DOI: 10.5061/dryad.44j0zpcq6
title: <em>Mycobacterium tuberculosis</em> triggers reduced inflammatory cytokine responses and virulence in mice lacking Tax1bp1

description: 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. 
description: Immunofluorescence microscopy: AMs were infected with
 fluorescent <em>Mtb</em> 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 <em>Mtb</em> 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 &gt; 20 mm<sup>2</sup>. 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. <em>Mtb</em> 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 <em>Mtb</em> that colocalized with LC3 or ubiquitin "spots", the select population module was used for the <em>Mtb</em> 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 <em>Mtb</em> at a MOI of 1, and 0.1 μg ml<sup>−1</sup> 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 <em>M. tuberculosis</em>, 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, <em>Mtb</em>, 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 <em>Mtb</em> 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.
description: # *Mycobacterium tuberculosis* triggers reduced inflammatory cytokine
 responses and virulence in mice lacking Tax1bp1
 [https://doi.org/10.5061/dryad.44j0zpcq6](https://doi.org/10.5061/dryad.44j0zpcq6) ## Description of the data and file structure Wild-type and Tax1bp1-/- alveolar macrophages were infected with fluorescent or wild-type *M. tuberculosis* (Mtb) in order to measure: (1) colocalization of Mtb and autophagy markers (Ubiquitin or LC3) at 8- and 24-hours post-infection by confocal microscopy. Nuclei were stained with DAPI. Autophagy markers were stained with primary antibodies and secondary antibodies conjugated to Alexa-Fluor 647. Mtb expressing ZsGreen was used in these experiments. or (2) host cell death by live cell imaging. Experiments were performed with propidium iodide to detect necrotic/late apoptotic cells in the red channel and caspace 3/7 CellEvent to detect apoptotic cells in the green channel. ### Files and variables #### File: 20231215_8hpi_24hpi_AM_UB_LC3_TIFF_-_Copy.zip **Description:** Microscopy images are included. The Excel files includes the sample legend and the percent colocalization between autophagy markers ubiquitin (UB) or LC3 and Mtb. Abbreviations include: AM alveolar macrophages, hpi hours post-infection. File names are labeled as follows: r## denotes the well row number in the 96-well plate; c## denotes the well column number in the 96-well plate; f## denotes the frame or x-y position number within the well; p## denotes the z-position in the well from the bottom z-position (p01) to the top (p04); ch1 denotes the blue fluorescence channel for DAPI; ch2 denotes the green fluorescence channel for ZsGreen-expressing Mtb; ch3 denotes the AlexaFluor647 channel for ubiquitin or LC3 staining. ### Files: Live_cell_imaging_for_Cell_Death_Mtb-infected_Dryad.zip and Live_cell_imaging_for_Cell_Death_uninfected_Dryad.zip **Description:** Microscopy images are included. The Excel files include a sample legend. Abbreviations include: AM alveolar macrophage, BMDM bone marrow-derived macrophage, IFN interferon. ### **File: FCS_files.zip** **Description:** Flow cytometry files are included. The folder name denotes the corresponding experiment. Abbreviations include: TAX Tax1bp1-/-, and WT wild-type. ### File: results_AB_122024.csv **Description:** Complete list of the changes in protein abundance in *Mtb*-infected AMs compared to mock-infected samples. The columns are identified as: * Protein: UnitprotKB protein name * Gene: gene encoding the protein * Description: protein description * Gene Ontology: Gene Ontology biological processes of the gene and protein * Organism: Mus musculus (mouse) denotes a murine protein and Mycobacterium tuberculosis (strain ATCC 35801 / TMC 107 / Erdman) denotes a *Mtb* protein * log2FC (TB-mock): log~2~fold change in protein abundance in the *Mtb*-infected vs. mock-infected alveolar macrophages measured across five independent biological replicate experiments * pvalue (TB-mock) and adj.pvalue (TB-mock): p-value and adjusted p-value of the log~2~fold change measured across five independent biological replicate experiments ## Code/software 1. Colocalization analysis of LC3, ubiquitin, and *Mtb* was performed with Harmony version 4.9 (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 &gt; 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. 