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 Pneumocystisspp. 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 indicated 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.

Methods
EV purification
Rats (n = 12) and mice (n = 6) were sacrificed after 9 weeks and 6 weeks of infection, respectively. Age-matched, uninfected, and immunosuppressed rodents were used as control groups (n = 12 rats, 6 mice). BALF was collected by instillation of cold 0.22 μm-filtered phosphate buffered saline (PBS; 10 mL for rats and 1 mL x 3 for mice) into the bronchiolar and alveolar spaces and gently collected. EV collection and purification were performed in 3 independent experiments and each isolation was used as a technical replicate for experiments described below. Cellular debris was removed by centrifugation at 3400 x g for 15 minutes.

EVs were purified using previously described methods (Lobb et al., 2015). Briefly, BALF was filtered using 100kDa Amicon Ultra (Millipore, Darmstadt, Germany). The flowthrough was collected as EV-depleted samples. Size exclusion chromatography (SEC) was performed on filtered BALF samples using qEV10 columns and the Automatic Fraction Collector (Izon Science, Medford, MA). Purified BALF EVs were concentrated by centrifugation at 190,000 x g for 2 hours at 4°C, and the pellet was resuspended in PBS. EV particles were quantified by nanoparticle tracking analysis (NTA) using a NanoSight NS300 (Malvern Panalytical, Malvern, UK). EV protein content was measured using Micro BCA Protein Assay Kit (Thermo Scientific, Rockford, IL).

Purified EVs were separated on a 4-12% Bis-Tris gel. The following steps were performed in 25 mM ammonium bicarbonate. Sections were excised, reduced with 25 mM dithiothreitol, alkylated with 55 mM iodoacetamide, and digested overnight with 10 ng/µL trypsin. The peptides were extracted and dried, then resuspended in 0.1% formic acid. Each sample was analyzed by nanoLC-MS/MS (Orbitrap Eclipse, Waltham, MA).