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Microbial pathogens have evolved mechanisms to overcome immune responses and successfully infect their host. Here, we studied how Listeria monocytogenes evades immune detection by peptidoglycan (PGN) modification. By analyzing L. monocytogenes muropeptides, we detected O-acetylated muramic acid residues. We identified an O-acetyltransferase gene, oatA, in the L. monocytogenes genome sequence. Comparison of PGN from parental and isogenic oatA mutant strains showed that the O-acetyltransferase OatA O-acetylates Listeria PGN. We also found that PGN O-acetylation confers resistance to different types of antimicrobial compounds targeting bacterial cell wall such as lysozyme, β-lactam antibiotics, and bacteriocins and that O-acetylation is required for Listeria growth in macrophages. Moreover, oatA mutant virulence is drastically affected in mice following intravenous or oral inoculation. In addition, the oatA mutant induced early secretion of proinflammatory cytokines and chemokines in vivo. These results suggest an important role for OatA in limiting innate immune responses and promoting bacterial survival in the infected host.

The innate immune system recognizes microbial products using germline-encoded receptors that initiate inflammatory responses to infection. The bacterial cell wall component peptidoglycan is a prime example of a conserved pathogen-associated molecular pattern (PAMP) for which the innate immune system has evolved sensing mechanisms. Peptidoglycan is a direct target for innate immune receptors and also regulates the accessibility of other PAMPs to additional innate immune receptors. Subtle structural modifications to peptidoglycan can influence the ability of the innate immune system to detect bacteria and can allow bacteria to evade or alter host defences. This Review focuses on the mechanisms of peptidoglycan recognition that are used by mammalian cells and discusses new insights into the role of peptidoglycan recognition in inflammation, metabolism, immune homeostasis and disease.

Lyme disease is a multisystem disorder caused by the spirochete Borrelia burgdorferi. A common late-stage complication of this disease is oligoarticular arthritis, often involving the knee. In ∼10% of cases, arthritis persists after appropriate antibiotic treatment, leading to a proliferative synovitis typical of chronic inflammatory arthritides. Here, we provide evidence that peptidoglycan (PG), a major component of the B. burgdorferi cell envelope, may contribute to the development and persistence of Lyme arthritis (LA). We show that B. burgdorferi has a chemically atypical PG (PGBb) that is not recycled during cell-wall turnover. Instead, this pathogen sheds PGBb fragments into its environment during growth. Patients with LA mount a specific immunoglobulin G response against PGBb, which is significantly higher in the synovial fluid than in the serum of the same patient. We also detect PGBb in 94% of synovial fluid samples (32 of 34) from patients with LA, many of whom had undergone oral and intravenous antibiotic treatment. These same synovial fluid samples contain proinflammatory cytokines, similar to those produced by human peripheral blood mononuclear cells stimulated with PGBb. In addition, systemic administration of PGBb in BALB/c mice elicits acute arthritis. Altogether, our study identifies PGBb as a likely contributor to inflammatory responses in LA. Persistence of this antigen in the joint may contribute to synovitis after antibiotics eradicate the pathogen. Furthermore, our finding that B. burgdorferi sheds immunogenic PGBb fragments during growth suggests a potential role for PGBb in the immunopathogenesis of other Lyme disease manifestations.

Secretion of extracellular vesicles (EVs), a process common to eukaryotes, archae, and bacteria, represents a secretory pathway that allows cell-free intercellular communication. Microbial EVs package diverse proteins and influence the host-pathogen interaction, but the mechanisms underlying EV production in Gram-positive bacteria are poorly understood. Here we show that EVs purified from community-associated methicillin-resistant Staphylococcus aureus package cytosolic, surface, and secreted proteins, including cytolysins. Staphylococcal alpha-type phenol-soluble modulins promote EV biogenesis by disrupting the cytoplasmic membrane; whereas, peptidoglycan cross-linking and autolysin activity modulate EV production by altering the permeability of the cell wall. We demonstrate that EVs purified from a S. aureus mutant that is genetically engineered to express detoxified cytolysins are immunogenic in mice, elicit cytolysin-neutralizing antibodies, and protect the animals in a lethal sepsis model. Our study reveals mechanisms underlying S. aureus EV production and highlights the usefulness of EVs as a S. aureus vaccine platform. Extracellular vesicles (EVs) influence host-pathogen interactions, but EV biogenesis in gram-positive bacteria remains elusive. Here authors characterize EVs from Staphylococcus aureus and show that phenol-soluble modulins and autolysins promote EV biogenesis by disrupting the membrane and cell wall.

