O-acetylation Of Peptidoglycan Research Articles

The mechanism of peptidoglycan O-acetylation in Gram-negative bacteria typifies bacterial MBOAT-SGNH acyltransferases.

Bacterial cell envelope polymers are commonly modified with acyl groups that provide fitness advantages. Many polymer acylation pathways involve pairs of membrane-bound O-acyltransferase (MBOAT) and SGNH family proteins. As an example, the MBOAT protein PatA and the SGNH protein PatB are required in Gram-negative bacteria for peptidoglycan O-acetylation. The mechanism for how MBOAT-SGNH transferases move acyl groups from acyl-CoA donors made in the cytoplasm to extracellular polymers is unclear. Using the peptidoglycan O-acetyltransferase proteins PatAB, we explore the mechanism of MBOAT-SGNH pairs. We find that the MBOAT protein PatA catalyzes auto-acetylation of an invariant Tyr residue in its conserved C-terminal hexapeptide motif. We also show that PatB can use a synthetic hexapeptide containing an acetylated tyrosine to donate an acetyl group to a peptidoglycan mimetic. Finally, we report the structure of PatB, finding that it has structural features that shape its activity as an O-acetyltransferase and distinguish it from other SGNH esterases and hydrolases. Taken together, our results support a model for peptidoglycan acylation in which a tyrosine-containing peptide at the MBOAT's C-terminus shuttles an acyl group from the MBOAT active site to the SGNH active site, where it is transferred to peptidoglycan. This model likely applies to other systems containing MBOAT-SGNH pairs, such as those that O-acetylate alginate, cellulose, and secondary cell wall polysaccharides. The use of an acyl-tyrosine intermediate for MBOAT-SGNH acyl transfer is also shared with AT3-SGNH proteins, a second major group of acyltransferases that modify cell envelope polymers.

Resistance to host antimicrobial peptides mediates resilience of gut commensals during infection and aging in Drosophila.

Resilience to short-term perturbations, like inflammation, is a fundamental feature of microbiota, yet the underlying mechanisms of microbiota resilience are incompletely understood. Here, we show that Lactiplantibacillus plantarum, a major Drosophila commensal, stably colonizes the fruit fly gut during infection and is resistant to Drosophila antimicrobial peptides (AMPs). By transposon screening, we identified L. plantarum mutants sensitive to AMPs. These mutants were impaired in peptidoglycan O-acetylation or teichoic acid D-alanylation, resulting in increased negative cell surface charge and higher affinity to cationic AMPs. AMP-sensitive mutants were cleared from the gut after infection and aging-induced gut inflammation in wild-type, but not in AMP-deficient flies, suggesting that resistance to host AMPs is essential for commensal resilience in an inflamed gut environment. Thus, our work reveals that in addition to the host immune tolerance to the microbiota, commensal-encoded resilience mechanisms are necessary to maintain the stable association between host and microbiota during inflammation.

Mechanism of Staphylococcus aureus peptidoglycan O-acetyltransferase A as an O-acyltransferase

The O-acetylation of exopolysaccharides, including the essential bacterial cell wall polymer peptidoglycan, confers resistance to their lysis by exogenous hydrolases. Like the enzymes catalyzing the O-acetylation of exopolysaccharides in the Golgi of animals and fungi, peptidoglycan O-acetyltransferase A (OatA) is predicted to be an integral membrane protein comprised of a membrane-spanning acyltransferase-3 (AT-3) domain and an extracytoplasmic domain; for OatA, these domains are located in the N- and C-terminal regions of the enzyme, respectively. The recombinant C-terminal domain (OatAC) has been characterized as an SGNH acetyltransferase, but nothing was known about the function of the N-terminal AT-3 domain (OatAN) or its homologs associated with other acyltransferases. We report herein the experimental determination of the topology of Staphylococcus aureus OatAN, which differs markedly from that predicted in silico. We present the biochemical characterization of OatAN as part of recombinant OatA and demonstrate that acetyl-CoA serves as the substrate for OatAN Using in situ and in vitro assays, we characterized 35 engineered OatA variants which identified a catalytic triad of Tyr-His-Glu residues. We trapped an acetyl group from acetyl-CoA on the catalytic Tyr residue that is located on an extracytoplasmic loop of OatAN Further enzymatic characterization revealed that O-acetyl-Tyr represents the substrate for OatAC We propose a model for OatA action involving the translocation of acetyl groups from acetyl-CoA across the cytoplasmic membrane by OatAN and their subsequent intramolecular transfer to OatAC for the O-acetylation of peptidoglycan via the concerted action of catalytic Tyr and Ser residues.

