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Peptidoglycan (PG) is an essential component in the cell wall of nearly all bacteria, forming a continuous, mesh-like structure, called the sacculus, around the cytoplasmic membrane to protect the cell from bursting by its turgor. Although PG synthases, the penicillin-binding proteins (PBPs), have been studied for 70 years, useful in vitro assays for measuring their activities were established only recently, and these provided the first insights into the regulation of these enzymes. Here, we review the current knowledge on the glycosyltransferase and transpeptidase activities of PG synthases. We provide new data showing that the bifunctional PBP1A and PBP1B from Escherichia coli are active upon reconstitution into the membrane environment of proteoliposomes, and that these enzymes also exhibit DD-carboxypeptidase activity in certain conditions. Both novel features are relevant for their functioning within the cell. We also review recent data on the impact of protein–protein interactions and other factors on the activities of PBPs. As an example, we demonstrate a synergistic effect of multiple protein–protein interactions on the glycosyltransferase activity of PBP1B, by its cognate lipoprotein activator LpoB and the essential cell division protein FtsN.

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 primary structural component of the bacterial cell wall is peptidoglycan, which is essential for viability and the synthesis of which is the target for crucial antibiotics 1 , 2 . Peptidoglycan is a single macromolecule made of glycan chains crosslinked by peptide side branches that surrounds the cell, acting as a constraint to internal turgor 1 , 3 . In Gram-positive bacteria, peptidoglycan is tens of nanometres thick, generally portrayed as a homogeneous structure that provides mechanical strength 4 – 6 . Here we applied atomic force microscopy 7 – 12 to interrogate the morphologically distinct Staphylococcus aureus and Bacillus subtilis species, using live cells and purified peptidoglycan. The mature surface of live cells is characterized by a landscape of large (up to 60 nm in diameter), deep (up to 23 nm) pores constituting a disordered gel of peptidoglycan. The inner peptidoglycan surface, consisting of more nascent material, is much denser, with glycan strand spacing typically less than 7 nm. The inner surface architecture is location dependent; the cylinder of B. subtilis has dense circumferential orientation, while in S. aureus and division septa for both species, peptidoglycan is dense but randomly oriented. Revealing the molecular architecture of the cell envelope frames our understanding of its mechanical properties and role as the environmental interface 13 , 14 , providing information complementary to traditional structural biology approaches. Using high-resolution atomic force microscopy of live cells, the authors present an updated view of the cell walls of both Staphylococcus aureus and Bacillus subtilis .

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.

Peptidoglycan is an essential structural component of the cell wall in the majority of bacteria, but the obligate intracellular human pathogen Chlamydia trachomatis was thought to be one of the few exceptions; here a click chemistry approach is used to label peptidoglycan in replicating C. trachomatis with novel d -amino acid dipeptide probes. The chlamydial anomaly resolved The sugar amino acid polymer peptidoglycan is an essential cell-wall component in most free-living bacteria. The Chlamydiales, Gram-negative parasites including the human pathogen Chlamydia trachomatis , were thought to be a rare exception: they encode genes for peptidoglycan biosynthesis and are susceptible to β -lactam antibiotics, yet attempts to detect chlamydial peptidoglycans had failed. Now this paradox, known as the 'chlamydial anomaly', has been resolved. This study, using a novel click chemistry technique to label peptidoglycans with D -amino acid dipeptide probes, demonstrates the presence of peptidoglycans in replicating C. trachomatis . Peptidoglycan (PG), an essential structure in the cell walls of the vast majority of bacteria, is critical for division and maintaining cell shape and hydrostatic pressure 1 . Bacteria comprising the Chlamydiales were thought to be one of the few exceptions. Chlamydia harbour genes for PG biosynthesis 2 , 3 , 4 , 5 , 6 , 7 and exhibit susceptibility to ‘anti-PG’ antibiotics 8 , 9 , yet attempts to detect PG in any chlamydial species have proven unsuccessful (the ‘chlamydial anomaly’ 10 ). We used a novel approach to metabolically label chlamydial PG using d -amino acid dipeptide probes and click chemistry. Replicating Chlamydia trachomatis were labelled with these probes throughout their biphasic developmental life cycle, and the results of differential probe incorporation experiments conducted in the presence of ampicillin are consistent with the presence of chlamydial PG-modifying enzymes. These findings culminate 50 years of speculation and debate concerning the chlamydial anomaly and are the strongest evidence so far that chlamydial species possess functional PG.

