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Proteins with a catalytically inactive LytM-type endopeptidase domain are important regulators of cell wall-degrading enzymes in bacteria. Here, we study their representative DipM, a factor promoting cell division in Caulobacter crescentus. We show that the LytM domain of DipM interacts with multiple autolysins, including the soluble lytic transglycosylases SdpA and SdpB, the amidase AmiC and the putative carboxypeptidase CrbA, and stimulates the activities of SdpA and AmiC. Its crystal structure reveals a conserved groove, which is predicted to represent the docking site for autolysins by modeling studies. Mutations in this groove indeed abolish the function of DipM in vivo and its interaction with AmiC and SdpA in vitro. Notably, DipM and its targets SdpA and SdpB stimulate each other’s recruitment to midcell, establishing a self-reinforcing cycle that gradually increases autolytic activity as cytokinesis progresses. DipM thus coordinates different peptidoglycan-remodeling pathways to ensure proper cell constriction and daughter cell separation.

Despite extensive studies on the curve-shaped bacterium Vibrio cholerae, the causative agent of the diarrheal disease cholera, its virulence-associated regulatory two-component signal transduction system VarS/VarA is not well understood. This pathway, which mainly signals through the downstream protein CsrA, is highly conserved among gamma-proteobacteria, indicating there is likely a broader function of this system beyond virulence regulation. In this study, we investigated the VarA-CsrA signaling pathway and discovered a previously unrecognized link to the shape of the bacterium. We observed that varA-deficient V. cholerae cells showed an abnormal spherical morphology during late-stage growth. Through peptidoglycan (PG) composition analyses, we discovered that these mutant bacteria contained an increased content of disaccharide dipeptides and reduced peptide crosslinks, consistent with the atypical cellular shape. The spherical shape correlated with the CsrA-dependent overproduction of aspartate ammonia lyase (AspA) in varA mutant cells, which likely depleted the cellular aspartate pool; therefore, the synthesis of the PG precursor amino acid meso-diaminopimelic acid was impaired. Importantly, this phenotype, and the overall cell rounding, could be prevented by means of cell wall recycling. Collectively, our data provide new insights into how V. cholerae use the VarA-CsrA signaling system to adjust its morphology upon unidentified external cues in its environment.

Peptidoglycan is the major structural component of the Staphylococcus aureus cell wall, in which it maintains cellular integrity, is the interface with the host, and its synthesis is targeted by some of the most crucial antibiotics developed. Despite this importance, and the wealth of data from in vitro studies, we do not understand the structure and dynamics of peptidoglycan during infection. In this study we have developed methods to harvest bacteria from an active infection in order to purify cell walls for biochemical analysis ex vivo. Isolated ex vivo bacterial cells are smaller than those actively growing in vitro, with thickened cell walls and reduced peptidoglycan crosslinking, similar to that of stationary phase cells. These features suggested a role for specific peptidoglycan homeostatic mechanisms in disease. As S. aureus missing penicillin binding protein 4 (PBP4) has reduced peptidoglycan crosslinking in vitro its role during infection was established. Loss of PBP4 resulted in an increased recovery of S. aureus from the livers of infected mice, which coincided with enhanced fitness within murine and human macrophages. Thicker cell walls correlate with reduced activity of peptidoglycan hydrolases. S. aureus has a family of 4 putative glucosaminidases, that are collectively crucial for growth. Loss of the major enzyme SagB, led to attenuation during murine infection and reduced survival in human macrophages. However, loss of the other three enzymes Atl, SagA and ScaH resulted in clustering dependent attenuation, in a zebrafish embryo, but not a murine, model of infection. A combination of pbp4 and sagB deficiencies resulted in a restoration of parental virulence. Our results, demonstrate the importance of appropriate cell wall structure and dynamics during pathogenesis, providing new insight to the mechanisms of disease.

The Gram-negative bacterium Serratia marcescens secretes many proteins that are involved in extracellular chitin degradation. This so-called chitinolytic machinery includes three types of chitinase enzymes and a lytic polysaccharide monooxygenase. An operon has been identified in S. marcescens, chiWXYZ, that is thought to be involved in the secretion of the chitinolytic machinery. Genetic evidence points to the ChiX protein being a key player in the secretion mechanism, since deletion of the chiX gene in S. marcescens led to a mutant strain blocked for secretion of all members of the chitinolytic machinery. In this work, a detailed structural and biochemical characterisation of ChiX is presented. The high-resolution crystal structure of ChiX reveals the protein to be a member of the LAS family of peptidases. ChiX is shown to be a zinc-containing metalloenzyme, and in vitro assays demonstrate that ChiX is an l-Ala d-Glu endopeptidase that cleaves the cross-links in bacterial peptidoglycan. This catalytic activity is shown to be intimately linked with the secretion of the chitinolytic machinery, since substitution of the ChiX Asp-120 residue results in a variant protein that is both unable to digest peptidoglycan and cannot rescue the phenoytype of a chiX mutant strain.

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.