Structural basis for the O-acetyltransferase function of the extracytoplasmic domain of OatA from Staphylococcus aureus

Many bacteria possess enzymes that modify the essential cell-wall polymer peptidoglycan by O-acetylation. This modification occurs in numerous Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus, a common cause of human infections. O-Acetylation of peptidoglycan protects bacteria from the lytic activity of lysozyme, a mammalian innate immune enzyme, and as such is important for bacterial virulence. The O-acetylating enzyme in Gram-positive bacteria, O-acetyltransferase A (OatA), is a two-domain protein consisting of an N-terminal integral membrane domain and a C-terminal extracytoplasmic domain. Here, we present the X-ray crystal structure at 1.71 Å resolution and the biochemical characterization of the C-terminal domain of S. aureus OatA. The structure revealed that this OatA domain adopts an SGNH-hydrolase fold and possesses a canonical catalytic triad. Site-specific replacement of active-site amino acids revealed the presence of a water-coordinating aspartate residue that limits esterase activity. This residue, although conserved in staphyloccocal OatA and most other homologs, is not present in the previously characterized streptococcal OatA. These results provide insights into the mechanism of acetyl transfer in the SGNH/GDSL hydrolase family and highlight important evolutionary differences between homologous OatA enzymes. Furthermore, this study enhances our understanding of PG O-acetyltransferases, which could guide the development of novel antibacterial drugs to combat infections with multidrug-resistant bacterial pathogens.

Insight into the Lytic Functions of the Lactococcal Prophage TP712.

The lytic cassette of Lactococcus lactis prophage TP712 contains a putative membrane protein of unknown function (Orf54), a holin (Orf55), and a modular endolysin with a N-terminal glycoside hydrolase (GH_25) catalytic domain and two C-terminal LysM domains (Orf56, LysTP712). In this work, we aimed to study the mode of action of the endolysin LysTP712. Inducible expression of the holin-endolysin genes seriously impaired growth. The growth of lactococcal cells overproducing the endolysin LysTP712 alone was only inhibited upon the dissipation of the proton motive force by the pore-forming bacteriocin nisin. Processing of a 26-residues signal peptide is required for LysTP712 activation, since a truncated version without the signal peptide did not impair growth after membrane depolarization. Moreover, only the mature enzyme displayed lytic activity in zymograms, while no lytic bands were observed after treatment with the Sec inhibitor sodium azide. LysTP712 might belong to the growing family of multimeric endolysins. A C-terminal fragment was detected during the purification of LysTP712. It is likely to be synthesized from an alternative internal translational start site located upstream of the cell wall binding domain in the lysin gene. Fractions containing this fragment exhibited enhanced activity against lactococcal cells. However, under our experimental conditions, improved in vitro inhibitory activity of the enzyme was not observed upon the supplementation of additional cell wall binding domains in. Finally, our data pointed out that changes in the lactococcal cell wall, such as the degree of peptidoglycan O-acetylation, might hinder the activity of LysTP712. LysTP712 is the first secretory endolysin from a lactococcal phage described so far. The results also revealed how the activity of LysTP712 might be counteracted by modifications of the bacterial peptidoglycan, providing guidelines to exploit the biotechnological potential of phage endolysins within industrially relevant lactococci and, by extension, other bacteria.

