Characterization of the PcCdc42 small G protein from Pneumocystis carinii, which interacts with the PcSte20 life cycle regulatory kinase.
Pneumocystis carinii (Pc) causes severe pneumonia in immunocompromised hosts. The binding of Pc trophic forms to alveolar epithelial cells 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 the potential role of such a G protein in Pneumocystis, 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 molecular mass of approximately 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 Saccharomyces cerevisiae (Sc) demonstrated that PcCdc42p was able to restore growth in an ScCdc42Delta yeast strain. Additional assays with purified PcCdc42 protein demonstrated GTP binding and intrinsic GTPase activity, which was partially but significantly suppressed by Clostridium difficile toxin B, characteristic of Cdc42 GTPases. Furthermore, PcCdc42 protein was also shown to bind to the downstream PCSte20 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 PcSte20, important in Pc life cycle regulation.
Krajicek
Bryan J
BJ
Thoracic Diseases Research Unit, Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota, USA.
Kottom
Theodore J
TJ
Villegas
Leah
L
Limper
Andrew H
AH
D000595:Amino Acid Sequence; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D045363:Pneumocystis carinii / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D006160:Guanosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D020559:Monomeric GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein