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The interplay between the human innate immune system and bacterial cell wall components is pivotal in understanding diseases such as Crohn’s disease and Lyme arthritis. Lyme disease, caused by Borrelia burgdorferi, is the most prevalent tick-borne illness in the United States, with a substantial number of cases reported annually. While antibiotic treatments are generally effective, approximately 10% of Lyme disease cases develop persistent arthritis, suggesting a dysregulated host immune response. We have previously identified a link between the immunogenic B. burgdorferi peptidoglycan (PG) and Lyme arthritis and showed that this pathogen sheds significant amounts of PG fragments during growth. Here, we synthesize these PG fragments, including ornithine-containing monosaccharides and disaccharides, to mimic the unique composition of Borrelia cell walls, using reproducible and rigorous synthetic methods. This synthetic approach allows for the modular preparation of PG derivatives, providing a diverse library of well-defined fragments. These fragments will serve as valuable tools for investigating the role of PG-mediated innate immune response in Lyme disease and aid in the development of improved diagnostic methods and treatment strategies.

Peptidoglycan recognition proteins (PGLYRPs) are implicated in the control of the intestinal microbiota; however, molecular requirements for peptidoglycan (PGN) binding and receptor signaling mechanisms remain poorly understood. Here we show that PGLYRP1 is a receptor for the disaccharide motif of lysine N-acetylglucosamine N-acetylmuramic tripeptide (GMTriP-K). PGLYRP1 is required for innate immune activation by GMTriP-K but not muramyl dipeptide (MDP). In macrophages, intracellular PGLYRP1 complexes with NOD2 and GEF-H1, both of which are required for GMTriP-K-regulated gene expression. PGLYRP1 localizes to the endoplasmic reticulum and interacts at the Golgi with NOD2 upon GMTriP-K stimulation. PGLYRP1 and dependent gene expression signatures are induced in both mouse intestinal inflammation and human ulcerative colitis. Importantly, PGLYRP1 activation by GMTriP-K can result in the protection of mice from TNBS-induced colitis. Mammalian PGLYRPs can function as intracellular pattern recognition receptors for the control of host defense responses in the intestine. Peptidoglycan recognition proteins (PGLYRPs) are implicated in the control of the intestinal microbiota. Here, combining in vitro and in vivo work, the authors show that PGLYRP-1 act as an intracellular pattern recognition receptor for the detection of peptidoglycan disaccharides that regulate host defense responses in the intestine.

O-GlcNAcylation is a dynamic and functionally diverse post-translational modification shown to affect thousands of proteins, including the innate immune receptor nucleotide-binding oligomerization domain-containing protein 2 (Nod2). Mutations of Nod2 (R702W, G908R and 1007 fs) are associated with Crohn’s disease and have lower stabilities compared to wild type. Cycloheximide (CHX)-chase half-life assays have been used to show that O-GlcNAcylation increases the stability and response of both wild type and Crohn’s variant Nod2, R702W. A more rapid method to assess stability afforded by post-translational modifications is necessary to fully comprehend the correlation between NLR stability and O-GlcNAcylation. Here, a recently developed cellular thermal shift assay (CETSA) that is typically used to demonstrate protein-ligand binding was adapted to detect shifts in protein stabilization upon increasing O-GlcNAcylation levels in Nod2. This assay was used as a method to predict if other Crohn’s associated Nod2 variants were O-GlcNAcylated, and also identified the modification on another NLR, Nod1. Classical immunoprecipitations and NF-κB transcriptional assays were used to confirm the presence and effect of this modification on these proteins. The results presented here demonstrate that CETSA is a convenient method that can be used to detect the stability effect of O-GlcNAcylation on O-GlcNAc-transferase (OGT) client proteins and will be a powerful tool in studying post-translational modification.

biosynthesis of cell wall peptidoglycan is essential for bacterial viability under many growth conditions and is a well-validated antibiotic target. Although generally not essential for bacterial fitness under standard laboratory growth conditions, peptidoglycan recycling can aid bacterial survival under host or antibiotic stress. Peptidoglycan consists of alternating sugars -acetylmuramic acid (Mur Ac) and -acetylglucosamine (Glc Ac) cross-linked by peptides. Recycling of these sugars can proceed via Glc Ac and glucosamine intermediates ( -type) or, in the case of Mur Ac, bypass these intermediates altogether ( -type). We serendipitously discovered that the pathogen and model organism assimilate 2-modified Mur Ac probes into their peptidoglycan despite lacking the -type machinery that is normally required for incorporation of these molecules. Our data suggest that unmodified and 2-modified Mur Ac incorporate into peptidoglycan via multiple pathways, the former preferentially via an -type route and the latter preferentially via a non- , non- -type route with Glc Ac but not glucosamine intermediates. These findings reveal metabolic flexibility in mycobacterial cell wall recycling that encompasses a previously undescribed pathway.