Peptidoglycan O-Acetylation as a Virulence Factor: Its Effect on Lysozyme in the Innate Immune System.

The peptidoglycan sacculus of both Gram-positive and Gram-negative bacteria acts as a protective mesh and provides structural support around the entirety of the cell. The integrity of this structure is of utmost importance for cell viability and so naturally is the first target for attack by the host immune system during bacterial infection. Lysozyme, a muramidase and the first line of defense of the innate immune system, targets the peptidoglycan sacculus hydrolyzing the β-(1→4) linkage between repeating glycan units, causing lysis and the death of the invading bacterium. The O-acetylation of N-acetylmuramoyl residues within peptidoglycan precludes the productive binding of lysozyme, and in doing so renders it inactive. This modification has been shown to be an important virulence factor in pathogens such as Staphylococcus aureus and Neisseria gonorrhoeae and is currently being investigated as a novel target for anti-virulence therapies. This article reviews interactions made between peptidoglycan and the host immune system, specifically with respect to lysozyme, and how the O-acetylation of the peptidoglycan interrupts these interactions, leading to increased pathogenicity.

Development of a High Throughput Screen for the Identification of Inhibitors of Peptidoglycan O-Acetyltransferases, New Potential Antibacterial Targets.

The O-acetylation of peptidoglycan occurs in many Gram-negative and most Gram-positive pathogens and this modification to the essential wall polymer controls the lytic activity of the autolysins, particularly the lytic transglycosylases, and inhibits that of the lysozymes of innate immunity systems. As such, the peptidoglycan O-acetyltransferases PatA/B and OatA are recognized as virulence factors. In this study, we present the high throughput screening of small compound libraries to identify the first known inhibitors of these enzymes. The fluorometric screening assay developed involved monitoring the respective O-acetyltransferases as esterases using 4-methylumbelliferylacetate as substrate. Pilot screens of 3921 compounds validated the usefulness of the HTS protocol. A number of potential inhibitors were identified amongst a total of 145,000 low molecular-weight compounds, some of which were common to both enzymes, while others were unique to each. After eliminating a number of false positives in secondary screens, dose response curves confirmed the apparent specificity of a benzothiazolyl-pyrazolo-pyridine as an inhibitor of Neisseria gonorrhoeae PatB, and several coumarin-based compounds as inhibitors of both this PatB and OatA from Staphylococcus aureus. The benzothiazolyl-pyrazolo-pyridine was determined to be a non-competitive inhibitor of PatB with a Ki of 126 µM. At 177 µg/mL and close to its solubility limit, this compound caused a 90% reduction in growth of N. gonorrhoeae, while growth of Escherichia coli, a bacterium that lacks PatB and, hence, does not produce O-acetylated peptidoglycan, was unaffected. These data provide preliminary proof of concept that peptidoglycan O-acetyltransferases would serve as useful antibacterial targets.

637. Β-Lactam (BL) Antibiotics Promote an IL-1β Response in Patients with Staphylococcus aureus Bacteremia (SaB)

