Pneumocystis carinii (PC) causes severe pneumonia in immunocompromised hosts. The binding of Pc trophic forms to alveolar epithelial cells (AECs) is a central feature of infection, inducing the expression and activation of PcSTE20, a gene participating in mating, proliferation, and pseudohyphal growth. In related fungi, STE20 proteins are generally activated by immediate upstream small G-proteins of the Cdc42-like family. PCCdc42 has not been previously described in Pneumocystis. To address this, PCCdc42 was cloned from a Pc cDNA library. Using the full-length 576-bp PCCdc42 cDNA sequence, a CHEF blot of genomic DNA yielded a single band, providing evidence that this gene is present as a single copy within the genome. The total length of PCCdc42 cDNA was 576-bp with an estimated MW of ~38 kDa. BLASTP analysis demonstrated greater than 80% homology with other fungal Cdc42p proteins. Northern analysis indicated equal mRNA expression in both cystic and trophic life forms. Heterologous expression of PCCdc42 in S. cerevisiae (Sc) demonstrated that PCCdc42p was able to restore growth in an Sccdc42Delta yeast strain. Additional assays with purified PCCdc42p demonstrated GTP binding and intrinsic GTPase activity which was abrogated by Clostridium difficile toxin B, characteristic of Cdc42 GTPases. Furthermore, PCCdc42p was shown to bind to the downstream PCSte20p kinase partner in the presence (but not the absence) of GTP. These data indicate that Pc possesses a Cdc42 gene expressing an active G-protein, which binds the downstream regulatory kinase PCSte20p important in PC life cycle regulation. Key words: Pneumocystis, Cdc42;, G-protein.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Reference||Annotation Extension|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Conditions||Strain||Source||Reference|