Biology subjects

Smulian, A. G.

Publications and source records attributed to Smulian, A. G..

3 recordsLinked to original sources

bioRxiv2025

Extracellular Vesicle-Mediated Delivery of Genetic Material for Transformation and CRISPR/Cas9-based Gene Editing in Pneumocystis murina

Pneumocystis species are obligate fungal pathogens that cause severe pneumonia, particularly in immunocompromised individuals. The absence of robust genetic manipulation tools has impeded our mechanistic understanding of Pneumocystis biology and the development of novel therapeutic strategies. Herein, we describe a novel method for the stable transformation and CRISPR/Cas9-mediated genetic editing of Pneumocystis murina utilizing extracellular vesicles (EVs) as a delivery vehicle. Building upon our prior investigations demonstrating EV-mediated delivery of exogenous material to Pneumocystis, we engineered mouse lung EVs to deliver plasmid DNA encoding reporter genes and CRISPR/Cas9 components. Our initial findings demonstrated successful in vitro transformation and subsequent expression of mNeonGreen and DhpsARS in P. murina organisms. Subsequently, we established stable in vivo expression of mNeonGreen in mice infected with transformed P. murina for a duration of up to 5 weeks. Furthermore, we designed and validated a CRISPR/Cas9 system targeting the P. murina Dhps gene, confirming its in vitro cleavage efficiency. Ultimately, we achieved successful in vivo CRISPR/Cas9-mediated homologous recombination, precisely introducing a DhpsARS mutation into the P. murina genome, which was confirmed by Sanger sequencing across all tested animals. Here, we establish a foundational methodology for genetic manipulation in Pneumocystis, thereby opening avenues for functional genomics, drug target validation, and the generation of genetically modified strains for advanced research and potential therapeutic applications. IMPORTANCEPneumocystis species are obligate fungal pathogens and major causes of pneumonia in immunocompromised individuals. However, their strict dependence on the mammalian lung environment has precluded the development of genetic manipulation systems, limiting our ability to interrogate gene function, study antifungal resistance mechanisms, or validate therapeutic targets. Here, we report the first successful approach for stable transformation and CRISPR/Cas9-based genome editing of Pneumocystis murina, achieved through in vivo delivery of engineered extracellular vesicles (EVs) containing plasmid DNA and encoding CRISPR/Cas9 components. We demonstrate sustained transgene expression and precise modification of the dhps locus via homology-directed repair. This modular, scalable platform overcomes a long-standing barrier in the field and establishes a foundation for functional genomics in Pneumocystis and other obligate, host-adapted microbes.

microbiology↗

bioRxiv2025

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

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 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, {beta}-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. ImportancePneumocystis 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.

microbiology↗

bioRxiv2022

Targeted curation of the gut microbial gene content modulating human cardiovascular disease

Despite the promise of the gut microbiome to forecast human health, few studies expose the microbial functions underpinning such predictions. To comprehensively inventory gut microorganisms and their gene content that control trimethylamine induced cardiovascular disease, we mined over 200,000 gut-derived genomes from cultivated and uncultivated microbial lineages. Creating MAGICdb (Methylated Amine Gene Inventory of Catabolism database), we designated an atherosclerotic profile for 6,341 microbial genomes that encoded metabolisms associated with heart disease. We used MAGICdb to evaluate diverse human fecal metatranscriptome and metaproteome datasets, demonstrating how this resource eases the recovery of methylated amine gene content previously obscured in microbiome datasets. From the feces of healthy and diseased subjects, we show MAGICdb gene markers predicted cardiovascular disease as effectively as traditional blood diagnostics. This functional microbiome catalog is a public, exploitable resource, enabling a new era of microbiota-based therapeutics.

genomics↗