DOI: 10.5061/dryad.bvq83bkj9
title: Data from: A host-adapted auxotrophic gut symbiont induces mucosal immunodeficiency (Part I)

description: Harnessing the microbiome to benefit human health requires an initial step
 in determining the identify and function of causative microorganisms that
 impact specific host physiologic functions. Here we show a functional
 screen of the bacterial microbiota from mice with low intestinal
 immunoglobulin A (IgA) levels; we identified a Gram-negative bacterium,
 proposed as Tomasiella immunophila that induces and degrades IgA in mouse
 intestine. Mice harboring T. immunophila are susceptible to infections and
 show poor mucosal repair. T. immunophila is auxotrophic for the bacterial
 cell wall amino sugar N-acetylmuramic acid (MurNAc). It delivers
 immunoglobulin-degrading proteases into outer membrane vesicles that
 preferentially degrade rodent antibodies with kappa but not lambda light
 chains. This work indicates a role for symbionts in immunodeficiency,
 which might be applicable to human disease. 
description: To screen for IgA-degrading bacteria from SIgA-Low mice
 (<em>15</em>), fecal samples were cultured anaerobically and
 plated on blood agar. Pools of bacterial colonies were then incubated with
 mouse monoclonal IgA and subjected to SDS-PAGE and immunoblot analysis.
 Pooled bacterial colonies showing IgA-degrading activity were tested
 individually again for cleavage activity. 16S rRNA gene sequencing
 revealed an IgA-degrading bacterium as a previously unknown species. Using
 the Genome Taxonomy Database Toolkit (GTDB-Tk), this bacterium was
 classified as the initial isolated representative of a previously
 undescribed genus within the <em>Muribaculaceae</em>
 family. Protologger (v1.3) was used to describe this novel taxon
 (<em>51</em>), which was proposed to be named
 <em>Tomasiella immunophila. </em>The bacterium and its outer
 membrane vesicles (OMVs) were visualized using transmission and scanning
 electron microscopy. Wild-type B6 mice were orally administered IgA-High
 fecal slurry with or without <em>T. immunophila</em> to test
 its in vivo IgA-degrading activity. The biological implications of
 <em>T. immunophila</em>-mediated degradation of SIgA in mice
 were assessed using the <em>Salmonella</em> Typhimurium
 vaccination/infection model (23), <em>Candida
 albicans</em> infection (<em>24, 25</em>), and the
 dextran sulfate sodium (DSS)-induced colitis model
 (<em>15</em>). Wild-type B6 mice were orally administered
 <em>T. immunophila</em> to evaluate its capability to induce
 IgA production. <em>Rag1<sup>-/-</sup></em> (no
 T/B cells) and <em>Pigr<sup>-/-</sup></em> (no
 luminal IgA/IgM) mice were colonized with <em>T.
 immunophila</em> to investigate the dependency of <em>T.
 immunophila</em> colonization on IgA. All mouse antibody isotypes
 and subclasses were incubated with <em>T. immunophila </em>and
 its OMVs in vitro to evaluate their susceptibility to degradation. The
 terminal cleavage sites of mouse IgG antibodies by T. immunophila were
 identified using N-terminal sequencing. All mouse antibody isotypes and
 subclasses, including those with kappa (κ) or lambda (λ) light chains,
 were incubated with <em>T. immunophila </em>and its outer
 membrane vesicles (OMVs) to assess their susceptibility to degradation in
 vitro. Recombinant mouse IgG1 antibodies with κ or λ light chains, mouse
 serum, and mouse fecal slurry were utilized to investigate the specificity
 of <em>T. immunophila</em>-mediated antibody cleavage.
