SIGNAL-TRANSDUCTION IN PNEUMOCYSTIS-CARINII - CHARACTERIZATION OF THEGENES (PCG1) ENCODING THE ALPHA-SUBUNIT OF THE G-PROTEIN (PCG1) OF PNEUMOCYSTIS-CARINII CARINII AND PNEUMOCYSTIS-CARINII RATTI

Pneumocystis carinii is a eukaryotic organism that causes pneumonia in immunocompromised hosts. The cell biology and life cycle of the organ ism are poorly understood primarily because of the lack of a continuou s in vitro cultivation system. These limitations have prevented invest igation of the organism's infectious cycle and hindered the rational d evelopment of new antimicrobial therapies and implementation of measur es to prevent exposure to the organism or transmission. The interactio n of P. carinii with its host and its environment may be critical dete rminants of pathogenicity and life cycle. Signal transduction pathways are likely to be critical in regulating these processes. G proteins a re highly conserved members of the pathways important in many cellular events, including cell proliferation and environmental sensing. To ch aracterize signal transduction pathways in P. carinii, we cloned a G-p rotein alpha subunit (G-alpha) of P. carinii carinii and P. carinii ra ni by PCR amplification and hybridization screening. The gene encoding the G-alpha was present in single copy on a 450-kb chromosome of P. c . carinii and on a 420-kb chromosome of P. c. ratti. The 1,062-bp G-al pha open reading frame is interrupted by nine introns. The predicted p olypeptide showed 29 to 53% identity with known fungal G-alpha protein s with greatest homology to Neurospora crassa Gna-2. Northern (RNA) bl ot analysis and immunoprecipitation demonstrated expression of the G-a lpha mRNA and protein in P. carinii isolated from heavily infected ani mals. Some alteration in the level of transcription was noted in short -term maintenance in starvation or rich medium. Characterization of si gnal transduction in P. carinii will permit a better understanding of the reproductive capacity and other cellular processes in this family of organisms that cannot be cultured continuously.