Peptidoglycan is the predominant stress-bearing structure in the cell envelope of most bacteria, and also a potent stimulator of the eukaryotic immune system. Obligate intracellular bacteria replicate exclusively within the interior of living cells, an osmotically protected niche. Under these conditions peptidoglycan is not necessarily needed to maintain the integrity of the bacterial cell. Moreover, the presence of peptidoglycan puts bacteria at risk of detection and destruction by host peptidoglycan recognition factors and downstream effectors. This has resulted in a selective pressure and opportunity to reduce the levels of peptidoglycan. In this review we have analysed the occurrence of genes involved in peptidoglycan metabolism across the major obligate intracellular bacterial species. From this comparative analysis, we have identified a group of predicted "peptidoglycan-intermediate" organisms that includes the Chlamydiae, Orientia tsutsugamushi, Wolbachia and Anaplasma marginale. This grouping is likely to reflect biological differences in their infection cycle compared with peptidoglycan-negative obligate intracellular bacteria such as Ehrlichia and Anaplasma phagocytophilum, as well as obligate intracellular bacteria with classical peptidoglycan such as Coxiella, Buchnera and members of the Rickettsia genus. The signature gene set of the peptidoglycan-intermediate group reveals insights into minimal enzymatic requirements for building a peptidoglycan-like sacculus and/or division septum. This article is protected by copyright. All rights reserved.

Background Increasing access to essential respiratory medicines and influenza vaccination has been a priority for over three decades. Their use remains low in low- and middle-income countries (LMICs), where little is known about factors influencing use, or about the use of influenza vaccination for preventing respiratory exacerbations. Methods We estimated rates of regular use of bronchodilators, inhaled corticosteroids and influenza vaccine, and predictors for use among 19 000 adults in 23 high-income countries (HICs) and LMIC sites. Results Bronchodilators, inhaled corticosteroids and influenza vaccine were used significantly more in HICs than in LMICs, after adjusting for similar clinical needs. Although they are used more commonly by people with symptomatic or severe respiratory disease, the gap between HICs and LMICs is not explained by the prevalence of chronic obstructive pulmonary disease or doctor-diagnosed asthma. Site-specific factors are likely to influence use differently. The gross national income per capita for the country is a strong predictor for use of these treatments, suggesting that economics influence under-treatment. Conclusion We still need a better understanding of determinants for the low use of essential respiratory medicines and influenza vaccine in low-income settings. Identifying and addressing these more systematically could improve the access and use of effective treatments.

The distribution of PBP5, the major D,D-carboxypeptidase in Escherichia coli, was mapped by immunolabelling and by visualization of GFP fusion proteins in wild-type cells and in mutants lacking one or more D,D-carboxypeptidases. In addition to being scattered around the lateral envelope, PBP5 was also concentrated at nascent division sites prior to visible constriction. Inhibiting PBP2 activity (which eliminates wall elongation) shifted PBP5 to midcell, whereas inhibiting PBP3 (which aborts divisome invagination) led to the creation of PBP5 rings at positions of preseptal wall formation, implying that PBP5 localizes to areas of ongoing peptidoglycan synthesis. A PBP5(S44G) active site mutant was more evenly dispersed, indicating that localization required enzyme activity and the availability of pentapeptide substrates. Both the membrane bound and soluble forms of PBP5 converted pentapeptides to tetrapeptides in vitro and in vivo, and the enzymes accepted the same range of substrates, including sacculi, Lipid II, muropeptides and artificial substrates. However, only the membrane-bound form localized to the developing septum and restored wild-type rod morphology to shape defective mutants, suggesting that the two events are related. The results indicate that PBP5 localization to sites of ongoing peptidoglycan synthesis is substrate dependent and requires membrane attachment.

To describe acupuncture and chiropractic use among patients with chronic musculoskeletal pain (CMP) at a health maintenance organization, and explore issues of benefit design and electronic medical record (EMR) capture.Cross-sectional survey.Kaiser Permanente members meeting EMR diagnostic criteria for CMP were invited to participate. The survey included questions about self-identified presence of CMP, use of acupuncture and chiropractic care, use of ancillary self-care modalities, and communication with conventional medicine practitioners. Analysis of survey data was supplemented with a retrospective review of EMR utilization data.Of 6068 survey respondents, 32% reported acupuncture use, 47% reported chiropractic use, 21% used both, and 42% used neither. For 25% of patients using acupuncture and 43% of those using chiropractic care, utilization was undetected by the EMR. Thirty-five percent of acupuncture users and 42% of chiropractic users did not discuss this care with their health maintenance organization (HMO) clinicians. Among chiropractic users, those accessing care out of plan were older (P.01), were more likely to use long-term opioids (P = .03), and had more pain diagnoses (P = .01) than those accessing care via clinician referral or self-referral. For acupuncture, those using the clinician referral mechanism exhibited these same characteristics.A majority of participants had used acupuncture, chiropractic care, or both. While benefit structure may materially influence utilization patterns, many patients with CMP use acupuncture and chiropractic care without regard to their insurance coverage. A substantial percentage of acupuncture and chiropractic use thus occurs beyond detection of EMR systems, and many patients do not report such care to their HMO clinicians.