 Additionally, antibodies from various species, including humans, were
 incubated with <em>T. immunophila</em>. Furthermore,
 recombinant chimeric IgG antibodies, consisting of mouse heavy chains with
 mouse (m-κ) or human κ light chains (h-κ), and human heavy chains with
 human or mouse κ light chains, were also incubated with T. immunophila to
 validate the light chain-dependent antibody cleavage.
description: # A host-adapted auxotrophic gut symbiont induces mucosal immunodeficiency
 **[Access of dataset on Dryad:](https://doi.org/10.5061/dryad.j0zpc86p8)**
 **[https://doi.org/10.5061/dryad.j0zpc86p8](https://doi.org/10.5061/dryad.j0zpc86p8),** raw data of main figure 2A, **[https://doi.org/10.5061/dryad.bvq83bkj9](https://doi.org/10.5061/dryad.bvq83bkj9),** all additional raw experimental data are presented in the main and supplementary figures of the manuscript **Summary of dataset contents:** Two key datasets are included to provide all the data utilized in the manuscript supporting the conclusion that the bacterium *Tomasiella immunophila* is responsible for reduced levels of intestinal immunoglobulin A (IgA) in wild-type B6 mice and explores its biological impact on the host. The first dataset consists of the NEXUS file used to construct the phylogenetic tree (Fig. 2A). This tree is pivotal as it visually demonstrates the distinct separation of the novel genus, *Tomasiella*, from closely related genera within the family *Muribaculaceae*. The second dataset encompasses all other experimental data presented in the manuscript. This includes results from an in vitro functional screening aimed at identifying IgA-degrading activity, specifically in mice exhibiting low IgA levels. The discovery of *Tomasiella immunophila* as a previously unidentified bacterium was rigorously validated through both in vitro biochemical assays and in vivo animal models. Together, these datasets provide comprehensive evidence supporting the manuscript's conclusion that *Tomasiella immunophila* plays a significant role in modulating intestinal IgA levels and elucidate its broader biological implications on the host. ## Description of the data and file structure [https://doi.org/10.5061/dryad.j0zpc86p8](https://doi.org/10.5061/dryad.j0zpc86p8): the NEXUS file for the tree from our manuscript, Figure 2A. [https://doi.org/10.5061/dryad.bvq83bkj9](https://doi.org/10.5061/dryad.bvq83bkj9): The data files include two separate zip files labeled as “Raw data for main figures” and “Raw data for supplemental figures”. Each zip files contains all the raw data for each panel of the figures (except Fig. 2A) in the manuscript **Key words and abbreviations used in the data files:** IgA-Low and IgA-High mice: wild-type C57BL/6 mice from the Jackson laboratory with spontaneous low or high levels of intestinal secretory immunoglobulin A (SIgA). BSA: Bovine serum albumin Ig: immunoglobulin MurNAc: N-Acetylmuramic acid GlcNAc: N-Acetylglucosamine DSS: dextran sodium sulfate TCEP: tris(2-carboxyethyl) phosphine NEM: N-Ethylmaleimide EDTA: Ethylenediaminetetraacetic acid EGTA: ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid Ti: Tomasiella immunophila OMV (s): outer membrane vesicle(s) 16S: 16S ribosomal RNA (rRNA) qPCR: quantitative real-time PCR CFU: colony formation unit FL: full-length Pigr: polymeric immunoglobulin receptor CH: constant region NC: negative control Sup: supernatant FMT: fecal microbiota transplantation ELISA: enzyme-linked immunosorbent assay SDS-PAGE: sodium dodecyl sulfate–polyacrylamide gel electrophoresis **Raw Data for main figures** This zip file contains all the source data for the main figures, and each figure has a separate folder with subfolders containing the raw data for each panel. Fig. 1, this folder contains the following subfolders: Fig. 1A, the source data for the immunoblot of IgA and Ponceau S staining of BSA for the upper and lower panels (including repeat experiment). The data presents the results from anaerobic and aerobic fecal cultures from IgA-Low mice housed