This proposal's long-term aim is to improve the treatment of Pneumocystis carinii infections in AIDS patients by providing a basis for improving therapeutics used to treat Pneumocystis pneumonia. Pneumocystis pneumonia is a common opportunistic infection and leading cause of death of AIDS patients. The applicant proposes to use Saccharomyces cerevisiae to determine the mechanism by which pentamidine, the primary therapeutic drug used to prevent and to treat Pneumocystis pneumonia, acts. In addition to determining the mechanism by which pentamidine acts, this work will identify mutations that cause and determine whether Saccharomyces is a useful model for P. carinii. The proposal relies upon the evolutionary relationship of S. cerevisiae and P. carinii. This relationship suggests that the cellular targets of pentamidine and the mechanisms of pentamidine resistance will be the same in both fungi. Genetic and metabolic experimental approaches are proposed that are feasible in S. cerevisiae but impossible in P. carinii, because the latter cannot be grown in pure culture or genetically manipulated. The primary genetic approach is characterization of pentamidine resistant yeast mutants. The pentamidine resistance genes will be cloned, sequenced, and disrupted to try to determine their functions. Homologous genes from P. carinii will be identified by molecular techniques and transferred to S. cerevisiae in which the corresponding gene has been disrupted to verify that they serve homologous functions. The metabolic effect of pentamidine on suspected cellular targets yeast respiration, nucleic acid metabolism, and vacuole function will be determined. The inhibitory effects of pentamidine analogs will be compared to those of pentamidine to determine whether the drugs have common cellular targets.
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