C. neoformans is the cause of 10% of all opportunistic infections in patients with AIDS. Therapy in these patients is complicated by the poor response rate, the need for chronic suppressive therapy, and intolerance to 5-fluorocytosine. Alternative and less toxic therapies would considerably enhance our ability to treat this disease. The proposed studies will begin with what is known of the genetics and biology of C. neoformans and begin to apply molecular biological techniques to address three areas of C. neoformans biology and pathobiology. Appreciation for the mechanisms of genetic control is paramount for the understanding of virulence in C. neoformans. One of our long-term goals is the dissection of the factors responsible for virulence in C. neoformans. In order to begin to understand these controls, we have developed a transformation system for C. neoformans. This allows for the first time the introduction of exogenous DNA into this pathogen. The current transformation system is not ideal as it appears to lack a true ARS and centromeric sequences. The proposed studies will seek these functions and should result in the production of vectors for the stable transformation of C. neoformans. Virulence in C. neoformans is associated with the ability to grow at 37degreesC, phenoloxidase activity, and the presence of capsule. The genes responsible for the production of capsule will be isolated by complementation of acapsular mutants. The phenoloxidase gene will be identified by its ability to form pigment on DOPA media. The genetic changes that underlie the formation of the sexual state are presumably initiated by secreted pheromones. The presence of these messengers will be tested by assessing morphologic and metabolic changes in response to extracts of opposite mating types. Genes for the pheromones and other sex-specific genes will be isolated by differential hybridization and the isolation of sterile mutants of C. neoformans. As sex pheromones result in G1 arrest of susceptible cells, they may provide novel targets for anticryptococcal therapy. Characterization of the pheromone receptors may eventually allow dissection of the signal transduction system in C. neoformans, which again may provide a therapeutic target. Even in the case that the pheromone-receptor axis is not a viable chemotherapeutic target, isolation of these genes and study of their control will be provide valuable insights into the gene regulation of C. neoformans.
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