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

ABSTRACT 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 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. IMPORTANCE Pneumocystis 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.

P neumocystis spp.are host obligate fungal pathogens that can cause a lethal pneumonia in humans and other mammals.Most species only infect a single mammalian species, and the Pneumocystis species infecting humans is Pneumocystis jirovecii with the resultant pneumonia called Pneumocystis jirovecii pneumonia (PjP).Historically associated with HIV/AIDS patients, PjP is now more commonly affecting cancer patients and individuals who have received organ transplants requiring immunesuppressing treatments.Among all hospitalizations for PjP, malignancy stands as the most prevalent predisposing factor, accounting for 46.0% to 55.7% of cases, followed by HIV at 17.8% (1,2).In rats, a model used in the present study, the pneumonia is termed Pneumocystis carinii pneumonia (PcP).These fungi are extracellular, stenoxe nous parasites that reside in the lung alveoli of mammals.Within the alveolar lumen, Pneumocystis spp.are thought to produce a biofilm-like system (3) with individual organisms forming a tight interdigitation with alveolar epithelial type I (ATI) cells, serving as an anchor for the pathogen clusters.Besides the fungal organisms, components of the Pneumocystis spp.-infected alveoli revealed by transmission electron micrographs (TEMs) include lamellar bodies, tubular myelin, and, notably, double-membraned vesicles (4,5).These vesicles have been thought to be filopodia serving for attachment, but there is disagreement as to their form and function, and no studies have been conducted to understand their purpose.
As is characteristic of host-obligate pathogens, Pneumocystis spp.have highly compact genomes with considerable loss of many biological pathways, making them highly dependent on the host for nutrients (6,7).These fungi have limited capacity for synthesizing amino acids, with only two enzymes present in the genome that are required for the synthesis of amino acids, which is insufficient for de novo synthesis of any of the 20 amino acids (6,7).In contrast, Schizosaccharomyces pombe (a phylo genetically close relative) contains all 54 enzymes required for amino acid synthesis.Additionally, Pneumocystis spp.do not contain biochemically detectable ergosterol, the major sterol contained within the cell membrane of most fungi.Cholesterol is the most prominent bulk sterol in Pneumocystis spp.and is considered to be transferred from the host (8)(9)(10).However, the mechanism of amino acid and cholesterol acquisition, as well as other nutrients, by Pneumocystis spp.remains unclear.
It is our contention that the long-observed vesicles accompanying Pneumocystis spp.infections are extracellular vesicles (EVs) that serve, in part, to shuttle essential nutrients that these fungi can no longer synthesize.
Fungal EVs have been described in terms of secretion, biological contents, and intercellular communication.Several fungi, such as Cryptococcus neoformans (11), Histoplasma capsulatam (12), Paracoccidioides brasiliensis (13), Malassezia sympodialis (14), Saccharomyces cerevisiae (15), and Aspergillus fumigatus (16), have shown secretion of EVs.Like those in mammals, these vesicles contain a diverse composition of pro teins, lipids, nucleic acids, and polysaccharides (17).Candida albicans secretes EVs that participate in community communication and are required for the proper formation of biofilms (18).Although the uptake of fungal EVs by host cells has been documented (19), there is limited evidence demonstrating the uptake of host EVs by fungal organisms.
In the mammalian lung, ATI cells are responsible for gas exchange with the capillaries and are the first line of defense against inhaled stimuli.In response to stimuli, ATI cells release EVs which modulate the lung environment (20,21).These mammalian EVs have been shown to contain DNA, RNA, and protein components that are involved in intercellular communication (22).Additionally, cholesterol and free amino acids have been found to be enriched in exosome and microvesicles from most tissues and cell types, including lungs, epithelial cells, and macrophages (23)(24)(25).
In the present work, nano-scale liquid chromatographic tandem mass spectrometry (nLC-MS/MS) analysis was used to characterize the proteome of bronchial alveolar lavage fluid (BALF)-derived EVs from P. carinii-infected rats and P. murina-infected mice.The Pneumocystis-specific major surface glycoprotein (Msg) and other Pneumocystis proteins were detected, indicating the potential source(s) of these EVs.These proteomic profiles support the hypothesis that P. carinii and P. murina may themselves release a population of EVs, while uptake studies revealed that P. carinii can uptake host-derived EVs.