Bacterial cells are surrounded by a dynamic cell wall which in part is made up of a mesh-like peptidoglycan (PG) layer that provides the cell with structural integrity and resilience. In Gram-positive bacteria, this layer is thick and robust, whereas in Gram-negative bacteria, it is thinner and flexible as the cell is supported by an additional outer membrane. PG undergoes continuous turnover, with degradation products being recycled to maintain cell wall homeostasis. Some Gram-negative species can bypass PG biosynthesis, relying instead on PG recycling to sustain growth and division. (hereafter ), the causative agent of Legionnaires' disease, encodes such recycling machinery within its genome. This study investigates the biochemical, genetic, and pathogenic roles of PG recycling in . Previously, we have shown that PG can be visualized in both model and native systems using a combination of -acetylmuramic acid (NAM) probes and PG recycling programs. Here, two PG recycling gene homologs in the genome ( ) and ( were identified and characterized; chemical biology strategies were used to rigorously track the incorporation of \"click\"-PG-probes. Deletion of abolished PG labeling, while genetic complementation restored labeling. Additionally, copper-free click chemistry with ultra-fast tetrazine-NAM probes enabled live-cell PG labeling. The data suggest that contributes to the pathogenicity of the organism, as deletion increased 's susceptibility to antibiotics and significantly reduced s ability to replicate in host alveolar macrophages. An intracellular replication assay demonstrated that while PG recycling is not essential for internalization, successful replication of within MH-S murine alveolar macrophages requires functional . These findings underscore the essential role of AmgK in 's intracellular survival, emphasizing the importance of PG recycling in pathogenicity, and establish a foundation for developing novel -specific antibiotic strategies.

Bordetella pertussis releases the monomeric peptidoglycan (PGN) fragment tracheal cytotoxin (TCT) due to inefficient recycling by the permease AmpG. Releasing this PGN is metabolically costly and potentially immune alarming and the benefits to B. pertussis are unclear. While TCT has been characterized as a potent NOD1 agonist capable of causing the extrusion of ciliated cells, in vitro, the consequences of its release have yet to be studied in vivo. Here we show that selective PGN release by B. pertussis biases host PGN sensing toward NOD1 and away from NOD2, suppressing IL-1β-driven inflammation and blunting adaptive immune recruitment. Mice infected with a TCT over-releasing strain (TCT(+)) exhibit reduced pulmonary immunopathology relative to wild type (WT) and a TCT-under-releasing strain (TCT(-)), despite similar bacterial burdens. NOD reporter assays demonstrate that TCT release enhances NOD1 activation and inversely correlates with NOD2 activation. Bulk transcriptomic analysis of infected lungs shows that B. pertussis PGN release dampens pro-inflammatory transcriptional programs. Single-cell transcriptomic determined Nod2 expression is limited to inflammatory myeloid subsets. IL-1 family genes were highly enriched in Nod2- but not Nod1 expressing alveolar macrophages. Upstream regulator analysis predicted IL-1β as a major driver of B. pertussis inflammation, which was enhanced by the absence of PGN release. Flow cytometry shows that PGN release skews macrophages polarization toward M2 and away from M1 in a NOD1 dependent manner. Finally, extracellular release of PGN and subsequent reduced IL-1 production facilitated the suppression fibroblast chemokine programs (e.g., CXCL13, CCL19), diminished recruitment of B and T cells, reduced iBALT formation, and limited immune memory development. Conversely, IL-1R1 deficiency impairs adaptive recruitment and bacterial clearance despite similar innate infiltration. Together, these data suggest PGN release by B. pertussis is an immune-evasion strategy, favoring NOD1 activation over NOD2, reducing IL-1–dependent fibroblast reprogramming, and curtailing chemokine-driven adaptive responses. Graphic Abstract B. pertussis can produce both NOD1 and NOD2 activating PGNs. Release of TCT promotes NOD1 activation and diminishes NOD2 activation. NOD2 activation in myeloid cells drives M1 polarization of macrophages and IL-1 family cytokine production. IL-1 family cytokines skew fibroblasts towards an inflammatory phenotype, leading to chemokine release, extracellular remodeling, and recruitment of lymphocytes. Therefore, TCT release tempers long-term immunity to B. pertussis.

Cytosolic innate immune sensing is critical for protecting barrier tissues. NOD1 and NOD2 are cytosolic sensors of small peptidoglycan fragments (muropeptides) derived from the bacterial cell wall. These muropeptides enter cells, especially epithelial cells, through unclear mechanisms. We previously implicated SLC46 transporters in muropeptide transport in Drosophila immunity. Here we focus on Slc46a2, which is highly expressed in mammalian epidermal keratinocytes, and show that it is critical for delivery of DAP-muropeptides and activation of NOD1 in keratinocytes, while the related transporter Slc46a3 is critical for responding to MDP, the NOD2 ligand. In a mouse model, Slc46a2 and Nod1 deficiency strongly suppressed psoriatic inflammation, while methotrexate, a commonly used psoriasis therapeutic, inhibited Slc46a2-dependent transport of DAP-muropeptides. Collectively these studies define SLC46A2 as a transporter of NOD1 activating muropeptides, with critical roles in the skin barrier, and identify this transporter as an important target for anti-inflammatory intervention. Competing Interest Statement A provisional patent on targeting SLC46s to inhibit inflammation in psoriasis and other auto-inflammatory diseases as been filed by some of the authors.