Study of Pneumocystis carinii : from Molecular Microbiology to Drug Discovery

卡氏肺孢子虫的主要细胞表面糖蛋白(MSG)在宿主与寄生虫的相互作用中起着至关重要的作用。我们发现编码MSG的基因是重复的、高度多态的，分布在所有14-15条染色体上，并从一个称为UCS的唯一表达位点表达，导致抗原变异以逃避宿主免疫系统。我们已经进一步证明，一个UCS位点是端粒的，抗原变异可能是通过该UCS位点与多个味精酶系之间的特定或同源重组而产生的。通过对端粒序列的筛选，克隆了除USCs外的其他端粒片段。无论是否用非MSG探针筛选，测序的非USCs端粒克隆也含有MSG基因(S)。这些端粒味精克隆的频繁出现表明，它们代表了定位于端粒区域的沉默味精酶系。该基因含有两个转录起始点。由于卡氏裂殖酵母与酵母菌的亲缘关系较近，因此，在芽殖酵母酿酒酵母和裂殖酵母庞贝裂殖酵母中，利用半乳糖苷酶基因融合的方法检测了USCs的启动子活性。在酿酒酵母中不能合成β-半乳糖苷酶，而能在庞氏葡萄球菌中合成β-半乳糖苷酶。经5‘RACE分析确定的转录本起始位点位于卡氏肺吸虫两个真实起始位点的上游。其启动子活性约为广泛应用的启动子nmtl的五分之一。卡氏肺孢子虫另外三个可能的启动子序列也在S.pombe中表达了一个报告基因。这些发现表明庞氏志贺氏菌可以作为卡氏肺孢子虫天然基因的异源表达宿主。

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.