P. carinii-infected rat BALF contain EVs
The presence of EVs within P. carinii-infected rat lungs was detected by TEM (Fig. 1A and  A*).These vesicles were distributed throughout the alveolar lumina, surrounding clusters of trophic cells, and asci.EVs were also observed between both the host ATI cells and P. carinii trophic cells.Additionally, EVs were seen within concave folds of trophic cells as well as between individual organisms.Furthermore, EVs with electron-dense outer layers are seen adjacent to P. carinii trophic and asci (Fig. 1B and C), suggesting that P. carinii, with a thickened cell wall, may be secreting these vesicles.
TEMs of EVs from infected rat lung BALF revealed a heterogenous size population of EVs ranging from 49 to 266 nm (Fig. 2A).The more sensitive nanoparticle tracking analysis showed BALF EVs isolated from uninfected (UI) and infected, immunosuppressed rats produced EVs mostly between 100 and 300 nm (Fig. 2B and C).These vesicles contained host mammalian proteins CD9 and TSG101, which are commonly used as EV markers for membrane and cytoplasmic proteins, respectively (Fig. 2D).

P. carinii and P. murina EV proteins
Characterizing the EV proteome derived from Pneumocystis spp.presents a challenge, as these fungi cannot be cultured and must be obtained from their host animal.In this study, we sought to identify the fungal EV proteome from infected host BALF by analyzing rat or mouse BALF EVs from immunosuppressed animals using nLC-MS/MS.P. carinii proteins were detected in infected (Pc+) rat BALF EVs (Table 1).These P. carinii proteins shared homology with the EV proteomic profiles of other fungal species, such as A. fumigatus, C. albicans, C. neoformans, H. capsulatum, and S. cerevisiae (15,16,(26)(27)(28); Table 1).Many of the proteins were heat shock proteins, which are commonly used as EV markers for mammalian vesicles and are shown to be contained within the fungal EV proteome (29).This further supports the hypothesis that P. carinii secretes EVs.
Similarly, we detected P. murina proteins in infected (Pm+) mouse BALF EVs (Table 2), which also showed homology to the EV proteome of other fungal species.Notably, both P. carinii and P. murina EVs contained Msg.Msg proteins are a superfamily of membranous proteins unique to the Pneumocystis genus and are found on the surface of all life cycle stages of these fungi.
The presence of homologous proteins from P. carinii and P. murina in BALF EVs from infected animals, along with the identification of Msg membrane proteins, suggests that both species produce and secrete EVs.

Host proteins from rat and mouse BALF EVs
BALF EVs from immunosuppressed rodents, both UI and infected, were subjected to nLC-MS/MS analysis.The results showed that UI rat BALF EVs contained 408 proteins, while infected Pc+ rat BALF EVs contained 261 proteins.Moreover, UI mouse BALF EVs had 355 proteins, and infected Pm+ mouse BALF EVs had 464 proteins.PANTHER tools (30) were used to assign the functional classifications of these proteins, resulting in the identification of Gene Ontology terms and biological processes (Table 3; Table S1).In both animals, the uninfected EV proteome was found to be enriched in cellu lar processes (GO:0009987).While in infected hosts, the BALF EV proteome showed enrichment in response to stimulus (GO:0050896) and immune system processes (GO:0002376).

