<em>Mycobacterium tuberculosis</em> triggers reduced inflammatory cytokine responses and virulence in mice lacking Tax1bp1

The dataset contains the imaging, proteomics analysis, and flow cytometry files that support our mansucript titled: Mycobacterium tuberculosis triggers reduced inflammatory cytokine responses and virulence in mice lacking Tax1bp1. Immunofluorescence microscopy images depict the staining of autophagy markers (ubiquitin and LC3) in primary wild-type and Tax1bp1-deficient murine alveolar macrophages infected with ZsGreen-expressing M. tuberculosis (Mtb). Fluorescence microscopy images depict the staining of uninfected control and Mtb-infected alveolar and bone marrow-derived macrophages with propidium iodide and Caspase-3/7 Green ReadyProbe as indicators of cell death. Images were obtained daily for 7 days after being infected with a wild-type non-fluorescent M. tuberculosis strain. Flow cytometry (FCS) files contain the analysis of AMs stained in BAL fluid from naive wild-type and Tax1bp1-deficient mice, and immune cells stained in the lungs from wild-type and Tax1bp1-deficient mice infected with ZsGreen-expressing Mtb. Histopathology files contain the following histopathology slide files of wild-type and Tax1bp1-deficient mice infected with: (1) Mtb for ubiquitin and Mtb immunohistochemistry in the lungs, (2) Mtb for H&E staining of the lungs, and (3) Mtb for myeloperoxidase immunostaining in the lungs, and (4) Listeria monocytogenes for H&E staining of the spleens. The proteomics file contains the complete list of changes in protein abundance during Mtb infection vs. mock-infection of primary murine alveolar macrophages. Methods Immunofluorescence microscopy: AMs were infected with fluorescent Mtb at a MOI of 2. At 8- and 24-hours post-infection, monolayers were washed with PBS, fixed with 4% PFA for 20 minutes, washed with PBS, and stained with anti-LC3 or anti-ubiquitin primary antibodies and AlexaFluor-647 conjugated secondary antibodies as previously described (Budzik et al., 2020 PMCID: PMC7030789). Images were obtained at 63x magnification from quadruplicate wells per condition, in 69 x/y positions, and 4 z positions (0 mm, 0.5 mm, 1 mm, and 1.5 mm) with an Opera Phenix microscope (Perkin Elmer). Colocalization analysis of LC3, ubiquitin, and Mtb was performed with Harmony (Perk Elmer) using the following analysis parameters. The four Z stack images in each x/y position were processed into a maximum projection. Nuclei were identified in the DAPI channel using Method B with a common threshold of 0.07 and an area threshold of > 20 mm2. Cytoplasm was identified in the AlexaFluor 647 channel using Method A with an individual threshold of 0.06. The find spot module was used to identify LC3 or ubiquitin "spots" in the AlexaFluor 647 channel using method C with a contrast setting of 0.42, uncorrected spot to region intensity of 3.8, and default radius. Mtb were identified in the AlexaFluor 488 channel using the find spot module method B with a detection sensitivity of 0.5 and splitting sensitivity of 0.5. To identify Mtb that colocalized with LC3 or ubiquitin "spots", the select population module was used for the Mtb population with the select by mask method. The percent colocalization was calculated for each well from all the images obtained in each well using the evaluation module. Live cell imaging experiments: AMs were uninfected or infected with Mtb at a MOI of 1, and 0.1 μg ml−1 of propidium iodide (LifeTechnologies) and two drops per milliliter of CellEvent Caspase-3/7Green ReadyProbes reagent (Invitrogen) were added to the media at the beginning of the infection to measure necrosis/late apoptosis and apoptosis, respectively. Fluorescence and phase contrast images were obtained at 20x magnification with a Keyence BZ-X 700 microscope. Images were obtained daily in three technical replicate wells per condition and at two positions in each well. Quantification of the number of necrotic and apoptotic cells was performed with ImageJ as described previously (Golovkine et al., 2023 PMCID: PMC1102773). Images were