2. Quantification of the number of necrotic and apoptotic cells was performed with ImageJ version 1.54f as described previously [(Golovkine et al., 2023 PMCID: ](https://sciwheel.com/work/citation?ids=14787233&pre=&suf=&sa=0&dbf=0)[PMC11027733](http://www.ncbi.nlm.nih.gov/pmc/articles/pmc11027733/)[)](https://sciwheel.com/work/citation?ids=14787233&pre=&suf=&sa=0&dbf=0). Images were converted to 8-bit (grayscale), binarized, and enumerated using the analyze particles module (size threshold 0.001-infinity). 3. Proteomics analysis. The GPF mass spectrometry data was searched against the Uniprot mouse database appended with the Erdman Mtb strain in Spectronaut to build a spectral library of detected peptides and proteins (Biognosys, version 19.0). Default settings, including trypsin digestion, variable modifications of methionine oxidation and N-termini acetylation, and fixed modification of cysteine carbamidomethylation, were used. DIA mass spectrometry data was then searched in Spectronaut against the GPF-generated spectral library. DIA runs were filtered to obtain a false discovery rate of 1% at the peptide spectrum match and protein level ([https://doi.org/10.1038/nmeth1019](https://doi.org/10.1038/nmeth1019)). Quantitative analysis was performed in the R statistical programming language using the artMS package (release 3.20) and quantitative packages from MSstats (version 3.20) (DOI: 10.1093/bioinformatics/btu305). For MSstats analysis, the normalization_method was set to equalizeMedians, and censored missing values were imputed by the Accelerated Failure Time modeStatistically significant changes were selected by applying a log2-fold-change (&gt;1.0 or&lt;−1.0) and an adjusted p value (&lt;0.05). ## Access information Other publicly accessible locations of the data: * Proteome XChange PRIDE Project Accession # PXD064244 * Histopathology files:  (1) Wild-type and Tax1bp1-/- mice infected with Mtb for H&E staining. Slides are available online at: [https://app.histowiz.com/shared_groups/e1bb0845-be2f-4174-9462-cfec419432da/slides](https://app.histowiz.com/shared_groups/e1bb0845-be2f-4174-9462-cfec419432da/slides). Images were analyzed by a board-certified veterinary pathology at Histowiz for Lesion Burden and Granloma/necrosis score. In the file names, D## denotes the day post-infection that the samples were obtained from the mice (for example, D21 is 21 days post-infection). WT denotes wild-type. TAX denotes Tax1bp1-deficient.  (2) Wild-type and Tax1bp1-/- mice infected with Mtb for myeloperoxidase staining, or ubiquitin and Mtb staining. Slides are available online at: [https://app.histowiz.com/shared_groups/1ef43319-292f-4932-8de9-5279bc4532c1/slides](https://app.histowiz.com/shared_groups/1ef43319-292f-4932-8de9-5279bc4532c1/slides) and [https://app.histowiz.com/shared_groups/1588e460-287c-41e4-bcdf-aee1b37f9e1c/slides](https://app.histowiz.com/shared_groups/1588e460-287c-41e4-bcdf-aee1b37f9e1c/slides). QuPath was used to calculate the percent of myeloperoxidase positive cells and overlap of Mtb and ubiquitin staining. In the file names, WT denotes wild-type. TAX denotes Tax1bp1-deficient. Myeloperox denotes myeloperoxidase staining. Ubiquitin denotes ubiquitin staining. Tuberculosis denotes Mtb staining.  (3) Wild-type and Tax1bp1-/- mice infected with Listeria for H&E staining (spleen and liver). Slides are available online at: [https://app.histowiz.com/shared_groups/b09a932e-f940-4e83-80c8-595f996bd040/slides](https://app.histowiz.com/shared_groups/b09a932e-f940-4e83-80c8-595f996bd040/slides). Images were analyzed by a board-certified veterinary pathology at Histowiz for lymphocyte depletion, coagulative necrosis, and microabscesses. WT denotes wild-type. TAX denotes Tax1bp1-deficient.