Functional Characterization and Localization of Pneumocystis carinii Lanosterol Synthase

Organisms in the genusPneumocystis are ubiquitous, opportunistic pathogenic fungi capable of causing a lethal pneumonia in immunocompromised mammalian hosts.Pneumocystis spp. are unique members of the fungal kingdom due to the absence of ergosterol in their cellular membranes. Although these organisms were thought to obtain cholesterol by scavenging, transcriptional analyses indicate thatPneumocystis carinii encodes gene homologs involved in sterol biosynthesis. To better understand the sterol pathway in these uncultivable fungi, yeast deletion strains were used to interrogate the function and localization ofP. carinii lanosterol synthase (ERG7 ). The expression of PcErg7p in anERG7 -null mutant of the yeastSaccharomyces cerevisiae did not alter its growth rate and produced a functional lanosterol synthase, as evidenced by the presence of lanosterol detected by gas chromatographic analysis in levels comparable to that produced by the yeast enzyme. Western blotting and fluorescence microscopy revealed that, like theS. cerevisiae Erg7p, the PcErg7p localized to lipid particles in yeast. Using fluorescence microscopy, we show for the first time the presence of apparent lipid particles inP. carinii and the localization of PcErg7p to lipid particles inP. carinii . The detection of lipid particles inP. carinii and their association with PcErg7p therein provide strong evidence that the enzyme serves a similar function inP. carinii . Moreover, the yeast heterologous system should be a useful tool for further analysis of theP. carinii sterol pathway.