DOI: 10.5061/dryad.p2ngf1vxr
title: Extracellular Vesicles from Pneumocystis carinii-infected rats impair fungal viability but are dispensable for macrophage functions

description: 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.
description: 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).
description: GENERAL INFORMATION 1. Title of Dataset: Extracellular Vesicles from
 Pneumocystis carinii-Infected Rats Impair Fungal Viability but are
 Dispensable for Macrophage Functions 2. Author Information A. Principal
 Investigator Contact Information Name: Melanie Cushion Institution:
 University of Cincinnati Address: Cincinnati, OH USA Email:
 B. Associate or Co-investigator Contact
 Information Name: Steven Sayson Institution: University of Cincinnati
 Address: Cincinnati, OH USA Email: 3. Date of
 data collection (single date, range, approximate date): 2020-2023
 SHARING/ACCESS INFORMATION 1. Licenses/restrictions placed on the data:
 CC0 1.0 Universal (CC0 1.0) Public Domain 2. Links to publications that
 cite or use the data: Sayson, S. G., Ashbaugh, A., & Cushion, M. T.
 (2024). Extracellular Vesicles from Pneumocystis carinii-infected rats
 impair fungal viability but are dispensable for macrophage functions.
 Microbiology Spectrum. 1. Links to other publicly accessible locations of
 the data: None 2. Links/relationships to ancillary data sets: None 3. Was
 data derived from another source? No A. If yes, list source(s): NA 4.
 Recommended citation for this dataset: Sayson, S. G., Ashbaugh, A., &
 Cushion, M. T. (2024). Data from: Extracellular Vesicles from Pneumocystis
 carinii-infected rats impair fungal viability but are dispensable for
 macrophage functions. Dryad Digital Repository.
 
 \#########################################################################
 DATA & FILE OVERVIEW 1. File List: A) Pc_rat1.xlsx B) Pc_rat2.xlsx C)
 Pm_mouse1.xlsx 1. For all datasets: Samples listed on sheet of *.xlsx.
 Naming scheme: {Species origin}_{infection status}{replicate} Rn = Rattus
 novegicus Mm = Mus musculus UI = Uninfected Pc/Pm = rodents infected with
 P. carinii or P. murina, as listed.
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: Pc_rat1.xlsx 1. Protein Pilot (Sciex;
 ) was
 used to generate peptide report from nLC-MS/MS raw data. 2. Table heading
 legend * %Cov(95) = The percentage of matching amino acids from identified
 peptides having confidence greater than or equal to 95% divided by the
 total number of amino acids in the sequence * Accessions = Protein
 assession number * Names = Name of the protein identified * Conf = The
 confidence for the peptide identification, expressed as a percentage *
 Sequence = The sequence of the peptide identified by the search *
 Modifications = Amino acid modifications found by the search for each
 peptide. * Cleavages = Indicates any atypical or missed cleavage sites *
 dMass = The difference in mass between the precursor molecular weight and
 the theoretical molecular weight of the matching peptide sequence * Obs MW
 = The experimentally measured monoisotopic mass for the precursor ion
 fragment for the peptide sequence, including modifications * Obs m/z = The
 experimentally measured monoisotopic m/z for the precursor ion fragment
 for the peptide sequence, including modifications * Theor MW = The
 theoretical monoisotopic mass for the precursor ion fragment for the
 peptide sequence, including modifications * Theor m/z = The theoretical
 monoisotopic m/z for the precursor ion fragment for the peptide sequence,
 including modifications * Theor z = The theoretical charge for the
 fragmentated ion, as calculated by the Paragon Algorithm * Sc = The score
 for the peptide; this is based on matching ions of various charge states *
 Spectrum = Denoted a particular MS/MS spectrum in this processing run *
 Acq Time = The retention time when the fragmentation spectrum was acquired
 * Intensity (Peptide) = The apex of the elution for a peptide in a given
 charge state in arbitrary units * PrecursorIntensityAcquisition =
 Precursor mass intensity in arbitrary units * Apex Time (Peptide) = The
 time of the apex of a peptide's elution * Elution Peak Width
 (Peptide) = The width of the peak corresponding to the detected peptide
 mass * MS2Counts = Sum of the fragment ions in arbitrary units BLANK cells
 indicate no data provided or detected.
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: Pc_rat2.xlsx and Pm_mouse1.xlsx 1. Proteome
 discoverer (Thermo Scientific;
 ) was used to generate peptide report from nLC-MS/MS raw data using the Sequest HT search algorithm. 2. Table heading legend * Description = Name of the protein identified * master protein = Identified Master * accession = Protein assession number * gene symbol = unique gene * Score Sequest HT = Composite Sequest HT core for all peptides corresponding the the identified protein * Exp. q-value = adjusted p-values found using an optimised FDR approach **(expand group to view)** * Annotated Sequence = Peptide sequence * Modifications = Amino acid modifications detected for each peptide. Detail in Suppl Table 2. * Master Protein = Accession of Master Protein * Position in Master Proteins = The amino acid sequence position in the identified protein corresponding to the peptide in question. * XCorr: Sequest HT = Individual peptide scores where 1.7-2 corresponds to a 95% FDR confidence and &gt;2 are 99% confidence. * \# Protein Groups = The total number of protein groups a particular protein belongs to * \# Proteins = The total number of proteins a particular protein belongs to * \# Missed Cleavages = Number of Thermolysin missed cleavages in the identified peptide * Confidence = FDR confidence of each peptide where medium and high represent &gt;95% and &gt;99% confidence, respectively * Theo. MH+ [Da] = calculated mass + H of the identified peptide * m/z [Da] = Detected mass to charge (m/z) of the identified peptide * DeltaM [ppm] = The mass difference between the theoretical m/z and the detected m/z in parts per million (ppm) * RT [Min] = The nanoLC retention time for the identified peptide BLANK cells indicate no data provided or detected.