Key Points All multicellular eukaryotes live in symbiotic associations with microorganisms, and the immune system accommodates host colonization by symbiotic microorganisms, maintains microbiota–host homeostasis and defends against pathogens. One family of antibacterial pattern recognition molecules — the peptidoglycan recognition proteins (PGRPs) — has evolved a variety of mechanisms to control host interactions with mutualistic, commensal and parasitic microorganisms to benefit both invertebrate and vertebrate hosts. PGRPs are antibacterial proteins of the innate immune system that are conserved from insects to mammals. In invertebrates, PGRPs function as soluble or cell-surface pattern recognition receptors and hydrolyse peptidoglycan, whereas in vertebrates they also directly kill bacteria. In Drosophila melanogaster , PGRPs are upstream pattern recognition molecules that activate the IMD and Toll pathways and induce the production of antimicrobial peptides, which control intestinal bacteria and defend against infections. PGRPs also control the level of pro-inflammatory peptidoglycan through their amidase activity. In mosquitoes, PGRPs not only defend the insect against bacterial infections, but also regulate symbiotic bacteria, as well as the host response to malaria parasites. In tsetse flies, PGRPs control endosymbiotic bacteria and trypanosome parasites. In squid, PGRPs control the winnowing and establishment of symbiotic luminescent bacteria in the squid light organ. In zebrafish, PGRPs protect the embryos from infections and enable their survival. In mammals, PGRPs control the acquisition and maintenance of normal gut microorganisms, which protect the host from enhanced inflammation, tissue damage and colitis. This Review discusses how invertebrate and vertebrate members of the PGRP family have developed an amazing variety of mechanisms to coordinate the host response to mutualistic, commensal and parasitic microorganisms. All animals, including humans, live in symbiotic association with microorganisms. The immune system accommodates host colonization by the microbiota, maintains microbiota–host homeostasis and defends against pathogens. This Review analyses how one family of antibacterial pattern recognition molecules — the peptidoglycan recognition proteins — has evolved a fascinating variety of mechanisms to control host interactions with mutualistic, commensal and parasitic microorganisms to benefit both invertebrate and vertebrate hosts.

Recent studies have revealed that the gut microbiota modulates brain development and behavior, but the underlying mechanisms are still poorly understood. Here, we show that bacterial peptidoglycan (PGN) derived from the commensal gut microbiota can be translocated into the brain and sensed by specific pattern-recognition receptors (PRRs) of the innate immune system. Using expression-profiling techniques, we demonstrate that two families of PRRs that specifically detect PGN (that is, PGN-recognition proteins and NOD-like receptors), and the PGN transporter PepT1 are highly expressed in the developing brain during specific windows of postnatal development in both males and females. Moreover, we show that the expression of several PGN-sensing molecules and PepT1 in the developing striatum is sensitive to manipulations of the gut microbiota (that is, germ-free conditions and antibiotic treatment). Finally, we used the PGN-recognition protein 2 (Pglyrp2) knockout mice to examine the potential influence of PGN-sensing molecules on brain development and behavior. We demonstrate that the absence of Pglyrp2 leads to alterations in the expression of the autism risk gene c-Met , and sex-dependent changes in social behavior, similar to mice with manipulated microbiota. These findings suggest that the central activation of PRRs by microbial products could be one of the signaling pathways mediating the communication between the gut microbiota and the developing brain.

