Development of a Molecular Toolbox for Pneumocystis

Abstract Pneumocystis jirovecii (Pj) is a fungal pathogen responsible for pneumonia (PjP) in immunocompromised individuals. Understanding the pathobiology of the organism and its infection is limited by the inability to cultivate pneumocystis outside the mammalian lung and a lack of molecular tools that would enable functional studies. The completion of the genomic sequencing of Pj and the closely related rodent pathogens P. carinii (Pc), and P. murina (Pm) found in rats and mice respectively, has allowed analysis of the genome and transcriptome under conditions of infection, however, the Pneumocystis toolbox still lacks tools enabling most functional rather than descriptive studies. The primary goal of this proposal is to develop a transformation system through the application of CRISPR/Cas9 gene editing technology and a plasmid mediated approach to this non-culturable pathogen. During the R61 phase of the proposal, we will introduce mutations associated with sulfa resistance into the folic acid synthase gene either by CRISPR/Cas9 gene editing or as a selectable marker on a plasmid introduced into the organism. We propose using extracellular vesicles (EVs) normally scavenged by Pm as transporters to introduce Cas9-sgRNA-dDNA or plasmids into Pm organisms. Successful transformation should manifest as organism proliferation despite the administration of sulfamethoxazole daily to immunosuppressed mice inoculated with transformed Pm organisms. In the second phase (R33), these techniques would be applied to demonstrate how these molecular tools would be used in functional studies addressing the biology and pathogenesis of Pneumocystis infection. We would demonstrate the utility of gene knock-out and gene knock-in using CRISPR and recombinant protein expression using plasmid -mediated transformation. This innovative methodology is poised to facilitate functional studies, by providing new and unique tools to explore the biology of these host-obligate pathogens.