcus neoformans, a human pathogenic fungus that is a major cause of mortality and morbidity in AIDS patients. Replicative aging leads to old C. neoformans cells, which are more resistant to clearing by the host cells, as well as antifungal treatment. The proposed studies are focused on continuing our prior studies by establishing mechanisms that convey the resilience of these old C. neoformans cells. The overarching hypothesis is that the observed resistance to phagocytic attack and antifungal drugs results from a combination of several physiological changes that occur in C. neoformans cells during replicative aging. These changes occur early in the replicative lifespan, and they synergize to further the selection and accumulation of old C. neoformans cells. In the past funding period, we made significant progress, including developing a high- throughput microfluidic chip device, which now enabled us to study morphology and protein expression in C. neoformans populations. Other discoveries obtained in the last funding cycle include knowledge about cell wall remodeling, altered mitochondrial function, and cloning of a novel efflux pump that is expressed in old C. neoformans cells. These findings now guide the proposed experimental approach. Four aims, each supported by comprehensive preliminary and published data, are proposed. Aim 1 will establish mechanisms how old C. neoformans cells are selected by macrophages. Aim 2 will seek to establish how altered mitochondrial function in old C. neoformans cells affects tolerance to antifungal drugs. In Aim 3, we propose to clone a mother enrichment mutant. Aim 4 will establish how vacuolar function of old C. neoformans cells affects tolerance to antifungal drugs.