Single-Cell RNA Sequencing Defines Developmental Progression and Reproductive Transitions ofPneumocystis carinii

Pneumocystisspecies are host-obligate fungal pathogens that cause severe pneumonia in immunocompromised individuals. Despite their clinical importance, their life cycle remains poorly understood, in part becausePneumocystisdepends on the host environment for most nutrients and requires sexual reproduction for survival, which occurs exclusivelyin vivo. This study presents the first single-cell RNA sequencing (scRNA-seq) atlas ofPneumocystis carinii, generated from isolated organisms recovered from the bronchoalveolar lavage fluid of infected rats to map the life cycle ofP. carinii. Transcriptomes from 87,716 cells were analyzed using the 10x 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 supports 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.