P. carinii actively uptake EVs
For the remaining functional assays, BALF EVs derived from rats were used due to the higher amount of EVs obtained from the lungs compared to mice and to reduce the numbers of mice required to perform comparable studies to safeguard the overarching ethical principle of animal welfare.EVs from UI and P. carinii-infected (Pc+) rats were labeled with the lipophilic membrane dye, PKH26, and incubated with fungal cells (Fig. 3).P. carinii incubated with PKH26-treated phosphate-buffered saline (PBS) shows no micelle formation of the dye (negative control; Fig. 3, first and third rows).P. carinii treated with PKH26-labeled UI EVs shows bright red punctate staining within fungal clusters (Fig. 3, fourth row).No red staining was observed in heat-killed (Δ80°C) P. carinii (Fig. 3, second row).These results indicate that EV uptake by P. carinii is an active process, which is lost upon cell death.When organisms were treated with Pc+ EVs, P. carinii displayed ubiquitous PKH26 staining in both live and dead P. carinii cells (Fig. 4), indicating that BALF EVs from infected animals are binding to fungal cells regardless of viability.This is likely due to EV secretion changes during infection and inflammation, which is supported by the Pc+ EV proteome.These EVs contain immune proteins which may bind pathogen-associated molecular patterns on the surface of P. carinii.
Pc+ EVs are detrimental to P. carinii viability P. carinii was treated with EVs from UI or Pc+ rats and assessed for viability over 7 days (Fig. 5A).UI EVs had no effect on viability compared to vehicle and negative control, ampicillin, which is used to test for bacterial growth.However, Pc+ EVs had a detrimental effect on viability, similar to that of the antifungal pentamidine.
Since Pc+ EVs were toxic to P. carinii, we sought to determine whether these EVs were detrimental to rat lung epithelial cells (RLE-6TN).RLE-6TN displayed no decreased viability when treated with UI or Pc+ EVs.These results indicate while EVs from infected rats were detrimental to P. carinii viability in vitro, they had no effect on the host epithelial cells.

EV-stimulated macrophages express pro-inflammatory cytokines
Rat alveolar macrophage cell line, NR8383, was stimulated with EVs from UI, infected (Pc+), or zymosan-treated (zymo) rats for 24 hours.Zymosan treatment was used as a positive control to induce a proinflammatory response in the lungs (31).Macro phages treated with any of the EV treatments expressed increased interleukin (Il)-1β, Il-6, and tumor necrosing factor alpha (Tnf⍺) mRNA (Fig. 6).Macrophages stimulated with zymosan-elicited EVs resulted in a more robust expression of Il-6 and Tnf⍺, compared to vehicle and UI EVs.However, there were no differences seen between rats treated with UI or Pc+ EVs, indicating that the cytokine expression was likely a response to EVs, as a whole, rather than the proteomic differences between UI and Pc+ EVs.

EVs do not increase macrophage phagocytosis or P. carinii killing
After the NR8383 macrophages were stimulated with vehicle, UI EVs, or Pc+ EVs for 24 hours, P. carinii was added to the wells.Macrophages and P. carinii were co-cultured for 24 hours to assess macrophage-mediated killing (Fig. 7A).Fungal cells were quantified by P. carinii dihydrofolate reductase (PcDhfr) copy number.Macrophages stimulated with EVs, regardless of the origin, did not result in significant killing and quantity of P. carinii.
To assess phagocytosis, vehicle, UI EVs, or Pc+ EV-stimulated macrophages were co-cultured with P. carinii for 4 hours (Fig. 7B).Macrophages were subjected to differential centrifugation at 300 × g.The ratio of P. carinii organisms per macrophage was quantified by qPCR using PcDhfr and RnGapdh.EV stimulation of macrophages did not significantly increase phagocytosis of P. carinii organisms.Perplexingly, phagocytosis was significantly reduced in Pc+ and Zymosan-treated BALF EVs.

