Pneumocystis carinii Pneumonia (PCP) is the major life-threatening opportunistic infection associated with AIDS. Recently presented molecular evidence from this group suggests that drug-resistant P. carinii is emerging. This could have serious public health implications. The evidence for drug resistance, however, is only indirect, since we are unable to reliably assess in vitro drug sensitivities of P. carinii clinical isolates. The goal of this project is to use yeast models to determine whether, and to what extent, specific mutations confer drug resistance. The investigators will focus on two genes - dihydropteroate synthase (DHPS), which is the target for sulfa and sultones, and cytochrome b, which is the target for atovaquone. Mutations in the P. carinii DHPS gene have been found which are associated with sulfa prophylaxis failures. Mutations in the P. carinii cytochrome b gene in patients failing atovaquone prophylaxis have been found as well. These mutations might confer resistance since they are similar to mutations that cause resistance to related compounds in other organisms.
The aims of the proposed project are to: Engineer clinically observed DHPS mutations into transgenic yeast (DHPS Saccharomyces cerevisiae expressing the P. carinii sp.f. hominis DHPS). Then the investigators will determine whether and to what extent these mutations confer resistance by assessing the effects of SMX on growth and on the DHPS activity of the purified recombinant protein. Construct yeast clones with mutations in their mitochondrially-encoded cytochrome b genes that are identical or similar to those found in P. carinii clinical isolates. The investigators will then determine how atovaquone affects a) the growth of the mutant strains in culture and b) electron transport in mitochondria isolated from these strains. Determine whether other sulfa drugs or hydroxynaphthoquinones retain efficacy against mutant targets both in whole yeast and in enzyme preparations. Ultimately, it is hoped that the results of this study will facilitate the rational use of antipneumocystis drugs.
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