Key Points NOD1 (nucleotide oligomerization domain-containing protein 1) and NOD2 are members of the NOD-like receptor family of proteins, which function to detect peptidoglycan and to stimulate host responses to limit bacterial infection. The link between NOD2 and the inflammatory bowel disease Crohn's disease highlights the importance of maintaining balanced innate immune responses through NOD signalling in response to the host microbiota at the intestinal mucosa. NOD proteins react to peptidoglycan fragments that enter into the host cytosol by a variety of mechanisms, including direct infection by cyto-invasive pathogens, delivery through bacterial outer membrane vesicles or type IV secretion systems, and through membrane oligopeptide transporters, including solute carrier family 15 member 4 (SLC15A4) and pH-sensing regulatory factor of peptide transporter 1 (PEPT1). Fragments of peptidoglycan can bind to NOD1 and NOD2, inducing their self-association through their interaction at the nucleotide-binding domain (NBD). Oligomerization leads to the recruitment of receptor-interacting protein 2 (RIP2), which regulates the activation of the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. NOD signalling results in several downstream effects, including cytokine production, recruitment of neutrophils and inflammatory macrophages, and initiation of type 2 immunity. Mice that are deficient in the NOD signalling pathway have altered abilities to fight off bacterial infection. Interestingly, several pathogens have developed mechanisms to evade NOD-mediated immunity, including mechanisms that modify peptidoglycan. Autophagy is also affected by NOD signalling. NOD proteins mediate the detection of bacteria, such as Shigella flexneri , in the cytosol of infected cells and, through their interaction with a key autophagy protein called ATG16L1 (autophagy-related protein 16-like 1), can bring the autophagy machinery to the site where the bacteria reside in the cytosol. The induction of autophagy wraps cytosolic bacteria in autophagosomes that subsequently fuse with lysosomes to degrade the bacteria. The interaction of ATG16L1 with NOD1 and NOD2 also regulates the ability of the NODs to drive inflammatory signalling. Indeed, ATG16L1 is a negative regulator of NOD signalling and reduces cytokine production in a manner that is independent of autophagy. Most studies using mouse colitis models have shown that NODs have a protective role in intestinal inflammation. NODs maintain intestinal homeostasis by a variety of mechanisms including fortification of the intestinal barrier and regulation of early inflammatory pathways, such as those governed by interleukin-17 (IL-17), to limit infection and to promote mucosal healing. NOD signalling is also thought to influence the gut microbiota, although there is still controversy as to whether NOD deficiency itself or the underlying inflammation mediates changes in the gut microbial communities. NOD proteins also affect the development of extra-intestinal diseases and cancer. Polymorphisms in the genes that encode NOD1 and NOD2 have been linked to asthma and atopy, graft-versus-host disease, the auto-inflammatory disease Blau syndrome, and cancer. In the case of cancer, NOD deficiency promotes carcinogenesis by providing an inflammatory microenvironment that is exacerbated by chemicals, such as dextran sodium sulphate (DSS), that induce epithelial injury. Nucleotide oligomerization domain-containing protein 1 (NOD1) and NOD2 are pattern-recognition receptors that detect bacterial peptidoglycan. Signalling through NODs initiates a variety of effector immune responses that seem to be crucial for maintaining immune homeostasis with the host microbiota. Indeed, mutations in NOD1 and NOD2 are associated with both intestinal and extra-intestinal disease. This Review summarizes our current understanding of the NODs. Entry of bacteria into host cells is an important virulence mechanism. Through peptidoglycan recognition, the nucleotide-binding oligomerization domain (NOD) proteins NOD1 and NOD2 enable detection of intracellular bacteria and promote their clearance through initiation of a pro-inflammatory transcriptional programme and other host defence pathways, including autophagy. Recent findings have expanded the scope of the cellular compartments monitored by NOD1 and NOD2 and have elucidated the signalling pathways that are triggered downstream of NOD activation. In vivo , NOD1 and NOD2 have complex roles, both during bacterial infection and at homeostasis. The association of alleles that encode constitutively active or constitutively inactive forms of NOD2 with different diseases highlights this complexity and indicates that a balanced level of NOD signalling is crucial for the maintenance of immune homeostasis.