DISCUSSION
Metabolism by Pneumocystis spp. is poorly understood, primarily due to the lack of a facilitative culture system (32).Much of our current knowledge of Pneumocystis spp.metabolism is based on inferences from genomic studies and coverage of genes from the relevant pathways (7,33).As with most obligate pathogens, many biosynthetic pathways have been lost, creating a reliance upon the host organism for essential nutrients.This situation poses significant challenges in establishing a suitable in vitro culture system and often restricts experimental studies to in vivo infection models.
Extracellular vesicles from mammalian tissues and cells are known to contain essential components found in Pneumocystis spp., such as cholesterol and free amino acids.We propose that P. carinii and P. murina take up EVs to supplement their metabolic require ments.To better understand the potential role played by the host EVs, we performed proteomic characterization of EVs from P. carinii-infected rat BALF.One challenge with this approach is that the EVs originated from both the rat and the pathogen.As such, we cannot rule out the possibility of host immunity leading to dissociation of fungal cells and presentation of these proteins.However, the identified P. carinii EV proteome contains homologs of proteins found in the EV proteomes of other fungi including A. fumigatus, C. albicans, C. neoformans, H. capsulatum, and S. cerevisiae (15,16,(26)(27)(28).Additionally, the presence of proteins from the Pneumocystis-unique superfamily of membrane proteins (Msg) within BALF EVs suggests that Pneumocystis is likely releasing EVs, as vesicles routinely contain membrane proteins from their originating cell.The presence of a putative serine protease, PNEG_02319, suggests the secretion of EVs from trophic forms of P. murina (34), yet no definitive asci markers were noted in the EV proteome.However, visual evidence of EVs with electron-dense cell walls suggests the release of EVs by both trophic and asci life stages of P. carinii (Fig. 1).While we cannot definitively state that Pneumocystis spp.secrete EVs, since the current in vitro culture system is far from optimal, we posit that the preponderance of evidence suggests that these organisms secrete EVs.These lines of evidence include TEM images of EVs in close proximity to P. carinii with electron-dense membranes, the Pneumocystis-infected BALF EV proteomes are similar to other fungal EV proteomes, and the presence of Msg surface proteins within these EVs.Further research is needed to identify the complete fungal EV proteome from P. carinii and P. murina.The reasons behind the release of extracellular vesicles by P. carinii and P. murina remain unclear, but fungal EVs have been shown to serve critical functions in other species.EVs from C. albicans and Pichia fermentans function in intercellular communication for the proper formation of biofilm (18,35).Moreover, Cryotococcus gattii releases EVs when phagocytosed by host macrophages, leading to long-distance communication and rapid fungal population proliferation (36).These fungal EVs may play important roles in intercellular communication to facilitate growth and survival of Pneumocystis within the host lungs.
Host-derived EVs displayed proteins involved in a variety of pathways including cellular and metabolic processes, cellular component organization, and biological regulation.Infected BALF EVs also include proteins involved in stress response and immune system processes, such as heat shock proteins.Heat shock proteins have been shown to be upregulated in A549 lung epithelial cells treated with P. carinii, in the lungs of Pneumocystis infection in rats, and in animal models of lung injury (37)(38)(39).The specific host-pathogen interactions of these host vesicles and their cargo with P. carinii and P. murina are currently unknown.
Neutrophils release EVs in response to A. fumigatus in vitro (40).These neutrophilderived EVs had antifungal properties and inhibited the growth of A. fumigatus hyphae.In the case of the phytopathogen Sclerotinia sclerotiorum, uptake of host plant EVs caused impaired growth and cell death of fungal cells (41).Additionally, Arabidopsis plant cells release EVs containing siRNAs that were taken up by fungal Botrytis cinerea cells and induced the silencing of virulence-associated genes (42).Based on these findings, it would be expected that EV host defense mechanisms have a detrimental role in Pneumocystis survival within the lungs.This notion is reinforced by the toxic effect observed on P. carinii viability in vitro when exposed to BALF EVs from P. carinii-infected rats.
On the contrary, we suspect that Pneumocystis may be also benefitting from the EV contents and utilizing them to supplement their metabolic requirements.Here, we observe host EV uptake by P. carinii.This vesicle uptake was lost in heat-killed P. carinii, demonstrating that EV import is an active process.EV uptake of mammalian-derived EVs may explain how Pneumocystis obtains cholesterol from its host.Considering that Pneumocystis spp.are deficient in many metabolic pathways, these EVs may also provide amino acids and other necessary components.However, treating P. carinii with EVs did not result in sustained growth or improvement in viability when maintained in a short-term in vitro system (43).This indicates that either EVs are dispensable for P. carinii metabolism or, as previously mentioned, P. carinii lacks a sustainable culture system, and the addition of EVs alone is not sufficient to overcome the limitations of this in vitro system.
Macrophages participate in host defense response and the clearance of various microorganisms.EVs released by neighboring cells can stimulate these macrophages, leading to their activation and polarization, ultimately promoting inflammation (44).When RAW 264.7 macrophages were stimulated with A. fumigatus EVs, they displayed increased killing and phagocytosis of A. fumagatus conidia (16).Similarly, A. flavus EVs induced M1 polarization of bone marrow-derived macrophages and production of TNF, IL-6, and IL-1β, resulting in increased phagocytosis and killing of A. flavus conidia in vitro (45).In this study, NR8383 macrophages expressed increased mRNA levels of pro-inflammatory cytokines Il-1β, Il-6, and Tnf⍺ when stimulated with EVs, regardless of whether they originated from uninfected or P. carinii-infected rat BALF.Additionally, macrophages stimulated with EVs did not exhibit increased killing or phagocytosis of P. carinii organisms.These findings are in contrast with macrophages stimulated with fungal EVs.However, our EVs contain a mixture of host and potentially P. carinii, EVs which may explain the contrasting response.
This study reveals the complex interactions between P. carinii and host EVs.Hostreleased EVs have a detrimental impact on P. carinii survival within the lungs, while also potentially benefiting the pathogen by supplementing its metabolic needs.Further research is needed to fully understand these interactions and their implications for P. carinii survival and metabolism within the host.This area of study shows promise for advancing our understanding of host-pathogen dynamics and exploring potential therapeutic approaches for Pneumocystis-related infections.Based on the observed EV uptake by P. carinii, it is conceivable that EVs could be packaged with transgenic nucleotide sequences, enzymes, or therapeutic agents for delivery.