BackgroundBL therapy has been associated with reduced SaB duration compared with non-BL therapy. It has been shown that patients with SaB who fail to generate increased serum IL-1β are at risk for prolonged SaB (> 4 days duration), a predictor of mortality. This suggests a major role for the IL-1β host response in prompt clearance of SaB. Furthermore, BL result in reduced peptidoglycan cross-linking, reduced peptidoglycan O-acetylation, and increased alpha-toxin expression, all of which have independently been shown to enhance IL-1β release. This study aims to show that BL therapy results in a more robust IL-1β host response compared with non-BL therapy to explain, in part, more rapid SaB clearance.MethodsFifty-nine patients (47 MRSA and 12 MSSA) with diverse SaB sources, including endovascular, extravascular (e.g., pneumonia), and catheter-related infections were included. In the first 48 hours, patients were treated with either BL, including oxacillin, ceftaroline, or cefazolin (n = 24), vs. non-BL vancomycin or daptomycin (n = 35). IL-1β concentrations were determined by ELISA on serum samples obtained on Days 1, 3 and Day 7 after bacteremia onset and compared between groups by Mann–Whitney U test.ResultsPatients in BL and non-BL groups had similar IL-1β concentrations on Day 1 of bacteremia (median BL 6.1 pg/mL vs. non-BL 2.8 pg/mL, P = 0.090). BL-treated patients had significantly higher IL-1β serum concentrations on Day 3 (median 7.54 mg/mL vs. 1.9 pg/mL; P = 0.007) and Day 7 (12.52 pg/mL vs. 1.56 pg/mL, P = 0.016) when compared with non-BL-treated patients. BL therapy resulted in 23% and 105% increase in IL-1β at Days 3 and 7, respectively, while non-BL treatment resulted in 32% and 44% reduction in IL-1β. The median duration of SaB was similar between BL and non-BL-treated patients (2.5 vs. 2.0 days, respectively, P = 0.590).ConclusionGiven that a lack of inflammasome-mediated IL-1β production is associated with prolonged SaB, the significant increases in IL-1β levels in patients treated with BL has important therapeutic implications. Previously observed reduced duration of MRSA bacteremia with the addition of BL to vancomycin may have its basis on enhancing IL-1β release. A therapeutic regimen of vancomycin or daptomycin in combination with BL to treat MRSA bacteremia and use of BL therapy in MSSA bacteremia is strongly advised to improve outcomes based on these results.Disclosures G. Sakoulas, Allergan: Consultant and Speaker, Consulting fee and Speaker honorarium. Sunovion: Speaker, Speaker honorarium. The Medicines Company: Speaker, Consulting fee. Paratek Pharmaceuticals: Consultant, Consulting fee. Cidara Therapeutics: Scientific Advisor, None. Arsanis Pharmaceuticals: Scientific Advisor, None. W. Rose, Merck: Grant Investigator, Research grant.

Mechanistic Pathways for Peptidoglycan O-Acetylation and De-O-Acetylation.

The post-synthetic O-acetylation of the essential component of bacterial cell walls, peptidoglycan (PG), is performed by many pathogenic bacteria to help them evade the lytic action of innate immunity responses. Occurring at the C-6 hydroxyl of N-acetylmuramoyl residues, this modification to the glycan backbone of PG sterically blocks the activity of lysozymes. As such, the enzyme responsible for this modification in Gram-positive bacteria is recognized as a virulence factor. With Gram-negative bacteria, the O-acetylation of PG provides a means of control of their autolysins at the substrate level. In this review, we discuss the pathways for PG O-acetylation and de-O-acetylation and the structure and function relationship of the O-acetyltransferases and O-acetylesterases that catalyze these reactions. The current understanding of their mechanisms of action is presented and the prospects of targeting these systems for the development of novel therapeutics are explored.

Use of Bioorthogonal N‐Acetylcysteamine (SNAc) Analogues and Peptidoglycan O‐Acetyltransferase B (PatB) to Label Peptidoglycan

