A host-adapted auxotrophic gut symbiont induces mucosal immunodeficiency (Part I)

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. 

Methods
To screen for IgA-degrading bacteria from SIgA-Low mice (15), 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 Muribaculaceae family. Protologger (v1.3) was used to describe this novel taxon (51), which was proposed to be named Tomasiella immunophila. 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 T. immunophila to test its in vivo IgA-degrading activity. The biological implications of T. immunophila-mediated degradation of SIgA in mice were assessed using the Salmonella Typhimurium vaccination/infection model (23), Candida albicans infection (24, 25), and the dextran sulfate sodium (DSS)-induced colitis model (15). Wild-type B6 mice were orally administered T. immunophila to evaluate its capability to induce IgA production. Rag1-/- (no T/B cells) and Pigr-/- (no luminal IgA/IgM) mice were colonized with T. immunophila to investigate the dependency of T. immunophila colonization on IgA. All mouse antibody isotypes and subclasses were incubated with T. immunophila 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 T. immunophila 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 T. immunophila-mediated antibody cleavage. Additionally, antibodies from various species, including humans, were incubated with T. immunophila. 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.