Pathogenic microbes have fine-tuned the structure and availability of their pathogen associated molecular patterns (PAMPs) to facilitate their life cycles. Interestingly, certain phylogenetically distant bacteria have independently evolved to release extracellular peptidoglycan (PGN) fragments, allowing recognition by the host immune system. Neisseria gonorrhoeae and Bordetella pertussis release specific muropeptides, despite the severe metabolic and immune alarming consequences. Both N. gonorrhoeae, a human-exclusive pathogen which colonizes the mucosal lining of the urogenital tract and B. pertussis, a human exclusive pathogen which colonizes the mucociliary cells of the respiratory tract, release a GlcNAc(beta1-4)-MurNAc(1,6-anhydro) DAP containing tetrapeptide PGN fragment, termed tracheal cytotoxin (TCT). TCT release is regulated by periplasmic membrane permease, AmpG, which mediates PGN recycling. Inefficient PGN recycling by AmpG in B. pertussis results in TCT release. Previous groups have investigated the deleterious effects of TCT in cell culture systems, but a gap remains in understanding the role of TCT in vivo.
Here, we demonstrate that excess TCT release during infection reduces pulmonary inflammation. Mice infected with a TCT over-releasing strain (TCT(+)) exhibited decreased lung immunopathology compared to wild-type or TCT-under releasing (TCT(-)) strains. Reporter assays revealed that TCT(+) enhanced NOD1 activation, while TCT(-) strains preferentially engaged NOD2. Profiling of PGN fragments preferentially released by the TCT(+) mutants revealed an enrichment availability of anhydrous muropeptides, while strains of B. pertussis which released less TCT, exhibited greater availability of MurNAc-Tripeptide containing muropeptides, predicted to engage NOD2 activation. In vivo, NOD2 knockout mice displayed attenuated lung inflammation, implicating NOD2 in pulmonary pathology. Transcriptomic analysis identified IL-1Β as being associated with expression of NOD2, but not NOD1. Consistently, increased TCT release inhibited IL1B expression. Single-cell RNA-seq analysis revealed that alveolar macrophages and neutrophils were the primary source of IL-1Β, while airway fibroblasts were the key responders to the cytokine. Upon infection, airway fibroblasts upregulated B and T cell attractant chemokines. Increased TCT release reduced recruitment of B cells and long-term TCT(+) infected mice showed decreased tertiary lymphoid structures compared to WT and TCT- infected mice, demonstrating a role for TCT in delaying the formation of the adaptive response. Infected IL1R1 KO mice demonstrated impaired B and CD4+ T cell recruitment and higher bacterial burdens, despite unchanged innate cell infiltration, underscoring the role of IL-1 in coordinating adaptive immunity. Together, these findings support a model in which TCT release biases the availability of NOD1 vs NOD2 stimulatory muropeptides to limit NOD2-mediated IL-1 production, blunting fibroblast activation and reducing chemokine-driven lymphocyte recruitment to impair the adaptive immune clearance of the pathogen.