Pneumocystis Biochemistry: Ubiquinones

University Of Cincinnati, Cincinnati OH

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Abstract

DESCRIPTION (Provided by the applicant): Pneumocystis carinii pneumonia (PcP) remains a prevalent AIDS-associated opportunistic infection. Anti-P. carinii drugs can produce adverse side effects in some patients and others can be ineffective due to the emergence of drug-resistant organisms. It is therefore important to have a broader armamentarium of alternative therapies for treating PcP. It is known that the anti-P, carinii drug atovaquone binds irreversibly to the cytochrome bc, complex as an analog of ubiquinone (CoQ). In P. carinii, the drug blocks mitochondrial electron transport resulting in the inhibition of respiration and ATP production. Correlations of mutations in the cytochrome b gene encoding CoQ binding sites have been described in some, but not all PcP atovaquone failure cases. Recently, it was shown that atovaquone also inhibited CoQ biosynthesis in P. carinii, identifying a second cellular process inhibited by this drug. Several other ubiquinone analogs also inhibited CoQ biosynthesis. Furthermore, it was demonstrated that the mitochondrlal and microsomal fractions of P. carinii both synthesize CoQ, and that only the process in microsomes was inhibited by atovaquone. The identification of a second cellular compartment with atovaquone sensitivity reveals another potential mechanism for drug-resistance. Four homologs, CoQ7 to CoQ10, were detected in P. carinii during metabolic radiolabeling experiments. The labeling patterns indicated that the specific activity decreased with increased length of the polyprenyl moiety, suggesting that the polyprenyl tail of completed CoQ molecules might have been elongated to form higher homologs. This mechanism has not been reported in any organism. Experiments in this proposed project will examine the possibility of distinct p-hydroxbenzoate (PHBA):polyprenyltransferase enzymes that transfer completed polyprenylchains with 7, 8, 9, and 10 isopentenyl units to PHBA for the synthesis of CoQ7, CoQ8, CoQ9 and CoQ10, respectively. Chain elongation of completed CoQ will also be examined. The specific aims of this project are (1) to identify CoQ synthesis in subceilular compartments and investigate whether electron transport occurs at the P. carinii cell surface, (2)to investigate the mechanisms by which four CoQ homologs are formed in the organism, (2) to test a number of compounds for their effects on P. carinii CoQ biosynthesis and growth in axenic cultures, and (4) develop atovaquone-resistant P. carinii populations to evaluate CoQ synthesis in sensitive and resistant organisms.