in three separate cages and incubated with monoclonal mouse IgA (upper panel) and BSA (lower panel) at 37°C for 6 hours. Fig. 1B, the source data of the immunoblot and the repeat. The data presents results from anaerobic fecal cultures of IgA-Low and IgA-High mice incubated with monoclonal mouse IgA at 37°C for specified durations. Fig. 1C, the source data for displaying results from incubating IgA with cultured colony #8 under various conditions (upper panel), along with PCR detection confirming the presence of T. immunophila (DNA gel in the lower panel). Fig. 1D,this folder includes 3 main components: 1. Raw reads folder: Contains 16S ribosomal RNA gene (rRNA) amplicon sequencing results from 5 subcultures of colony #8 ("Negative #1", "Negative #2", "Positive #1", "Positive #2", and "Negative #3"), grown on blood agar plates sourced from different vendors. 2. Raw Analysis folder: This folder contains the analyzed dataset of the 16S rRNA amplicon sequencing results for the 5 samples mentioned above. Within the "Raw Analysis" folder, there are:  out_table: OUT analysis results without rarefying. rarefied_otu_table: OUT analysis results with rarefying. rarefied_otu_table_L2 to rarefied_otu_table_L7: OUT analysis results at 6 taxonomic levels (species through phylum), where L7 corresponds to species and L2 to phylum. 3. A spreadsheet designed for visualizing data (between high and low IgA), specifically showing the abundance of different bacteria across the 5 subcultures of colony #8. Fig. 1E, the source data of the immunoblot detecting IgA degradation after being incubated with colony #8 cultured with various antibiotics (upper panel), along with PCR detection confirming the presence of T. immunophila (DNA gel in the lower panel). Fig. 1F, the source data of the immunoblot showing three Muribaculaceae sp. strain X isolates incubated with IgA (upper panel) and BSA (Ponceau S staining in the lower panel). Fig. 1G, the source data of fecal IgA levels of individual mouse in IgA-High and IgA-Low groups measured by ELISA (upper panel), along with PCR detection confirming the presence of T. immunophila (DNA gel in the lower panel). Fig. 2, this folder contains the following subfolders: Fig. 2A, the Adobe Illustrator file of the phylogenomic tree. The link for the raw data of this tree is: [https://datadryad.org/stash/dataset/doi:10.5061/dryad.j0zpc86p8](https://datadryad.org/stash/dataset/doi:10.5061/dryad.j0zpc86p8) Fig. 2B, the source and processed images of gram-stained T. immunophila cultured without (Left panel) and with 5 µg/mL of MurNAc. Fig. 2C, the source images of T. immunophila grown on blood agar plates supplemented with indicated additives. Fig. 2D, two files with DNA sequences of the genes in the predicated MurNAc transport pathway in Tomasiella immunophila and Tannerella forsythia, and a spreadsheet containing detailed information on all the genes for creating the pathway schematic. Fig. 2E, the source images from transmission electron microscopy and scanning electron microscopy for Tomasiella immunophila and its outer membrane vesicles (OMVs). Fig. 2F, the source data of the immunoblot and the repeat. This data shows the levels of IgA after being incubated with T. immunophila culture, supernatant of culture, OMVs, and supernatant of OMVs. Fig. 2G, the source data of the immunoblot and the repeat. This data shows the levels of IgA after being incubated with intact T. immunophila OMVs and OMVs disrupted by sonication or 2% Tween 20. Fig. 3, this folder contains the following subfolders: Fig. 3A, a spreadsheet containing the source data of fecal IgA ELISA. This dataset includes the fecal IgA levels of IgA-Low and IgA-High mice before and after cohousing for 14 days. Fig. 3B, the source data of DNA gel for detecting T. immunophila (Ti, upper panel) and total bacteria (16S, lower panel) in feces of IgA-Low and IgA-High mice before and after cohousing for 14 days. Fig. 3C, the source data of DNA gel for detecting T. immunophila (Ti, upper panel) and total bacteria (16S, lower