Peptidoglycan performs the essential role of resisting turgor in the cell walls of most bacteria. It determines cell shape, and its biosynthesis is the target for many important antibiotics. The fundamental chemical building blocks of peptidoglycan are conserved: repeating disaccharides cross-linked by peptides. However, these blocks come in many varieties and can be assembled in different ways. So beyond the fundamental similarity, prodigious chemical, organizational and architectural diversity is revealed. Here, we track the evolution of our current understanding of peptidoglycan and underpinning technical and methodological developments. The origin and function of chemical diversity is discussed with respect to some well-studied example species. We then explore how this chemistry is manifested in elegant and complex peptidoglycan organization and how this is interpreted in different and sometimes controversial architectural models. We contend that emerging technology brings about the possibility of achieving a complete understanding of peptidoglycan chemistry, through architecture, to the way in which diverse species and populations of cells meet the challenges of maintaining viability and growth within their environmental niches, by exploiting the bioengineering versatility of peptidoglycan.

The shape of a bacterial cell is maintained by its peptidoglycan sacculus that completely surrounds the cytoplasmic membrane. During growth the sacculus is enlarged by peptidoglycan synthesis complexes that are controlled by components linked to the cytoskeleton and, in Gram-negative bacteria, by outer-membrane regulators of peptidoglycan synthases. Cell division is achieved by a large assembly of essential cell division proteins, the divisome, that coordinates the synthesis and hydrolysis of peptidoglycan during septation. Coccal species such as Staphylococcus aureus grow exclusively by synthesis and cleavage of a cross-wall. Ovococci like Streptococcus pneumoniae elongate at a central growth zone resulting in their lancet-shape. Rod-shaped species elongate either at the side-wall coordinated by the MreB cytoskeleton, like Escherichia coli or Bacillus subtilis, or at the poles like Corynebacterium glutamicum. Bacteria have different mechanisms to achieve bent or helical cell shape, involving cytoskeletal proteins, periplasmic flagella or peptidoglycan hydrolases, and to form branched, filamentous cell chains. Peptidoglycan enzymes and cytoskeletal proteins are validated targets for antimicrobial compounds. Recent approaches applying structure-based inhibitor design, high-throughput screening assays and whole cell assays have identified a large number of novel inhibitors of cytoskeletal proteins and enzymes of the peptidoglycan biosynthesis pathway.

For both males and females, the pathway to diagnosis and treatment of lung disease often begins with the reporting of respiratory symptoms to their physician. Males and females with impaired lung function may experience the same symptoms but perceive and/or report them differently.

The pneumococcus is an important Gram-positive pathogen, which shows increasing resistance to antibiotics, including β-lactams that target peptidoglycan assembly. Understanding cell-wall synthesis, at the molecular and cellular level, is essential for the prospect of combating drug resistance. As a first step towards reconstituting pneumococcal cell-wall assembly in vitro, we present the characterization of the glycosyltransferase activity of penicillin-binding protein (PBP)2a from Streptococcus pneumoniae. Recombinant full-length membrane-anchored PBP2a was purified by ion-exchange chromatography. The glycosyltransferase activity of this enzyme was found to differ from that of a truncated periplasmic form. The full-length protein with its cytoplasmic and transmembrane segment synthesizes longer glycan chains than the shorter form. The transpeptidase active site was functional, as shown by its reactivity towards bocillin and the catalysis of the hydrolysis of a thiol-ester substrate analogue. However, PBP2a did not cross-link the peptide stems of glycan chains in vitro. The absence of transpeptidase activity indicates that an essential component is missing from the in vitro system.

Chronic obstructive pulmonary disease (COPD) is underdiagnosed. One barrier to diagnosis is the limited availability of spirometry testing, but in adults at risk for COPD, a normal pre-bronchodilator (pre-BD) peak expiratory flow (PEF) may rule out clinically significant COPD.To identify post-BD airway obstruction using data from 13 708 individuals agedor=40 years from the PLATINO and BOLD studies.We evaluated different cut-off points of pre-BD. The PEF was obtained from a diagnostic-quality spirometer (not a mechanical PEF meter). At least one of the following COPD risk factors was present in 77% of the subjects: chronic respiratory symptoms; exposure to tobacco smoke, biomass smoke or dust in the workplace; or a previous diagnosis of asthma, COPD, emphysema or chronic bronchitis.Although the positive predictive value was low as expected, a pre-BD PEF ofor=70% predicted effectively ruled out Stages III and IV COPD of the Global Initiative for Chronic Obstructive Lung Disease. Among those with at least one risk factor, only 12% would require confirmatory spirometry using this criterion.Adding PEF measurement to a screening questionnaire may rule out severe to very severe COPD without the need for pre- and post-BD spirometry testing. Confirmation is needed from a study using inexpensive PEF meters or pocket spirometers with a staged screening protocol.