Sunday, May 18, 2025
5/18/2025
Alveolar organoids and metabolomic-driven cultivation of Pneumocystis species - The proposed studies focus on establishing ex vivo growth systems for the host-obligate fungal genus Pneumocystis, particularly P. jirovecii and its surrogate model, P. carinii. Pneumocystis species (spp.) infect the lungs of immune-deficient mammals, including humans, where a life-threatening pneumonia can develop (PjP). The primary impediment for Pneumocystis spp. research of almost any kind is the absence of a growth system outside the lungs. These fungi have reduced genomes and lack genes for essential metabolic functions and rely on their hosts for nutrients. The challenge for successful growth relies on identifying these essential nutrients and a suitable environmental milieu replicating conditions within the lung alveoli, their microhabitat. Two recent technologies offer new strategies to address these heretofore unsolved requirements. The assembled team proposes two approaches for ex vivo growth: a cell-free system guided by metabolomics and establishment of alveolar organoid cultures. The first approach involves a rational metabolomics strategy to identify essential nutrients for P. carinii growth. Metabolic Flux Balance Analysis (FBA) will guide the optimal composition and replenishment schedule for these nutrients providing an iterative process leading to an optimized culture medium. The second strategy aims to recreate the fungi's specific niche by constructing rat and human alveolar organoids. Preliminary studies support the feasibility of these innovative approaches, and the assembled team provides expertise in metabolomics, in vitro culture systems, and organoid construction. The R61 phase specific aims are to: (1) Conduct metabolite profiling and FBA to identify critical nutrients for P. carinii growth in a cell-free culture. The surrogate rat model of PjP will provide sufficient organisms for these interrogations, not possible with clinically obtained samples of P. jirovecii and (2) Develop alveolar organoids using rat-derived lung cells to support P. carinii's life cycle and growth. Pneumocystis spp. are mostly species specific, and the alveolar organoids developed from rat lung tissue and infected with P. carinii, are based on this knowledge. The optimized cell-free medium and the establishment of rat alveolar organoids provide the necessary tools for transition to the R33 phase, which is to adapt these systems for ex vivo growth of P. jirovecii. These aims include evaluation of the life cycle kinetics of P. carinii in both systems followed by use of this knowledge to evaluate these parameters for sustained growth of P. jirovecii in the optimized cell free medium and human alveolar organoids. This comprehensive plan encompasses metabolomics, FBA, organoid cultures, and detailed assessments of viability, growth, and life cycle progression for both P. carinii and P. jirovecii, showcasing a multidisciplinary and innovative approach to understanding and cultivating these fungi. Access of the scientific community to research tools generated from this work will support new research on Pneumocystis pathogenesis and virulence and medical treatments against PjP.
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