In Gram (−) bacteria, peptidoglycan O‐acetyltransferase B (PatB) is responsible for the O‐acetylation of peptidoglycan to prevent degradation by lysozyme and other lytic transglycosylases generated by its host. In this work, PatB was exploited as a tool to incorporate bioorthogonal functionality on the carbohydrate backbone of the peptidoglycan by using a variety of donors, either p‐nitrophenol (pNP) or N‐acetylcysteamine (SNAc). Using tri‐N‐acetylglucosamine (GlcNAc)3 as a peptidoglycan mimic, a variety of functionalities were installed, including an alkyne and azide handle, on this mimic. Further studies in vitro showed that these modifications have a protective effect on isolated peptidoglycan from B. subtilis, E. coli, P. putida, and V. parahaemolyticus against lysozyme degradation. Intrigued by these results, an alkyne handle was subsequently installed on whole bacterial cells of B. subtilis using the SNAc alkyne derivative and the carbohydrate backbone was fluorescently labeled via click chemistry. Altering the peptidoglycan post‐synthetically with these derivatives will aid in the isolation and identification of immune stimulatory fragments from bacteria, as well as in the development of antibiotics and immune modulators.Support or Funding InformationThe Delaware COBRE and INBRE programs supported this project with a grant from the NIGMS (5 P30GM110758‐02, P20GM104316‐01A1 and P20GM103446) from the NIH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In vitro characterization of the antivirulence target of Gram-positive pathogens, peptidoglycan O-acetyltransferase A (OatA).

The O-acetylation of the essential cell wall polymer peptidoglycan occurs in most Gram-positive bacterial pathogens, including species of Staphylococcus, Streptococcus and Enterococcus. This modification to peptidoglycan protects these pathogens from the lytic action of the lysozymes of innate immunity systems and, as such, is recognized as a virulence factor. The key enzyme involved, peptidoglycan O-acetyltransferase A (OatA) represents a particular challenge to biochemical study since it is a membrane associated protein whose substrate is the insoluble peptidoglycan cell wall polymer. OatA is predicted to be bimodular, being comprised of an N-terminal integral membrane domain linked to a C-terminal extracytoplasmic domain. We present herein the first biochemical and kinetic characterization of the C-terminal catalytic domain of OatA from two important human pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Using both pseudosubstrates and novel biosynthetically-prepared peptidoglycan polymers, we characterized distinct substrate specificities for the two enzymes. In addition, the high resolution crystal structure of the C-terminal domain reveals an SGNH/GDSL-like hydrolase fold with a catalytic triad of amino acids but with a non-canonical oxyanion hole structure. Site-specific replacements confirmed the identity of the catalytic and oxyanion hole residues. A model is presented for the O-acetylation of peptidoglycan whereby the translocation of acetyl groups from a cytoplasmic source across the cytoplasmic membrane is catalyzed by the N-terminal domain of OatA for their transfer to peptidoglycan by its C-terminal domain. This study on the structure-function relationship of OatA provides a molecular and mechanistic understanding of this bacterial resistance mechanism opening the prospect for novel chemotherapeutic exploration to enhance innate immunity protection against Gram-positive pathogens.

Peptidoglycan O-acetylation is functionally related to cell wall biosynthesis and cell division in Streptococcus pneumoniae.

The peptidoglycan is a rigid matrix required to resist turgor pressure and to maintain the cellular shape. Itis formed by linear glycan chains composed of N-acetylmuramic acid-(β-1,4)-N-acetylglucosamine (MurNAc-GlcNAc) disaccharides associated through cross-linked peptide stems. The peptidoglycan is continually remodelled by synthetic and hydrolytic enzymes and by chemical modifications, including O-acetylation of MurNAc residues that occurs in most Gram-positive and Gram-negative bacteria. This modification is a powerful strategy developed by pathogens to resist to lysozyme degradation and thus to escape from the host innate immune system but little is known about its physiological function. In this study, we have investigated to what extend peptidoglycan O-acetylation is involved in cell wall biosynthesis and cell division of Streptococcus pneumoniae. We show that O-acetylation driven by Adr protects the peptidoglycan of dividing cells from cleavage by the major autolysin LytA and occurs at the septal site. Our results support a function for Adr in the formation of robust and mature MurNAc O-acetylated peptidoglycan and infer its role in the division of the pneumococcus.