panel) in feces of IgA-Low mice before and after vancomycin treatment for 14 days. Fig. 3D, a spreadsheet containing the source data of fecal IgA ELISA. This dataset includes the fecal IgA levels of IgA-Low mice before and after vancomycin treatment for 14 days. Fig. 3E, a spreadsheet containing the source data for fecal IgA ELISA. This dataset includes the fecal IgA levels of 2 groups of wild-type B6 mice, both before and after being gavaged with IgA-High fecal slurry, with and without T. immunophila. Fig. 3F, the source data of DNA gel for detecting T. immunophila (Ti) and total bacteria (16S) in feces of 2 groups of wild-type B6 mice, both before and after being gavaged with IgA-High fecal slurry, with and without T. immunophila. Fig. 3G, the source data of DNA gel for detecting T. immunophila (Ti, upper panel) and total bacteria (16S, lower panel) in luminal contents along the gut of IgA-Low mice. Fig. 3H, the source and processed fluorescence in situ hybridization images illustrating the localization of T. immunophila in the gut of IgA-Low mice. Fig. 3I, a spreadsheet the source data of IgA ELISA results of luminal contents along the gut of IgA-Low and IgA-High mice. Fig. 3J, the image for the schematic representation of the Salmonella vaccination/infection experiment (Created using BioRender.com) and the publication license. Fig. 3K, a spreadsheet the source data of IgA ELISA results of 4 groups of wild-type B6 mice including: group #1, mice vaccinated with PBS and gavaged with IgA-High fecal slurry; group #2, mice vaccinated with PBS and gavaged with IgA-High fecal slurry plusT. immunophila; group #3, mice vaccinated with killed Salmonella and gavaged with IgA-High fecal slurry; group #4, mice vaccinated with killed Salmonella and gavaged with IgA-High fecal slurry plus T. immunophila. Fig. 3L, a spreadsheet containing the source data of Salmonella infection experiment for plotting and a Prism GraphPad file for the plots were created. Fig. 3M, a spreadsheet containing the source data of Salmonella CFUs for plotting. This dataset includes Salmonella CFU data (Day 1 to Day 7 post infection) of 4 groups of wild-type B6 mice including: group #1, mice vaccinated with PBS and gavaged with IgA-High fecal slurry; group #2, mice vaccinated with PBS and gavaged with IgA-High fecal slurry plusT. immunophila; group #3, mice vaccinated with killed Salmonella and gavaged with IgA-High fecal slurry; group #4, mice vaccinated with killed Salmonella and gavaged with IgA-High fecal slurry plusT. immunophila. Fig. 4, this folder contains the following subfolders: Fig. 4A, a spreadsheet containing the source data of fecal IgA ELISA. This dataset includes the fecal IgA levels of wild-type B6 mice that were gavaged with PBS or T. immunophila at the indicated times. Fig. 4B-D, the raw data for flow cytometry analysis of IgA binding to T. immunophila (Ti) and various bacterial species including Bacteroides thetaiotaomicron (Bt), Bacteroides fragilis (Bf), Enterococcus faecalis (Ef), Muribaculum intestinale (Mi), 2 Muribaculaceae sp. isolates from IgA-Low mice (Ms1 and Ms2). This folder also includes a spreadsheet, a PowerPoint file, and a GraphPad file pertaining to flow cytometry analysis. Fig. 4E, a spreadsheet containing qPCR data for the analysis of T. immunophila copy numbers in wild-type B6 and Rag1 knockout mice before and after gavage with IgA-Low fecal slurry. Fig. 4F, a spreadsheet containing qPCR data for the analysis of T. immunophila copy numbers in wild-type B6 and Pigr knockout mice before and after gavage with IgA-High fecal slurry plus T. immunophila. Folder labeled “qPCR Raw data for Fig. 4E and F”, all the raw data for qPCR from Bio-Rad CFX Connect Real-Time PCR Detection System. Fig. 5, this folder contains the following subfolders: Fig. 5A, the source data of stain-free SDS-PAGE and the repeat. The data shows the levels of mouse immunoglobulins being incubated with T. immunophila at 37 °C for 6 hours. Fig. 5B, this folder contains files of the amino acid sequences