Bacterial and host enzymes modulate the inflammatory response produced by the peptidoglycan of the Lyme disease agent
Authors: McCausland Joshua W. , Kloos Zachary A. , Irnov Irnov , Sonnert Nicole D. , Zhou Junhui , Putnick Rachel , Mueller Elizabeth A. , Steere Alan C. , Palm Noah W. , Grimes Catherine L. , Jacobs-Wagner Christine 
Published: January 2025

The spirocheteBorrelia burgdorfericauses Lyme disease. In some patients, an excessive, dysregulated proinflammatory immune response can develop in joints leading to persistent arthritis. In such patients, persistence of antigenicB. burgdorferipeptidoglycan (PGBb) fragments within joint tissues may contribute to the immunopatho-genesis, even after appropriate antibiotic treatment. In liveB. burgdorfericells, the outer membrane shields the polymeric PGBbsacculus from exposure to the immune system. However, unlike most diderm bacteria,B. burgdorferireleases PGBbturnover products into its environment due to the absence of recycling activity. In this study, we identified the released PGBbfragments using a mass spectrometry-based approach. By characterizing the L,D-carboxypeptidase activity ofB. burgdorferiprotein BB0605 (renamed DacA), we found that PGBbturnover largely occurs at sites of PGBbsynthesis. In parallel, we demonstrated that the lytic transglycosylase activity associated with BB0259 (renamed MltS) releases PGBbfragments with 1,6-anhydro bond on theirN-acetylmuramyl residues. Stimulation of human cell lines with various synthetic PGBbfragments revealed that 1,6-anhydromuramyl-containing PGBbfragments are poor inducers of a NOD2-dependent immune response relative to their hydrated counterparts. We also showed that the activity of the humanN-acetylmuramyl-L-alanine amidase PGLYRP2, which reduces the immunogenicity of PGBbmaterial, is low in joint (synovial) fluids relative to serum. Altogether, our findings suggest that MltS activity helpsB. burgdorferievade PG-based immune detection by NOD2 during growth despite shedding PGBbfragments and that PGBb-induced immunopathology likely results from host sensing of PGBbmaterial from dead (lysed) spirochetes. Additionally, our results suggest the possibility that natural variation in PGLYRP2 activity may contribute to differences in susceptibility to PG-induced inflammation across tissues and individuals.Author summaryDuring bacterial infection, the presence of peptidoglycan—a polymeric element of bacterial cell walls—triggers a host inflammatory response. Although generally protective during acute phases, inflammation, when chronic, can contribute to disease development. Recent work has suggested that the persistence of pro-inflammatory peptidoglycan derived from the Lyme disease spirocheteBorrelia burgdorferiin joints may contribute to persistent arthritis in some patients despite appropriate antibiotic therapy. Interestingly,B. burgdorferisheds peptidoglycan turnover products into the environment during growth. Here, we show that these shed products from live spirochetes are poor effectors of an immune response by the human NOD2 immune receptor due to the formation of an anhydro bond on theN-acetyl-muramic residue during peptidoglycan hydrolysis by aB. burgdorferilytic transglycosylase. We also show that humanN-acetylmuramyl-L-alanine amidase activity, which abrogates the NOD2-dependent response to the immunogenic peptidoglycan isolated from lysedB. burgdorferi, is low in human joint fluids relative to serum. Based on our findings, we propose that immunopathogenesis by peptidoglycan material more likely derives from lysed spirochetes (killed by an immune attack or antibiotics) than live ones and that the level of human peptido-glycan hydrolytic enzymes across tissues and individuals influences susceptibility to chronic inflammation.