converted to 8-bit (grayscale), binarized, and enumerated using the analyze particles module (size threshold 0.001-infinity). FACS experiments: For the characterization and infection of sorted AMs from the BAL, AMs in BAL cells were stained, and the SiglecF+ CD11c+ AMs were gated as previously described (DOI: 10.21769/BioProtoc.3302). For the measurement of immune cell recruitment in the lungs of wild-type and Tax1bp1-deficient mice infected with Zs-Green expressing M. tuberculosis, lung homogenates were stained with two antibody panels. The first panel detected alveolar macrophages, recruited monocyte-derived cells, and neutrophils. The second panel detected T cells, B cells, and NK cells. The samples were fixed with paraformaldehyde and analyzed by FACS. Histopathology: The specified organs were fixed in paraformaldehyde. Organs were thin-sectioned and stained with H&E, and antibodies for myeloperoxidase, Mtb, or ubiquitin. In the slide file names, Tax1bp1 is abbreviated TAX, and wild-type is abbreviated WT. D21 and D50 denote post-infection day 21 and 50. Proteomics sample preparation: AMs were infected with Mtb at a MOI of 10. At 24 hours post-infection, monolayers were lysed. A bicinchoninic acid assay (Pierce) was performed to measure protein concentration in cell lysate supernatants. 1 mg of each clarified lysate was reduced by the addition of dithiothreitol (DTT) to a 5 mM final concentration for 30 min at room temperature and alkylated by the addition of iodoacetamide to 10 mM final concentration for 30 min at room temperature in the dark. Remaining alkylating agent was quenched by the addition of DTT to 10 mM final concentration for 30 min at room temperature in the dark. The samples were diluted with 100 mM ammonium bicarbonate, pH 8.0, to reduce the urea concentration to below 2M. Samples were incubated with sequencing grade modified trypsin (Promega) at a 1:25 enzyme:protein ratio and LysC (Wako) at a 1:100 enzyme:protein ratio overnight at 37C with rotation. Prior to desalting, the sample pH was reduced to approximately 2.0 by the addition of 10% trifluoroacetic acid (TFA) to a final concentration of 1% trifluoroacetic acid. Peptides were desalted using 50 mg SepPak C18 solid-phase extraction cartridges (Waters). The columns were activated with 3 ml of 80% acetonitrile (ACN) 0.1% TFA, and equilibrated with 3 ml of 0.1% TFA. Peptide samples were applied to the columns, and the columns were washed with 3 ml of 0.1% TFA. Peptides were eluted with 1.1 ml of 40% ACN, 0.1% TFA. 10 µg of peptides analyzed by LC-MS/MS for global protein abundance measurement. Liquid chromatography and mass spectrometry Following digestion, peptides were separated on a PepSep reverse-phase C18 column (1.9 μm particles, 15 cm, 150 mm ID) (Bruker) with a gradient of 3–28% buffer B (0.1% formic acid in 80% acetonitrile) over buffer A (0.1% formic acid in water) over 67 minutes, an increase to 40% B in 5 minutes, and held at 95% B for 8 minutes. Eluting peptide cations were analyzed by electrospray ionization on an Orbitrap Exploris (Thermo Fisher Scientific). For DIA analysis, MS1 scans of peptide precursors were performed at 120,000 resolution (200 m/z) over a scan range of 350-1050 m/z, with an AGC target of 250% and a max injection time of 100 ms. MS2 scans were collected over 350-950 m/z in 15 m/z isolation windows with a 0.5 m/z overlap. Maximum injection time was set to auto, with AGC set to standard, and an MS2 resolution of 15,000. Higher energy collisional dissociation (HCD) was performed at a NCE of 28%. Six gas-phase fractions (GPF) (https://doi.org/10.1038/s41467-020-15346-1) -DIA runs were collected from a pooled sample of all conditions. tSIM MS1 scans were performed at 120,000. tMS2 scans were collected at 30,000 fragment resolution, an AGC target of 1e6, a maximum ion injection time of 60 ms, and a NCE of 26. Data was collected using 4m/z precursor isolation windows in a staggered-window pattern, with each GPF fraction covering an approximately 100 m/z range. Proteomics RAW files are accessible on the ProteomeXChange Accession # PDX064244.