Study of Pneumocystis carinii : from Molecular Microbiology to Drug Discovery

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The major cell surface glycoprotein (MSG) of Pneumocystis carinii plays a crucial role in the host-parasite interaction. We have discovered that genes encoding MSGs are repeated, highly polymorphic, distributed among all of the 14-15 chromosomes, and are expressed from a unique expression site termed UCS,leading to antigenic variation to evade the host immune system. We have shown further that a UCS site is telomeric and that antigenic variation may be generated by site-specific or homologous recombination between the UCS site and multiple MSG repertoires. Telomeric fragments other than the UCS were cloned by screening with the telomere sequence. Non-UCS telomeric clones that were sequenced also contained MSG gene (s) regardless of having been screened with non-MSG probes. Frequent occurrences of these telomeric MSG clones indicate that they represent silent MSG repertoires localized in the telomere regions. The UCS contained the two transcription start sites. The promoter activity of UCS was examined using a UCS-lacZ (beta-galactosidase gene) fusion in the budding yeast Saccharomyces cerevisiae and the fission yeast Shizosaccharomyces pombe because P.carinii is phylogenically close to yeast. UCS allowed beta-galactosidase synthesis in S.pombe but not in S.cerevisiae. The transcript start sites determined by 5'RACE analysis are located slightly upstream of the two authentic start sites in P.carinii. The promoter activity of UCS itself is about one fifth of that of a widely used promoter of S.pombe, nmtl. Three other putative promoter sequences of P.carinii also expressed a reporter gene in S.pombe. These findings indicate that Shizosaccharomyces pombe is useful as a heterologous expression host for native Pneumocystis carinii genes.