of mouse IgG constant regions, an alignment file analyzed using GeneDoc software, and several PDF files containing raw data from the N-terminal sequencing of the fragments shown in Fig. 5A and Fig. S10 of the manuscript. Fig. 5C, the source data of the immunoblot and the repeat, detecting recombinant mouse antibodies (Including wild-type, K95A/K96A mutant, and K92A/K95A/K96A mutant) after incubation with. immunophila. Fig. 5D, the source data of stain-free SDS-PAGE and the repeats, detecting mouse IgG (full-length, F(ab’)2, and Fc fragment) after incubation with T. immunophila. Fig. 5E, the source data of stain-free SDS-PAGE and the repeats, detecting mouse IgGs subclasses (IgG1, IgG2a, IgG2b, and IgG3) with kappa (κ) or lambda (λ) light chains after incubation with T. immunophila. Fig. 5F, the source data of stain-free SDS-PAGE and the repeat, detecting recombinant mouse IgG1 with κ, λ1, or λ2 light chains after incubation with T. immunophila. Fig. 5G, the source data of stain-free SDS-PAGE and the repeats, detecting recombinant mouse IgM with κ or λ chains after incubation with T. immunophila. Fig. 5H, the source data of the immunoblots and the repeats, detecting various protein levels in mouse serum (IgG, IgA, IgM, κ light chain, λ light chain, and albumin) after incubation with T. immunophila. Fig. 5I, the source data of the immunoblots and the repeat, detecting various protein levels in mouse fecal slurry (IgA, λ light chain, secretory component, trypsin 2, and mucin 2) after incubation with T. immunophila and its OMVs. Fig. 6 The data in this folder labeled “Data in Fig. 6” contains the following subfolders: Fig. 6A, the source data of stain-free SDS-PAGE, detecting IgG/IgY antibodies from various species after incubation with T. immunophila. Fig. 6B, the source data for stain-free SDS-PAGE, detecting hamster IgG1 and IgG2 antibodies harboring either κ or λ1 light chains after incubation with T. immunophila. Fig. 6C, the source data for stain-free SDS-PAGE, detecting all isotypes and subclasses of human antibodies after incubation with T. immunophila. The mouse IgG1 was used as a positive control. Fig. 6D, the source data for the immunoblot includes the use of anti-mouse IgG antibodies to detect recombinant chimeric IgG antibodies. These antibodies comprise mouse heavy chain with mouse (m-κ) or human κ light (h-κ) chains, as well as human heavy chain with human or mouse κ light chains, after incubation with T. immunophila. Fig. 6E, the source data for the immunoblot includes the use of anti-human IgG antibodies to detect recombinant chimeric IgG antibodies. These antibodies comprise mouse heavy chain with mouse (m-κ) or human κ light (h-κ) chains, as well as human heavy chain with human or mouse κ light chains, after incubation with T. immunophila. The folder labeled “Fig. 6 repeat” contains all the repeated data for Fig. 6, encompassing panels A through E. **Raw data for supplemental figures** This zip file contains all the source data for the supplementary figures, and each figure has a separate folder containing the raw data for all panels. Fig. S1, this folder includes the image for Fig. S1A (Workflow for screening IgA-degrading bacteria from IgA-Low mice, created with BioRender.com) and the publication license, the source data for immunoblots of Fig. S1B and Fig. S1C (screening results of IgA-degrading bacteria from IgA-Low mice), the source data for the DNA gel of Fig. 1D (verification of 3 isolates of Sutterella sp.), the source data for the immunoblots of Fig. S1E (3 isolates of Sutterella sp. incubated with IgA), Fig. S1F (3 isolates of Sutterella sp. incubated with a secretory component of Pigr), and Fig. S1G (5 isolates of colony #8 incubated with IgA), and the image of Fig. S1H (Schematic diagram of isolating a pure culture with IgA-degrading activity from colony #8, created with BioRender.com) along with the publication license. Fig. S2, this folder includes the source data for immunoblots of Fig. S2A (Mouse IgA/IgG incubated with OMVs purified from