Animals
Male Sprague-Dawley rats (125-150 g; 5-6 weeks) were infected with P. carinii as previously described (46).The rats were housed under barrier conditions with autoclaved food and bedding sterilized in cages equipped with sterile microfilter lids.Rats were immunosuppressed by weekly subcutaneous injections of methylprednisolone (4 mg/kg) and intranasally dosed twice over 2 weeks with 2 × 10 6 organisms of P. carinii.Animals continued with 9 weeks of immunosuppression to permit the development a heavy fungal burden.
Corticosteroid-immunosuppressed C3H/HeN male mice (20-25 g; 5-6 weeks) were infected with P. murina through exposure to P. murina-infected and immunosuppressed mice.Mice were immunosuppressed by adding dexamethasone (4 µg/L) to acidified drinking water and housed with the infected "seed mice" for 2 weeks.A fulminate infection was developed over 6 weeks.The study focused solely on male rodents to minimize the impact of hormonal fluctuations related to the estrous cycle, ensuring a more controlled experimental setting.Investigations involving female rodents are planned for future research endeavors.
Pneumocystis infection was confirmed and quantified by homogenization of the lung tissue and stained with Diff-Quik.The animals were monitored daily, and those showing signs of cachexia or respiratory distress were euthanized using an approved method by the AVMA Panel on Euthanasia.These studies were conducted following the guidelines outlined in the 8th edition of the Guide for the Care and Use of Laboratory Animals (National Academies Press, Washington, DC, USA) and in AAALAC-accredited laboratories under the supervision of veterinarians.Additionally, all procedures adhered to the regulations set forth by the Institutional Animal Care and Use Committee at the Veterans Affairs Medical Center, Cincinnati, OH, USA.

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 PBS (10 mL for rats and 1 mL × 3 for mice) into the bronchiolar and alveolar spaces and gently collected (47).EV collection and purification were performed in three independent experiments, and each isolation was used as a technical replicate for experiments described below.Cellular debris was removed by centrifugation at 3,400 × g for 15 minutes.
EVs were purified using previously described methods (48).Briefly, BALF was filtered using 100-kDa 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 × g for 2 hours at 4°C, and the pellet was resuspended in PBS.EV particles were quantified by nanoparticle tracking analysis using a NanoSight NS300 (Malvern Panalytical, Malvern, UK).EV protein content was measured using Micro BCA Protein Assay Kit (Thermo Scientific, Rockford, IL).

