Pneumocystis carinii is an opportunistic fungal pathogen which causes severe pneumonia in patients with impaired immunity such as patients with AIDS. The life cycle of P. carinii is poorly understood. In fungi related to P. carinii, nutrient deprivation and dessication are the major stimuli for the release of pheromone mating factors which activate a mitogen-activated protein kinase (MAPK), resulting in mitotic cell cycle arrest, conjugation and meiosis, and increased pathogenicity. MAPK is the essential molecule in the pheromone-induced signal transduction cascade which controls these events. As an essential molecule regulating an organism's life cycle, MAPK activity is highly regulated. Dual phosphorylations are requiring for activation, and variable MAPK activity and expression are restricted to certain life cycle forms. Our initial studies indicate that P. carinii possesses a MAPK which is closely related to fungal pheromone-induced MAPKs. We hypothesize that the pheromone-induced mitogen-activated protein kinase (MAPK) signal transduction cascade regulates cellular differentiation and proliferation of P. carinii organisms. As an essential component in the life cycle of fungi, investigation of the P. carinii MAPK will result in an enhanced understanding of the P. carinii life cycle, pathogenicity, and lead to development of novel therapeutic agents to treat P. carinii pneumonia. To evaluate these hypotheses, we will undertake several parallel investigations. MAPK activity and expression in separated life cycle forms will be accessed to determine whether P. carinii MAPK is differentially regulated over the P. carinii life cycle. We will identify and characterize the P. carinii pheromone receptors and separate P. carinii mating types by FACS. We will access whether nutrient deprivation, hypoxia, or temperature changes results in mitotic cell cycle arrest leading to meiosis and formation of the cyst, and whether inhibitors of the MAPK cascade block these effects. We will also investigate if isolated mating types undergo haploid mitosis or if mixing of mating types is essential for meiosis and formation of the cyst. Through these investigations, we hope to gain important insights into the life cycle regulation and pathogenicity ofP. carinii.
