Extracellular vesicles from Pneumocystis carinii-infected rats impair fungal viability but are dispensable for macrophage functions

ABSTRACTPneumocystis 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 that 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 that Pneumocystis may release EVs. Notably, EV uptake by P. carinii indicated their potential involvement in nutrient acquisition and 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. carinii’s 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.IMPORTANCEPneumocystis spp. are fungal pathogens that can cause severe pneumonia in mammals, relying heavily on the host for essential nutrients. The absence of an in vitro culture system poses challenges in understanding their metabolism, and the acquisition of vital nutrients from host lungs remains unexplored. Extracellular vesicles (EVs) are found near Pneumocystis spp., and it is hypothesized that these vesicles transport nutrients to the pathogenic fungi. Pneumocystis proteins within the EVs showed homology to other fungal EV proteomes, suggesting that Pneumocystis spp. release EVs. While EVs did not significantly enhance P. carinii growth in vitro, P. carinii displayed active uptake of these vesicles. Moreover, EVs induced proinflammatory cytokine production in macrophages without compromising their ability to combat P. carinii. These findings provide valuable insights into EV dynamics during host-pathogen interactions in Pneumocystis pneumonia. However, the precise underlying mechanisms remain uncertain. This research also raises the potential for engineered EVs in therapeutic applications.