O-Acetylation of Peptidoglycan Limits Helper T Cell Priming and Permits Staphylococcus aureus Reinfection

Humans do not usually develop effective immunity to Staphylococcus aureus reinfection. Using a murine model that mimics human infection, we show that lack of protective immunity to S.aureus systemic reinfection is associated with robust interleukin-10 (IL-10) production and impaired protective Th17 responses. In dendritic cell co-culture assays, priming with S.aureus promotes robust Tcell proliferation, but limits Th cells polarization and production of IL-1β and other cytokines important for Th1 and Th17 differentiation. We show that O-acetylation of peptidoglycan, a mechanism utilized by S.aureus to block bacterial cell wall breakdown, limits the induction of pro-inflammatory signals required for optimal Th17 polarization. IL-10 deficiency in mice restores protective immunity to S.aureus infection, and adjuvancy with a staphylococcal peptidoglycan O-acetyltransferase mutant reduces IL-10, increases IL-1β, and promotes development of IL-17-dependent, Th cell-transferable protective immunity. Overall, our study suggests a mechanism whereby S.aureus modulates cytokines critical for induction of protective Th17 immunity.

Impact of cell wall peptidoglycan O-acetylation on the pathogenesis of Staphylococcus aureus in septic arthritis

Staphylococcus aureus (S. aureus) is one of the most common pathogen causing septic arthritis. To colonize the joints and establish septic arthritis this bacterium needs to resist the host innate immune responses. Lysozyme secreted by neutrophils and macrophages is an important defense protein present in the joint synovial fluids. S. aureus is known to be resistant to lysozyme due to its peptidoglycan modification by O-acetylation of N-acetyl muramic acid. In this study we have investigated the role of O-acetylated peptidoglycan in septic arthritis. Using mouse models for both local and hematogenous S. aureus arthritis we compared the onset and progress of the disease induced by O-acetyl transferase mutant and the parenteral wild type SA113 strain. The disease progression was assessed by observing the clinical parameters including body weight, arthritis, and functionality of the affected limbs. Further X-ray and histopathological examinations were performed to monitor the synovitis and bone damage. In local S. aureus arthritis model, mice inoculated with the ΔoatA strain developed milder disease (in terms of knee swelling, motor and movement functionality) compared to mice inoculated with the wild type SA113 strain. X-ray and histopathological data revealed that ΔoatA infected mice knee joints had significantly lesser joint destruction, which was accompanied by reduced bacterial load in knee joints. Similarly, in hematogenous S. aureus arthritis model, ΔoatA mutant strain induced significantly less severe clinical septic arthritis compared to its parental strain, which is in accordance with radiological findings. Our data indicate that peptidoglycan O-acetylation plays an important role in S. aureus mediated septic arthritis.

Peptidoglycan O-acetylation increases in response to vancomycin treatment in vancomycin-resistant Enterococcus faecalis

Vancomycin resistance is conferred upon vancomycin-resistant enterococci (VRE) through the replacement of peptidoglycan (PG) stem terminal d-Ala-d-Ala with d-Ala-d-Lac. The d-Ala-d-Lac incorporation can affect both the fitness and virulence of VRE. Here we comprehensively investigate the changes to PG composition in vancomycin-resistant Enterococcus faecalis following the growth in presence of vancomycin using liquid chromatography-mass spectrometry. Using high-resolution mass spectrometry, 104 unique muropeptides fragments were identified and the relative abundance of each fragment was accurately quantified by integrating the ion current of a selected ion using extracted-ion chromatogram. The analysis indicates reduced PG cross-linking, increased carboxypeptidase activities, increased N-deacetylation, and increased O-acetylation in VRE when grown in the presence of vancomycin. We found that O-acetylation preferentially occurred on muropeptides fragments with reduced cross-linking with a pentapeptide stem that terminated in d-Ala-d-Lac. These findings show that O-acetylation preferentially occurred in regions of the cell wall with reduced PG cross-linking on PG units that have stems terminating in d-Ala-d-Lac, serving as markers to prevent both the PG-stem modification by carboxypeptidases and the cell wall degradation by autolysins. Accurate quantitative PG composition analysis provided compositional insights into altered cell wall biosynthesis and modification processes in VRE that contribute to lysozyme resistance and enhanced virulence for VRE grown in the presence of vancomycin.

