Several drugs that interact with membrane sterols or inhibit their syntheses are effective in clearing fungal infections but Pneumocystis is not cleared by many of these therapies. P. carinii normally synthesizes distinct delta7 C28 and C29 24-alkylsterols and some rare 24-alkyllanosterol derivatives, but the major fungal sterol ergosterol is not among them. Several Pneumocystis 24-alkylsterols are not characteristic of any other clinically important microbe. Currently, there is confusion with respect to P. carinii sterol biosynthesis because the components that are synthesized de novo have not been distinguished from those that are scavenged and those that are scavenged and then metabolized by P. carinii enzymes. Direct biochemical experiments are required to address this lack of information. The goals of this project are to identify sterols synthesized by P. carinii and to elucidate the major pathways by which they are formed. The hypotheses to be tested are: (A) the acetate-mevalonate pre-lanosterol pathway is functional, (B) the major post-lanosterol pathway goes through alkylation at C-24 of the sterol side chain prior to demethylation of C-14 of the sterol nucleus, (C) the P. carinii sterol delta24(28) reductase catalyzes the formation of methyl and ethyl groups in the beta configuration at C-24 of the sterol side chain, and (D) metabolism of the lanosterol nucleus first involves removal of one of two methyl groups at C-4, followed by demethylation at C-14, and then again at C-4; demethylation at C-14 precedes double bond isomerization at C-8 to C-7.
The specific aims of this project are to: (1) demonstrate the de novo pathway from acetate to lanosterol using radiolabeled precursors, (2) determine the rates of lanosterol metabolism using recombinant enzymes, and elucidate branched pathways using P. carinii homogenates with radiolabeled precursors and inhibitors known to block specific reactions in sterol synthesis, (3) analyze the stereochemical features of the P. carinii recombinant sterol delta24(28) reductase products by nuclear magnetic resonance spectroscopy, and (4) determine the sequence of reactions in sterol nucleus metabolism. Because Pneumocystis pneumonia remains a major opportunistic infection in AIDS patients worldwide, and because various drug resistant infectious microbes are emerging, there is a need to develop a greater variety of drugs that can clear the infection. To improve therapy and public health, it is critical to better understand the nature of this fungus. Since reactions in sterol biosynthesis are excellent targets for the development of antimycotic drugs, it is important to identify the sterols actually synthesized by Pneumocystis and to understand how this pathogen produces these vital membrane components.
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