{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T11:15:44Z","timestamp":1791371744919,"version":"4.3.1"},"reference-count":59,"publisher":"American Society for Microbiology","issue":"10","license":[{"start":{"date-parts":[[2025,10,7]],"date-time":"2025-10-07T00:00:00Z","timestamp":1759795200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,10,7]],"date-time":"2025-10-07T00:00:00Z","timestamp":1759795200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01HL146266"],"award-info":[{"award-number":["R01HL146266"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000738","name":"U.S. Department of Veterans Affairs","doi-asserted-by":"publisher","award":["1I01BX004441- 01"],"award-info":[{"award-number":["1I01BX004441- 01"]}],"id":[{"id":"10.13039\/100000738","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2025,10,7]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      <jats:italic toggle=\"yes\">Pneumocystis<\/jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">Pneumocystis<\/jats:italic>\n                      depends on the host environment for most nutrients and requires sexual reproduction for survival, which occurs exclusively\n                      <jats:italic toggle=\"yes\">in vivo<\/jats:italic>\n                      . This study presents the first single-cell RNA sequencing (scRNA-seq) atlas of\n                      <jats:italic toggle=\"yes\">Pneumocystis carinii<\/jats:italic>\n                      , generated from isolated organisms recovered from the bronchoalveolar lavage fluid of infected rats to map the life cycle of\n                      <jats:italic toggle=\"yes\">P. carinii<\/jats:italic>\n                      . Transcriptomes from 87,716 cells were analyzed using the 10\u00d7 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, \u03b2-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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      <jats:italic toggle=\"yes\">Pneumocystis<\/jats:italic>\n                      species (spp.) are clinically significant fungal pathogens that cannot be sustainably cultured\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">P. carinii<\/jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      . 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.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.01277-25","type":"journal-article","created":{"date-parts":[[2025,8,25]],"date-time":"2025-08-25T13:07:31Z","timestamp":1756127251000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":5,"title":["Single-cell RNA sequencing defines developmental progression and reproductive transitions of\n                    <i>Pneumocystis carinii<\/i>"],"prefix":"10.1128","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-5840-1890","authenticated-orcid":false,"given":"Aaron W.","family":"Albee","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01e3m7079","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Internal Medicine, University of Cincinnati, College of Medicine","place":["Cincinnati, USA"]},{"name":"The Veterans Affairs Medical Center","place":["Cincinnati, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7461-0704","authenticated-orcid":true,"given":"Steven G.","family":"Sayson","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01e3m7079","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Internal Medicine, University of Cincinnati, College of Medicine","place":["Cincinnati, USA"]},{"name":"The Veterans Affairs Medical Center","place":["Cincinnati, 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