Accumulation of Peptidoglycan O-Acetylation Leads to Altered Cell Wall Biochemistry and Negatively Impacts Pathogenesis Factors of Campylobacter jejuni

Campylobacter jejuni is a leading cause of bacterial gastroenteritis in the developed world. Despite its prevalence, its mechanisms of pathogenesis are poorly understood. Peptidoglycan (PG) is important for helical shape, colonization, and host-pathogen interactions in C. jejuni. Therefore, changes in PG greatly impact the physiology of this organism. O-acetylation of peptidoglycan (OAP) is a bacterial phenomenon proposed to be important for proper cell growth, characterized by acetylation of the C6 hydroxyl group of N-acetylmuramic acid in the PG glycan backbone. The OAP gene cluster consists of a PG O-acetyltransferase A (patA) for translocation of acetate into the periplasm, a PG O-acetyltransferase B (patB) for O-acetylation, and an O-acetylpeptidoglycan esterase (ape1) for de-O-acetylation. In this study, reduced OAP in ΔpatA and ΔpatB had minimal impact on C. jejuni growth and fitness under the conditions tested. However, accumulation of OAP in Δape1 resulted in marked differences in PG biochemistry, including O-acetylation, anhydromuropeptide levels, and changes not expected to result directly from Ape1 activity. This suggests that OAP may be a form of substrate level regulation in PG biosynthesis. Ape1 acetylesterase activity was confirmed in vitro using p-nitrophenyl acetate and O-acetylated PG as substrates. In addition, Δape1 exhibited defects in pathogenesis-associated phenotypes, including cell shape, motility, biofilm formation, cell surface hydrophobicity, and sodium deoxycholate sensitivity. Δape1 was also impaired for chick colonization and adhesion, invasion, intracellular survival, and induction of IL-8 production in INT407 cells in vitro. The importance of Ape1 in C. jejuni biology makes it a good candidate as an antimicrobial target.

Understanding the Structure-Function Relationship of Lysozyme Resistance in Staphylococcus aureus by Peptidoglycan O-Acetylation Using Molecular Docking, Dynamics, and Lysis Assay.

Lysozyme is an important component of the host innate defense system. It cleaves the β-1,4 glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine of bacterial peptidoglycan and induce bacterial lysis. Staphylococcus aureus (S. aureus), an opportunistic commensal pathogen, is highly resistant to lysozyme, because of the O-acetylation of peptidoglycan by O-acetyl transferase (oatA). To understand the structure-function relationship of lysozyme resistance in S. aureus by peptidoglycan O-acetylation, we adapted an integrated approach to (i) understand the effect of lysozyme on the growth of S. aureus parental and the oatA mutant strain, (ii) study the lysozyme induced lysis of exponentially grown and stationary phase of both the S. aureus parental and oatA mutant strain, (iii) investigate the dynamic interaction mechanism between normal (de-O-acetylated) and O-acetylated peptidoglycan substrate in complex with lysozyme using molecular docking and molecular dynamics simulations, and (iv) quantify lysozyme resistance of S. aureus parental and the oatA mutant in different human biological fluids. The results indicated for the first time that the active site cleft of lysozyme binding with O-acetylated peptidoglycan in S. aureus was sterically hindered and the structural stability was higher for the lysozyme in complex with normal peptidoglycan. This could have conferred reduced survival of the S. aureus oatA mutant in different human biological fluids. Consistent with this computational analysis, the experimental data confirmed decrease in the growth, lysozyme induced lysis, and lysozyme resistance, due to peptidoglycan O-acetylation in S. aureus.