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.

The peptidoglycan (murein) sacculus is a unique and essential structural element in the cell wall of most bacteria. Made of glycan strands cross-linked by short peptides, the sacculus forms a closed, bag-shaped structure surrounding the cytoplasmic membrane. There is a high diversity in the composition and sequence of the peptides in the peptidoglycan from different species. Furthermore, in several species examined, the fine structure of the peptidoglycan significantly varies with the growth conditions. Limited number of biophysical data on the thickness, elasticity and porosity of peptidoglycan are available. The different models for the architecture of peptidoglycan are discussed with respect to structural and physical parameters.

Peptidoglycan is an essential polymer that forms a protective shell around bacterial cell membranes. Peptidoglycan biosynthesis is the target of many clinically used antibiotics, including the β-lactams, imipenems, cephalosporins, and glycopeptides. Resistance to these and other antibiotics has prompted interest in an atomic-level understanding of the enzymes that make peptidoglycan. Representative structures have been reported for most of the enzymes in the pathway. Until now, however, there have been no structures of any peptidoglycan glycosyltransferases (also known as transglycosylases), which catalyze formation of the carbohydrate chains of peptidoglycan from disaccharide subunits on the bacterial cell surface. We report here the 2.1-Å crystal structure of the peptidoglycan glycosyltransferase (PGT) domain of Aquifex aeolicus PBP1A. The structure has a different fold from all other glycosyltransferase structures reported to date, but it bears some resemblance to λ-lysozyme, an enzyme that degrades the carbohydrate chains of peptidoglycan. An analysis of the structure, combined with biochemical information showing that these enzymes are processive, suggests a model for glycan chain polymerization.

The cell wall of Gram-positive bacteria is a complex assemblage of glycopolymers and proteins. It consists of a thick peptidoglycan sacculus that surrounds the cytoplasmic membrane and that is decorated with teichoic acids, polysaccharides, and proteins. It plays a major role in bacterial physiology since it maintains cell shape and integrity during growth and division; in addition, it acts as the interface between the bacterium and its environment. Lactic acid bacteria (LAB) are traditionally and widely used to ferment food, and they are also the subject of more and more research because of their potential health-related benefits. It is now recognized that understanding the composition, structure, and properties of LAB cell walls is a crucial part of developing technological and health applications using these bacteria. In this review, we examine the different components of the Gram-positive cell wall: peptidoglycan, teichoic acids, polysaccharides, and proteins. We present recent findings regarding the structure and function of these complex compounds, results that have emerged thanks to the tandem development of structural analysis and whole genome sequencing. Although general structures and biosynthesis pathways are conserved among Gram-positive bacteria, studies have revealed that LAB cell walls demonstrate unique properties; these studies have yielded some notable, fundamental, and novel findings. Given the potential of this research to contribute to future applied strategies, in our discussion of the role played by cell wall components in LAB physiology, we pay special attention to the mechanisms controlling bacterial autolysis, bacterial sensitivity to bacteriophages and the mechanisms underlying interactions between probiotic bacteria and their hosts.

Tuberculosis (TB), caused by the intracellular pathogen Mycobacterium tuberculosis, remains one of the leading causes of mortality across the world. There is an urgent requirement to build a robust arsenal of effective antimicrobials, targeting novel molecular mechanisms to overcome the challenges posed by the increase of antibiotic resistance in TB. Mycobacterium tuberculosis has a unique cell envelope structure and composition, containing a peptidoglycan layer that is essential for maintaining cellular integrity and for virulence. The enzymes involved in the biosynthesis, degradation, remodelling and recycling of peptidoglycan have resurfaced as attractive targets for anti-infective drug discovery. Here, we review the importance of peptidoglycan, including the structure, function and regulation of key enzymes involved in its metabolism. We also discuss known inhibitors of ATP-dependent Mur ligases, and discuss the potential for the development of pan-enzyme inhibitors targeting multiple Mur ligases.