Biology subjects

Sayson, S. G.

Publications and source records attributed to Sayson, S. G..

4 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↗

bioRxiv2024

Pneumocystis murina Promotes Inflammasome Formation and NETosis during Pneumocystis Pneumonia

Pneumocystis jirovecii pneumonia (PjP) poses a serious risk to individuals with compromised immune systems, such as individuals with HIV/AIDS or undergoing immunosuppressive therapies for cancer or solid organ transplants. Severe PjP triggers excessive lung inflammation, resulting in lung function decline and consequential alveolar damage, potentially culminating in acute respiratory distress syndrome. Non-HIV patients face a 30-60%mortality rate, emphasizing the need for a deeper understanding of inflammatory responses in PjP. Prior research emphasized macrophages in Pneumocystis infections, neglecting neutrophils role in tissue damage. Consequently, the overemphasis on macrophages led to an incomplete understanding of the role of neutrophils and inflammatory responses. In the current investigation, our RNAseq studies on a murine surrogate model of PjP revealed heightened activation of the NLRP3 inflammasome and NETosis cell death pathways in their lungs. Immunofluorescence staining confirmed Neutrophil Extracellular Trap (NET) presence in the lungs of the P. murina-infected mice, validating our findings. Moreover, isolated neutrophils exhibited NETosis when directly stimulated with P. murina. While isolated NETs did not compromise P. murina viability, our data highlight the potential role of neutrophils in promoting inflammation during P. murina pneumonia through NLRP3 inflammasome assembly and NETosis. These pathways, essential for inflammation and pathogen elimination, bear the risk of uncontrolled activation leading to excessive tissue damage and persistent inflammation. This pioneering study is the first to identify the formation of NETs and inflammasomes during Pneumocystis infection, paving the way for comprehensive investigations into treatments aimed at mitigating lung damage and augmenting survival rates for individuals with PjP. IMPORTANCEPneumocystis jirovecii pneumonia (PjP) affects individuals with weakened immunity, such as HIV/AIDS, cancer, and organ transplant patients. Severe PjP triggers lung inflammation, impairing function and potentially causing acute respiratory distress syndrome. Non-HIV individuals face a 30-60% mortality rate, underscoring the need for deeper insight into PjPs inflammatory responses. Past research focused on macrophages in managing Pneumocystis infection and its inflammation, while the role of neutrophils was generally overlooked. In contrast, our findings in P. murina-infected mouse lungs showed neutrophil involvement during inflammation and increased expression of NLRP3 inflammasome and NETosis pathways. Detection of neutrophil extracellular traps further indicated their involvement in the inflammatory process. Although beneficial in combating infection, unregulated neutrophil activation poses a potential threat to lung tissues. Understanding the behavior of neutrophils in Pneumocystis infections is crucial for controlling detrimental reactions and formulating treatments to reduce lung damage, ultimately improving the survival rates of individuals with PjP.

immunology↗

bioRxiv2023

Extracellular Vesicles from Pneumocystis carinii-Infected Rats Impair Fungal Viability but are Dispensable for Macrophage Functions

Pneumocystis spp. are host obligate fungal pathogens that can cause severe pneumonia in mammals and rely heavily on their host for essential nutrients. The lack of a sustainable in vitro culture system poses challenges in understanding their metabolism and the acquisition of essential nutrients from host lungs remains unexplored. Transmission electron micrographs show Extracellular Vesicles (EVs) are found near Pneumocystis spp. within the lung. We hypothesized that EVs transport essential nutrients to the fungi during infection. To investigate this, EVs from P. carinii and P. murina infected rodents were biochemically and functionally characterized. These EVs contained host proteins involved in cellular, metabolic, and immune processes as well as proteins with homologs found in other fungal EV proteomes, indicating Pneumocystis may release EVs. Notably, EV uptake by P. carinii indicated their potential involvement in nutrient acquisition and indicate a possibility for using engineered EVs for efficient therapeutic delivery. However, EVs added to P. carinii in vitro, did not show increased growth or viability, implying that additional nutrients or factors are necessary to support their metabolic requirements. Exposure of macrophages to EVs increased proinflammatory cytokine levels, but did not affect macrophages ability to kill or phagocytose P. carinii. These findings provide vital insights into P. carinii and host EV interactions, yet the mechanisms underlying P. cariniis survival in the lung remain uncertain. These studies are the first to isolate, characterize, and functionally assess EVs from Pneumocystis-infected rodents, promising to enhance our understanding of host-pathogen dynamics and therapeutic potential.

microbiology↗