Assay for peptidoglycan O-acetyltransferase: A potential new antibacterial target

The O-acetylation of peptidoglycan occurs at the C-6 hydroxyl group of muramoyl residues in many human pathogens, both gram positive and gram negative, such as Staphylococcus aureus and species of Campylobacter, Helicobacter, Neisseria, and Bacillus, including Bacillus anthracis. The process is a maturation event being catalyzed either by integral membrane O-acetylpeptidoglycan transferase (Oat) of gram-positive bacteria or by a two-component peptidoglycan O-acetyltransferase system (PatA/PatB) in gram-negative cells. Here, we describe the development of the first in vitro assay for any peptidoglycan O-acetyltransferase using PatB from Neisseria gonorrhoeae as the model enzyme. This assay is based on the use of chromogenic p-nitrophenyl acetate as the donor substrate and chitooligosaccharides as model acceptor substrates in place of peptidoglycan. The identity of the O-acetylated chitooligosaccharides was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rates of transacetylations were determined spectrophotometrically by monitoring p-nitrophenol release after accounting for both spontaneous and enzyme-catalyzed hydrolysis of the acetate donor. Conditions were established for use of the assay in microtiter plate format, and its applicability was demonstrated by determining the first Michaelis–Menten kinetic parameters for PatB. The assay is readily amenable for application in the high-throughput screening for potential inhibitors of peptidoglycan O-acetyltransferases that may prove to be leads for novel classes of antibiotics.

De‐O‐acetylation of peptidoglycan regulates glycan chain extension and affects in vivo survival of Neisseria meningitidis

Peptidoglycan O-acetylation is a modification found in many bacteria. In Gram-positive pathogens, it contributes to virulence by conferring resistance to host lysozyme. However, in Gram-negative pathogens, its contribution to physiology and virulence is unknown. We examined the contribution of patA, patB and ape1 to peptidoglycan O-acetylation in the major human pathogen Neisseria meningitidis (Nm). Using genetic expression of all possible combinations of the three genes in Escherichia coli and Nm, we confirmed that PatA and PatB were required for PG O-acetylation, while ApeI removed the O-acetyl group. ApeI was active on all O-acetylated muropeptides produced by PatA and PatB during heterologous expression in E. coli and was also active on several PG structures in vitro. Interestingly, in Nm, ApeI was found to preferentially de-O-acetylate muropeptides with tripeptide stems (GM3), suggesting that its activity is highly regulated. Accordingly, de-O-acetylation of GM3 regulated glycan chain elongation and cell size. Additionally, the virulence of Nm lacking ApeI was drastically reduced suggesting that regulation of glycan chain length by O-acetylation contributes to bacterial fitness in the host. Altogether, our results suggest that ApeI represents an attractive target for new drug development.

Dual Role for the O-Acetyltransferase OatA in Peptidoglycan Modification and Control of Cell Septation in Lactobacillus plantarum

Until now, peptidoglycan O-acetyl transferases (Oat) were only described for their peptidoglycan O-acetylating activity and for their implication in the control of peptidoglycan hydrolases. In this study, we show that a Lactobacillus plantarum mutant lacking OatA is unable to uncouple cell elongation and septation. Wild-type cells showed an elongation arrest during septation while oatA mutant cells continued to elongate at a constant rate without any observable pause during the cell division process. Remarkably, this defect does not result from a default in peptidoglycan O-acetylation, since it can be rescued by wild-type OatA as well as by a catalytic mutant or a truncated variant containing only the transmembrane domain of the protein. Consistent with a potential involvement in division, OatA preferentially localizes at mid-cell before membrane invagination and remains at this position until the end of septation. Overexpression of oatA or its inactive variants induces septation-specific aberrations, including asymmetrical and dual septum formation. Overproduction of the division inhibitors, MinC or MinD, leads to cell filamentation in the wild type while curved and branched cells are observed in the oatA mutant, suggesting that the Min system acts differently on the division process in the absence of OatA. Altogether, the results suggest that OatA plays a key role in the spatio-temporal control of septation, irrespective of its catalytic activity.