Computational protocol: Hookworm Secreted Extracellular Vesicles Interact With Host Cells and Prevent Inducible Colitis in Mice

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Protocol publication

[…] For the proteomic analysis of EVs from N. brasiliensis, 50 µg of protein of the ODG fractions with a density of 1.06–1.10 g/ml (fractions 7–9) were loaded on a 12% SDS-PAGE gel and electrophoresed at 100 V until the protein marker reached 2/3 of the total run length (approximately for 1.5 h). Each lane was sliced into 10 pieces, which were subjected to trypsin digestion as described previously (). The final digest supernatant was removed from the gel slices, and residual peptides were removed from the gel slices by washing three times with 0.1% trifluoroacetic acid for 45 min at 37°C. Peptide samples were combined into 5 tubes per lane, resulting in total 15 samples for mass spectrometry analysis. Samples were desalted and concentrated using Zip-Tip® and kept at −80°C until use.Samples were reconstituted in 10 µl of 5% formic acid. Six microliters of sample was injected onto a 50 mm 300 µm C18 trap column (Agilent Technologies, Santa Clara, CA, USA) and desalted for 5 min at 30 µl/min using 0.1% formic acid (aq). Peptides were then eluted onto an analytical nano HPLC column (150 mm × 75 µm 300SBC18, 3.5 µm, Agilent Technologies) at a flow rate of 300 nl/min and separated using a 95 min gradient of 1–40% buffer B (90/10 acetonitrile/0.1% formic acid) followed by a steeper gradient of 40–80% buffer B in 5 min. The mass spectrometer (ABSCIEX 5600+) operated in information-dependent acquisition mode, in which a 1-s TOF MS scan from 350–1,400 m/z was performed, and for product ion ms/ms 80–1,400 m/z ions observed in the TOF-MS scan exceeding a threshold of 100 counts and a charge state of +2 to +5 were set to trigger the acquisition of product ion. Analyst 1.6.1 (ABSCIEX) software was used for data acquisition and analysis.For the analysis of the EV mass spectrometry data, a database was built using the N. brasiliensis genome (PRJEB511) with the common repository of adventitious proteins (cRAP) appended to it. Database search was performed using Mascot Versions 2.4 (Matrix Science Ltd., London, UK) and X!Tandem, MS-GF+, OMSSA, and Tide search engines using SearchGUI (). The same parameters were used as described in Ref. ().The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD009165 and 10.6019/PXD009165. A final list of parasite-specific proteins resulted by combining the different fractions and removing hits for common contaminants from the cRAP database, considering only proteins containing at least two validated unique peptides matching N. brasiliensis gene models. Proteins were functionally classified according to Gene Ontology categories using the software Blast2GO basic version 4.0.7 (). Putative signal peptides and transmembrane domain(s) were predicted using the programs CD-Search tool () and SignalP (). Structural comparison of proteomic datasets was performed by all-vs-all blast in NCBI Blast + executables (v2.7.1). [...] Murine SI organoids were produced from intestinal crypts of a female C57 Bl6/J mouse according to previous reports () with some modifications. Briefly, murine SI crypts were dissociated with Gentle Cell Dissociation reagent (Stemcell Technology Inc., Vancouver, BC, Canada). Approximately 500 crypts were seeded in 50 µl of Matrigel (Corning Inc., New York, NY, USA) in a 24-well plate and cultured in Intesticult Organoid Growth Medium (Stemcell Technology Inc.).Imaging was performed as described in Eichenberger et al. () with minor modifications. Briefly, to investigate internalization of EVs in the SI epithelium layer, 30–50 million PKH26 (Sigma-Aldrich) -labeled EVs in 3–5 µl were injected into the central lumen of individual organoids and cultured for 3 h at 37 and 4°C, respectively. Washed organoids were fixed and autofluorescence was quenched with 50 mM NH4Cl in PBS (for 30 min at RT) and 100 mM glycine in PBS (for 5 min). Cell nuclei were stained with Hoechst dye (Invitrogen, Carlsbad, CA, USA) and images were visualized on a laser scanning confocal microscope (Zeiss 780 NLO, Zeiss, Oberkochen, Germany). Confocal image deconvolution was performed in ImageJ using the plugins “Diffraction PSF 3D” for PSF calculation and “DeconvolutionLab” with the Tikhonov–Miller algorithm for 2D deconvolution (). […]

Pipeline specifications

Software tools ImageJ, Diffraction PSF 3D, DeconvolutionLab
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Caenorhabditis elegans, Rattus norvegicus, Homo sapiens, Nippostrongylus brasiliensis, Trichuris muris
Diseases Celiac Disease, Colitis, Gastrointestinal Diseases, Hookworm Infections, Infection, Parasitic Diseases, Inflammatory Bowel Diseases