Transmission electron microscopy
Lung samples were prepared for TEM using previously published methods (49).Briefly, lung samples were fixed in 3% glutaraldehyde/3% acrolein in 0.1 M cacodylate buffer (pH 7.3).Specimens were fixed at room temperature overnight and then post-fixed in 2% OsO 4 in 0.1 M cacodylate buffer at room temperature for 1 hour.After dehydration in acetone, samples were then embedded in ultralow viscosity plastic.Thin sections were stained with uranyl acetate and lead citrate.
SEC-purified EVs were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) for 30 minutes.Samples were visualized by whole mounting vesicles onto a formvar/car bon-coated 200 grid copper mesh and counterstained with uranyl acetate.

Western blot analysis
Purified EVs were lysed using radioimmunoprecipitation assay lysis buffer.Two micrograms of EV protein content were separated on a 4%-12% Bis-Tris gel (Thermo Scientific, Rockford, IL) and then blotted onto a PVDF membrane.Nonspecific binding was blocked by Starting Block T20 PBS Blocking Buffer (Thermo Scientific, Rockford, IL) for 1 hour followed by primary antibodies targeting EV membrane protein, CD9 (Abcam, Waltham, MA; 1:10,000), or EV cytoplasmic protein, TSG101 (Abcam, Waltham, MA; 1:10,000), and incubated for 1 hour.Horseradish peroxidase-conjugated anti-rabbit secondary antibody (Invitrogen, Eugene, OR; 1:50,000) was applied to the membrane and incubated for 1 hour.Membranes were washed for 15 minutes three times between incubation periods with PBS + 0.1% Tween 20.Signal was detected using SuperSig nal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Waltham, MA) and imaged using an Invitrogen iBright CL1000.

Mass spectrometry
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 and then resuspended in 0.1% formic acid.Each sample was analyzed by nanoLC-MS/MS (Orbitrap Eclipse, Waltham, MA), and the peptides were matched against a P. carinii + Rattus norvegicus or P. murina + Mus musculus UniProt database (50; accessed June 2020) using Proteome discoverer v2.4 and the Sequest HT search algorithm (Thermo Scientific, Waltham, MA).Proteomic analysis was performed on three independent EV isolations, and data were concatenated for coverage of the proteome.PANTHER tools (30; Release 17.0) were used for functional classifications to identify Gene Ontology terms and biological processes.

Uptake assays
P. carinii-infected rat BALF EVs were used for the remaining functional assays, as described below, due to the greater yield of rat BALF EVs obtained compared to those obtained in mice and to reduce the amount of animals required for functional studies.
P. carinii-infected rat BALF EVs or control BALF EVs (2-µg protein equivalent) were stained with PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich, St. Louis, MO) for 5 minutes and then quenched with 10% bovine serum albumin.PKH26-labeled EVs were layered onto 0.971 M sucrose and centrifuged at 190,000 × g for 2 hours at 4°C to remove excess PKH26 dye.The resulting pellet was resuspended in RPMI 1640 (Gibco, Waltham, MA).
P. carinii (2 × 10 6 ) were incubated at 37°C 5% CO 2 for 2 hours to allow cells to resume metabolic function.Dead cells were produced by heat killing the cells at 80°C for 20 minutes and used as control.PKH26-labeled EVs (2-µg protein equivalent) were added to live or dead P. carinii.Cells were incubated at 37°C 5% CO 2 for 24 hours.The cells were washed in PBS and fixed in 3.7% formaldehyde in PBS for 15 minutes.Cells were attached to slides using CytoSpin 2 (Thermo Shandon, Kalamazoo, MI) at 1,000 rpm for 10 minutes.Samples were blocked in 5% goat serum for 1 hour and then incubated with anti-Msg antibodies and anti-rabbit-Alexa Fluor 488 for 1 hour each.Cells were washed between incubations with PBS + 0.1% Tween 20 three times for 15 minutes.Samples were imaged using Olympus IX83 inverted microscope.

