A common characteristic of parasites is their ability to utilize many host molecules with a concomitant abbreviation of their own metabolism. However, recent studies in lipid metabolism of parasites have demonstrated that some organisms synthesize low of 'sparking' levels of 'metabolic' lipids which cannot be substituted by host lipids for parasite-specific enzymatic reactions and membrane integrity. 'Metabolic' lipids occur in low concentrations but have molecular structures that are very different from those of the host. Preliminary data suggest that Pneumocystis carinii scavenges many host lipids, but careful analyses of individual lipid classes indicate that the parasite contains lipids that are compositionally distinct. Thus, it is hypothesized that (1) the parasite transports and utilizes host lipids for the bulk of its own membrane lipids and (2) it de novo synthesizes and/or tailors unique lipids to fit the specifications of its vital functions. One goal of this project is to determine whether or not P. carinii synthesizes 'metabolic' lipids by employing radiolabeled precursors together with an inhibitor compound. Detection and identification of radiolabeled metabolites resulting from the block will be evidence that the parasite has retained the pathway leading to the formation of that lipid. Another goal of this project is to determine the extent to which P. carinii tailors its unique lipids. Analyses of changes in radiolabeled or fluorescent compounds presented to the organism will provide information on these metabolic capacities. Pneumocystis organisms will be analyzed for the presence of specific lipases that may be involved in lipid tailoring. Since P. carinii lives in an environment rich in lung surfactant lipids and its bulk lipids reflect those of the host, we further hypothesize that (3) it has a growth requirement for pre-made lipids. Thus, specific lipids that are taken up and incorporated into the lipids of the organism in vitro will be identified in this project by employing radioisotope methods, fluorescence-activated cell sorter techniques, quantitative microfluorimetry and time-resolved digital image analysis. Processes occurring in mixed populations, preparations of enriched or pure life-cycle stages, and individual organisms will be examined. Fluorescent lipids will be instilled into the lungs of infected rats to examine the uptake and metabolism of these compounds by the organisms in situ. A better understanding of lipid uptake and metabolism of P. carinii should provide insights into the nutrition of the parasite which in turn would aid in the eventual formulation of axenic media for the continuous cultivation of this important human parasite. Transport of dietary lipids, reactions in the synthesis of 'metabolic' lipids, and reactions in the tailoring of parasite-specific lipids provide attractive targets for drug development.
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