T. immunophila (Ti), Bacteroides thetaiotaomicron (Bt), and Muribaculum intestinale (Mi)) and Fig. 2B (Mouse IgA incubated with intact Ti OMVs, and OMVs treated with sonication or 2% Tween 20). Fig. S3, this folder includes 4 spreadsheets containing the source data of fecal IgA ELISA for plotting Fig. S3A, Fig. S3D, Fig. S3E, and Fig. S3G. It also includes all the source data of DNA gels for detecting Ti, total bacteria (16S) or 5 isolates (S1 through S5) of colony #8 in fecal samples of the mouse groups in Fig. S3A, Fig. S3D, Fig. S3E, and Fig. S3G. All details are included in the spreadsheets and DNA gel images. Fig. S4, this folder contains 2 subfolders containing source and processed images detecting total bacteria and T. immunophila in the cecum and colon of IgA-High mice. Fig. S5, this folder contains a subfolder containing the raw qPCR data from the Bio-Rad qPCR thermocycler, a spreadsheet showing how we analyzed and processed the raw qPCR data for plotting the standard curve in Fig. S5A and the copy numbers of T. immunophila in Fig. 5B, and a raw image of a DNA gel demonstrating the specificity of the qPCR primers for T. immunophila, using fecal samples from various mouse lines as indicated. Fig. S6, this folder contains a spreadsheet with 2 tabs. One tab contains the source data of fecal IgA ELISA results for Fig. S6B, illustrating the fecal IgA levels of wild-type B6 mice before and after being gavaged with IgA-High fecal slurry, with or without T. immunophila. It also includes the fecal IgA levels in these mice after infection with Candida albicans for 21 days. The other tab contains the source data of CFU assays for Candida albicans in fecal samples from wild-type mice infected with Candida albicans. Fecal samples were collected at various indicated timepoints that were included in the spreadsheet. Fig. S7, this folder contains a subfolder with raw images of histology data (Hematoxylin and eosin staining of the mouse gut as indicated) from Fig. S7B, along with two spreadsheets: one for Fig. S7A (body weights of two groups of mice with treatment as indicated) and another for Fig. S7C (crypt numbers in the mouse gut after DSS treatment). Fig. S8, this folder contains a subfolder of the raw data for flow cytometry analysis of IgA binding to T. immunophila (Ti) and various bacterial species including Bacteroides thetaiotaomicron (Bt), Bacteroides fragilis (Bf), Enterococcus faecalis (Ef), Muribaculum intestinale (Mi), 2 Muribaculaceae sp. isolates from IgA-Low mice (Ms1 and Ms2), and 2 spreadsheets for plotting Fig. S8B and Fig. S8C.this folder contains a subfolder of the raw data for flow cytometry analysis of IgA binding to *T. immunophila *(Ti) and various bacterial species including *Bacteroides thetaiotaomicron *(Bt), *Bacteroides fragilis *(Bf),* Enterococcus faecalis *(Ef),* Muribaculum intestinale *(Mi),* *2* Muribaculaceae *sp. isolates from IgA-Low mice (Ms1 and Ms2). This subfolder also includes a spreadsheet, a PowerPoint file, and a GraphPad file pertaining to flow cytometry analysis. Additionally, the folder also includes 2 spreadsheets for plotting Fig. S8B and Fig. S8C. Fig. S9, this folder includes a spreadsheet containing the source data of amino acid levels in the cecum and colon of IgA-Low and IgA-High mice. Fig. S10, this folder includes the source data stain-free SDS-PAGE detecting fusion proteins harboring a fragment of mouse IgG1 constant region (wild-type and K95A/K96A mutant) after being incubated with T. immunophila. This folder also includes two PDF documents for the raw data of the N-terminal sequencing of the fragments indicated in the SDS-PAGE. Fig. S11, this folder includes the source data for two immunoblots detecting IgA after incubation with T. immunophila cultures and OMVs under various culture conditions (Fig. S11A), as well as after incubation with T. immunophila alone, T. immunophila plus microbes from IgA-High fecal slurry, or microbes from IgA-High fecal slurry only (Fig. S11B). Fig. S12, this folder includes the source data