Macrophage killing and phagocytosis assays
Macrophages were incubated with EVs as described above.After the 24-hour stimulation period, P. carinii (2 × 10 6 nuclei) were added to the wells.To assess the killing of P. carinii by macrophages, the cells were collected after 24 hours.DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen).Fungal organisms were quantified by PcDhfr copy number using TaqMan Fast Advanced Master Mix (Applied Biosciences, Waltham, MA) for qPCR on a ABI 7500 Fast Real-Time PCR system (Applied Biosciences, Waltham, MA).Serial dilutions of known P. carinii counts were used to generate the standard curve.

FIG 1 P
FIG 1 P. carinii-infected rat lungs contain abundant EVs.(A and A*) Trophic (Tr) forms were seen within the alveolar space and tightly adhered to the alveolar epithelial type I (AT1) cells.Trophic and asci (Asc) were not adhered to type II cells (AT2).Large population of EVs (arrowhead) were seen in the alveolar lumen.Scale, (A) 5 µm and (A*) 1 µm.P. carinii trophic cells (B) and asci (C) have EVs with electron-dense outer membrane.Scale, (B and C) 1 µm.

FIG 5
FIG 5 BALF EVs do not improve growth of P. carinii.Luminescence data are represented as luminescence compared to the day 0. (A) P. carinii were treated with controls or EVs (2-µg protein equivalent).ATP levels were detected by luminescence using ATPlite.EVs from Pc-infected (Pc+) rats were detrimental to P. carinii viability.(B) Rat lung epithelial cells (RLE-6TN) were treated with vehicle or EVs.EVs do not have a detrimental effect on host cell viability.Statistical analyses, analysis of variance (ANOVA).*P < 0.05 compared to vehicle at each timepoint; † P < 0.05 compared to vehicle effect.

FIG 6
FIG 6 Macrophages stimulated with EVs express pro-inflammatory cytokine mRNA.NR8383 macro phages (4 × 10 4 ) were stimulated with vehicle or EVs (2-µg protein equivalent) for 24 hours.EVs from zymosan-treated rats were used as control for a proinflammatory response.Total RNA was extracted from the cells and synthesized to cDNA.Reverse transcriptase-quantitative polymerase chain reaction revealed an upregulation of Il-1β, Il-6, and Tnf⍺ in EV treated samples, regardless of whether they originated from UI or P. carinii (Pc+)-infected rats.Statistical analyses, ANOVA.*P < 0.05 compared to vehicle; † P < 0.05 compared to UI EVs.

FIG 7
FIG 7 Macrophages stimulated with EVs do not increase P. carinii killing or phagocytosis.EVs from zymosan-treated rats were used as control for a proinflammatory response.NR8383 macrophages were stimulated with EVs for 24 hours, and then, P. carinii (2 × 10 6 nuclei) were added to the wells.(A) To assess macrophages killing of P. carinii, wells were collected after 24-hour coculture.DNA was extracted, and fungal organisms were quantified by qPCR using PcDhfr copy number compared to a standard curve.(B) Phagocytosis was assessed by collecting the co-culture after 4 hours.Macrophages were washed and differentially centrifuged at 300 × g, and then, DNA was extracted and used for qPCR.Phagocytosis was quantified by comparing the ratio of PcDhfr per RnGapdh.Statistical analyses, one-way ANOVA.*P < 0.05 compared to vehicle.

TABLE 1
Pneumocystis carinii EV proteomic profile from P. carinii-infected rat lungs identified by nLC-MS/MS analysis a

TABLE 3
Functional enrichment of biological processes within uninfected and infected rat-and mouse-derived BALF EVs a a Data represent percentage of genes matched against the total number of process hits.FIG 3 P. carinii actively uptake BALF EVs.PKH26-labeled EVs (2 µg) were incubated with either 2.5 × 10 6 live or heat-killed (Δ80°C) P. carinii for 16 hours.Cells were fixed and probed with antibodies directed against Msg.No PKH26 micelle formation was seen in PBS-labeled samples.Δ80°C P. carinii treated with PKH26-labeled UI EVs showed no uptake.Live P. carinii treated with PKH26-labeled UI EVs showed bright punctate staining within clusters.Scale, 20 µm.Research Article Microbiology Spectrum February 2024 Volume 12 Issue 2 10.1128/spectrum.03653-236