PubMed2025
UNLABELLED: Pneumocystis species are host-obligate fungal pathogens that cause severe pneumonia in immunocompromised individuals. Despite their clinical importance, their life cycle remains poorly understood, in part because Pneumocystis depends on the host environment for most nutrients and requires sexual reproduction for survival, which occurs exclusively in vivo. This study presents the first single-cell RNA sequencing (scRNA-seq) atlas of Pneumocystis carinii, generated from isolated organisms recovered from the bronchoalveolar lavage fluid of infected rats to map the life cycle of P. carinii. Transcriptomes from 87,716 cells were analyzed using the 10× Genomics platform, revealing 13 transcriptionally distinct clusters representing key developmental stages, including biosynthetically active trophic forms, mating-competent intermediates, and asci undergoing sporulation. These states were characterized by expression of MAPK signaling components, β-glucan-modifying enzymes, and spore-associated genes, respectively. The scRNA-seq data support previous evidence that these host-obligate fungi undergo sexual reproduction and provide new insights into the gene expression patterns associated with different life cycle phases. Biomarkers associated with ascus formation identified by scRNA-seq were validated by RT-qPCR, showing decreased expression levels in ascus-depleted populations treated with anidulafungin, a drug that halts ascus formation. More broadly, this approach provides a strategy for studying the full life cycles of fungal pathogens that cannot be continuously cultured. IMPORTANCE: Pneumocystis species (spp.) are clinically significant fungal pathogens that cannot be sustainably cultured in vitro due to their host-obligate nature. This longstanding limitation has impeded progress in understanding their life cycle and identifying therapeutic vulnerabilities. Here, we apply scRNA-seq to P. carinii isolated directly from infected rat lungs, generating the first transcriptional map of its developmental progression. Our results define discrete gene expression states associated with trophic growth, mating activation, and ascus formation and provide transcriptional evidence for a structured life cycle, clarifying key developmental transitions and identifying potential regulatory targets for therapeutic intervention. Importantly, this study demonstrates that scRNA-seq can resolve the developmental biology of host-restricted fungal pathogens that cannot be cultured in vitro. This approach offers a generalizable framework for investigating other unculturable or obligate microbial pathogens directly within their native host environments, where traditional experimental tools are limited.