Determining the Meiotic Regulators of the Opportunistic Fungal Pathogen, Pneumocystis carinii

Joshua Burgess

Pneumocystis carinii is an opportunistic fungal pathogen that most commonly effects individuals with advanced AIDS or other immunosuppressive afflictions or treatments. P. carinii causes a severe and persistent pneumonia in these patients that can be treated, but is also commonly re-activated. P. carinii has a highly variable but significant mortality rate of between 10-50% dependent upon the origin of the patients immunosuppression. Perhaps, most interestingly, P. carinii is entirely intractable in cell-free culture, therefore little is known about its niche, life-cycle, virulence factors, or basic cellular processes. In Dr. Andy Limper's lab, we have several interesting and essential, yet diverse, pursuits in understanding the broad applications of how P. carinii infects, propagates, and damages host tissues. My work is specifically targeted at understanding the manner in which the life cycle of P. carinii progresses through its cyst and trophic form to initiate and progress through meiosis. Life cycle progression in Pneumocystis is a highly contested issue because without the ability to culture the fungus ex-vivo, the manner in which the fungus invades, infects, and damages the host tissue will remain mostly conjecture. Specifically, through the use of the Pneumocystis genome project, we have been able to locate and characterize homologues of two genes, PCRan1 and PCMei2, known to be involved in the temporal determination of the initiation of meiosis in Schizosaccharomyces pombe. As a result, we have taken these sequence homologues and determined the entire ORF. Using this sequence, we have confirmed the presence, activation, and modulation of these genes in vivo, and expressed them in a heterologous system to determine probable function. Future work involves the biochemical characterization of these genes to determine localization patterns, kinase activities, active domains, and further binding partners, such as an as-of-yet uncharacterized RNA-binding partner believed to be involved in the promotion of localization to the nucleus. In addition, further experiments include the planned use of RNAi to determine the putative role of PCRan1 in the inhibition of meiosis, and the possible effects of dysregulation of maintenance of the haploid genotype. The greater aim of each of these further experiments is to ascertain the mechanisms by which Pneumocystis maintains its life cycle progression in vivo, gaining further insight into this enigmatic, yet significant, fungal infection of the immunocompromised host.