Organisms in the genus Pneumocystis 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 that Pneumocystis 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 of P. carinii lanosterol synthase (ERG7). The expression of PcErg7p in an ERG7-null mutant of the yeast Saccharomyces 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 the S. 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 in P. carinii and the localization of PcErg7p to lipid particles in P. carinii. The detection of lipid particles in P. carinii and their association with PcErg7p therein provide strong evidence that the enzyme serves a similar function in P. carinii. Moreover, the yeast heterologous system should be a useful tool for further analysis of the P. carinii sterol pathway.
Members of the fungal genus Pneumocystis colonize healthy mammalian hosts
without causing apparent disease, but colonization in immunocompromised hosts
may result in a potentially fatal pneumonia known as Pneumocystis pneumonia.
Although Pneumocystis are fungi, this genus has characteristics that make it atypical
among other fungi. Pneumocystis do not appear to synthesize the major fungal sterol,
ergosterol, and biochemical analyses have shown that they utilize cholesterol rather
than ergosterol as the bulk sterol. Pneumocystis carinii appears to scavenge exogenous sterols, including cholesterol, from its mammalian host. As a result, it has long been held that their ability to scavenge cholesterol from their hosts, and their inability to undergo sterol biosynthesis, makes them resistant to antifungal drugs that target ergosterol or ergosterol biosynthesis. However, genome scans and in vitro assays indicate the presence of sterol biosynthetic genes within the P. carinii genome, and targeted inhibition of these enzymes resulted in reduced viability of P. carinii,
suggesting that these enzymes are functional within the organism. Heterologous
expression of P. carinii sterol genes, along with biochemical analyses of the lipid
content of P. carinii cellular membranes, have provided an insight into sterol
biosynthesis and the sterol-scavenging mechanisms used by these fungi.