for immunoblots detecting protein levels (IgA, secretory component, λ-light chain, and J chain) in purified SIgA after incubation with T. immunophila cultures (Fig. S12A) and OMVs (Fig. S12B) under various culture conditions. This folder also includes the source data for immunoblots detecting IgA, secretory component, and λ-light chain in mouse feces after gavaged with T. immunophila OMVs. An SDS-PAGE image is also included to show carmine levels in feces before and after gavage of OMVs into mice. Fig. S13, this folder includes the source data of stain-free SDS-PAGE for Fig. S13A (IgG1, IgG2a and IgG2b with κ or λ light chains incubated with T. immunophila in the presence or absence of TCEP) Fig. S13B (recombinant IgG1 harboring κ, λ1 or λ2 light chains incubated with T. immunophila in the presence or absence of TCEP). Fig. S14, this folder includes the source data of stain-free SDS-PAGE for Fig. S14A (mouse IgG1 incubated with T. immunophila in the presence of various protease inhibitors as indicated) and Fig. S14C (recombinant fusion proteins harboring indicated mouse IgG constant region fragments incubated with T. immunophila in the presence of NEM, EDTA, or EGTA). This folder also includes the source data of immunoblots for detecting mouse IgA or mouse IgG after incubation with T. immunophila in the presence of NEM, EDTA or EGTA (Fig. 14B) Fig. S15, this folder contains the source data of SDS-PAGE and immunoblots detecting mouse IgG (Fig. S15, A and B) and IgA (Fig. S15, C and D) after incubation with T. immunophila at 37°C for the specified times, as indicated. This folder also contains the source data of SDS-PAGE (Fig. S15, upper panel) and immunoblots (lower panel) detecting mouse IgG and IgA after incubated with T. immunophila at different temperatures and durations. Fig. S16, this folder contains the source data of immunoblots for all isotypes and subclasses of mouse antibodies, following incubation with T. immunophila and OMVs purified from T. immunophila cultures under three different conditions. Fig. S17, this folder includes the source data of a stain-free SDS-PAGE showing mouse IgG1 and IgA incubated with T. immunophila culture and 2 OMV preparations (Fig. S17A), the source data of the immunoblot (Fig. S17B) for samples in Fig. S17A, the source data of an SDS-PAGE stained with PAGE-Blue detecting T. immunophila OMV proteins (Fig. S17C), the source data of the immunoblot (Fig. S17D) detecting mouse IgA after incubation with T. immunophila (Ti) or Muribaculum intestinale (Mi). Fig. S18, this folder includes the source data of the immunoblots detecting the expression of mCherry- or GST-fusion proteins 23 candidate proteases from T. immunophila in HEK 293T (Fig. S18A) and E. coli (Fig. S18B) cells. This folder also includes the source data of the immunoblots detecting mouse IgA (Fig. S18, C, D, and G) or IgG1 (Fig. S18, E, F, and H) incubated with HEK 293T or E. coli cell lysates with overexpressed candidate proteases from T. immunophila. Fig. S19, this folder contains the source data for two stain-free SDS-PAGE. One is for detecting rat IgG subclasses after incubation with T. immunophila (Fig. S19A), and the other is for detecting IgA from C57BL/6 and BALB/c mice, as well as rat IgA, after incubation with T. immunophila (Fig. S19B). Fig. S20, this folder includes a file containing sequences of mouse and human IgG heavy chain constant regions and the alignment result analyzed with the software GeneDoc (Fig. S20A). This folder also includes the source data of the SDS-PAGE (Fig. S20B) and immunoblot (Fig. S20C) for detecting human IgG1, IgG2, IgG3, and IgG4 after incubation with T. immunophila with or without TCEP. The repeats for Fig. S20B and Fig. S20C are also included. Fig. S21, this folder includes all the subjects for the figures in Fig. S21 used to illustrate the proposed mechanism of proteolytic cleavage of mouse immunoglobulins by T. immunophila. All the figures in Fig. S21 were created using BioRender.com, and the publication licenses are also included.