Plant innate immunity relies on successful detection of microbe-associated molecular patterns (MAMPs) of invading microbes via pattern recognition receptors (PRRs) at the plant cell surface. Here, we report two homologous rice (Oryza sativa) lysin motif-containing proteins, LYP4 and LYP6, as dual functional PRRs sensing bacterial peptidoglycan (PGN) and fungal chitin. Live cell imaging and microsomal fractionation consistently revealed the plasma membrane localization of these proteins in rice cells. Transcription of these two genes could be induced rapidly upon exposure to bacterial pathogens or diverse MAMPs. Both proteins selectively bound PGN and chitin but not lipopolysaccharide (LPS) in vitro. Accordingly, silencing of either LYP specifically impaired PGN-or chitin-but not LPS-induced defense responses in rice, including reactive oxygen species generation, defense gene activation, and callóse deposition, leading to compromised resistance against bacterial pathogen Xanthomonas oryzae and fungal pathogen Magnaporthe oryzae. Interestingly, pretreatment with excess PGN dramatically attenuated the alkalinization response of rice cells to chitin but not to flagellin; vice versa, pretreatment with chitin attenuated the response to PGN, suggesting that PGN and chitin engage overlapping perception components in rice. Collectively, our data support the notion that LYP4 and LYP6 are promiscuous PRRs for PGN and chitin in rice innate immunity.

Recognition of microbial patterns by host pattern recognition receptors is a key step in immune activation in multicellular eukaryotes. Peptidoglycans (PGNs) are major components of bacterial cell walls that possess immunity-stimulating activities in metazoans and plants. Here we show that PGN sensing and immunity to bacterial infection in Arabidopsis thaliana requires three lysin-motif (LysM) domain proteins. LYM1 and LYM3 are plasma membrane proteins that physically interact with PGNs and mediate Arabidopsis sensitivity to structurally different PGNs from Gram-negative and Gram-positive bacteria. lym1 and lym3 mutants lack PGN-induced changes in transcriptome activity patterns, but respond to fungus-derived chitin, a pattern structurally related to PGNs, in a wild-type manner. Notably, lym1, lym3, and lym3 lym1 mutant genotypes exhibit supersusceptibility to infection with virulent Pseudomonas syringae pathovar tomato DC3000. Defects in basal immunity in lym3 lym1 double mutants resemble those observed in lym1 and lym3 single mutants, suggesting that both proteins are part of the same recognition system. We further show that deletion of CERK1, a LysM receptor kinase that had previously been implicated in chitin perception and immunity to fungal infection in Arabidopsis, phenocopies defects observed in lym1 and lym3 mutants, such as peptidoglycan insensitivity and enhanced susceptibility to bacterial infection. Altogether, our findings suggest that plants share with metazoans the ability to recognize bacterial PGNs. However, as Arabidopsis LysM domain proteins LYM1, LYM3, and CERK1 form a PGN recognition system that is unrelated to metazoan PGN receptors, we propose that lineage-specific PGN perception systems have arisen through convergent evolution.

Listeria monocytogenes is a human intracellular pathogen that is able to survive in the gastrointestinal environment and replicate in macrophages, thus bypassing the early innate immune defenses. Peptidoglycan (PG) is an essential component of the bacterial cell wall readily exposed to the host and, thus, an important target for the innate immune system. Characterization of the PG from L. monocytogenes demonstrated deacetylation of N-acetylglucosamine residues. We identified a PG N-deacetylase gene, pgdA, in L. monocytogenes genome sequence. Inactivation of pgdA revealed the key role of this PG modification in bacterial virulence because the mutant was extremely sensitive to the bacteriolytic activity of lysozyme, and growth was severely impaired after oral and i.v. inoculations. Within macrophage vacuoles, the mutant was rapidly destroyed and induced a massive IFN-β response in a TLR2 and Nod1-dependent manner. Together, these results reveal that PG N-deacetylation is a highly efficient mechanism used by Listeria to evade innate host defenses. The presence of deacetylase genes in other pathogenic bacteria indicates that PG N-deacetylation could be a general mechanism used by bacteria to evade the host innate immune system.