Extracellular Vesicles from Pneumocystis carinii-infected rats impair fungal viability but are dispensable for macrophage functions
Data files
Jan 12, 2024 version files 6.11 MB
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Pc_rat1.xlsx
3.67 MB
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Pc_rat2.xlsx
198.52 KB
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Pm_mouse1.xlsx
2.24 MB
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README.md
6.42 KB
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 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.
README
GENERAL INFORMATION
Title of Dataset: Extracellular Vesicles from Pneumocystis carinii-Infected Rats Impair Fungal Viability but are Dispensable for Macrophage Functions
Author Information
A. Principal Investigator Contact Information
Name: Melanie Cushion
Institution: University of Cincinnati
Address: Cincinnati, OH USA
Email: cushiomt@ucmail.uc.eduB. Associate or Co-investigator Contact Information
Name: Steven Sayson
Institution: University of Cincinnati
Address: Cincinnati, OH USA
Email: Steven.Sayson@uc.eduDate of data collection (single date, range, approximate date): 2020-2023
SHARING/ACCESS INFORMATION
Licenses/restrictions placed on the data: CC0 1.0 Universal (CC0 1.0) Public Domain
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.
Links to other publicly accessible locations of the data: None
Links/relationships to ancillary data sets: None
Was data derived from another source? No
A. If yes, list source(s): NARecommended 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. https://doi.org/10.5061/dryad.p2ngf1vxr
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DATA & FILE OVERVIEW
- File List:
A) Pc_rat1.xlsx
B) Pc_rat2.xlsx
C) Pm_mouse1.xlsx
- 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.
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DATA-SPECIFIC INFORMATION FOR: Pc_rat1.xlsx
Protein Pilot (Sciex; https://sciex.com/products/software/proteinpilot-software) was used to generate peptide report from nLC-MS/MS raw data.
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.
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DATA-SPECIFIC INFORMATION FOR: Pc_rat2.xlsx and Pm_mouse1.xlsx
Proteome discoverer (Thermo Scientific; https://www.thermofisher.com/us/en/home/industrial/mass-spectrometry/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-software/multi-omics-data-analysis/proteome-discoverer-software.html) was used to generate peptide report from nLC-MS/MS raw data using the Sequest HT search algorithm.
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 >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 >95% and >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.
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).