Step Of Cell Wall Biosynthesis Research Articles

Targeting MurB from Helicobacter pylori: insights from virtual screening, molecular docking and molecular dynamics simulation

ABSTRACT Helicobacter pylori is a gram-negative, helical-shaped bacteria found in the mucus lining of the stomach of humans responsible for causing sores or an ulcer. H. pylori is becoming a multi-drug resistant bacterium, which in turn demands the need for development of alternative treatment strategies for the proper management of diseases. The peptidoglycan biosynthesis pathway is critical for the synthesis of cell walls and for the survival of H. pylori. There are several crucial enzymes in this biosynthesis pathway which can act as significant drug targets against H. pylori. One of these important enzymes is UDP-N-acetylenolpyruvoylglucosamine reductase (MurB), which catalyses an important committed step of cell wall biosynthesis. In the present study, we performed a high throughput virtual screening using the Enamine HTSC library against the MurB enzyme. The molecules were also screened for druglike properties by ADME screening, toxicity, and tumorigenic nature, followed by MD simulations. We found that Z102621114_1 and Z228235240_1 ligand molecules can act as potential inhibitors of MurB protein. Our results suggest, these molecules may be able to meet the demand for novel antimicrobials to treat H. pylori medication resistance. The subsequent experimental investigations can increase their potential as a novel agents against H. pylori infections. Research highlights Helicobacter pylori infects 50% of the Global population. Helicobacter pylori infection is associated with gastric cancer. MurB is a key drug target protein as it is responsible for cell wall synthesis. Z102621114_1 and Z228235240_1 were identified as potential inhibitors of MurB of Helicobacter pylori.

Mechanism of action of pseudopteroxazole and pseudopterosin G: Diterpenes from marine origin.

Pseudopteroxazole (Ptx) and the pseudopterosins are marine natural products with promising antibacterial potential. While Ptx has attracted interest for its antimycobacterial activity, pseudopterosins are active against several clinically relevant pathogens. Both compound classes exhibit low cytotoxicity and accessibility to targeted synthesis, yet their antibacterial mechanisms remain elusive. In this study, we investigated the modes of action of Ptx and pseudopterosinG (PsG) in Bacillus subtilis employing an unbiased approach that combines gel-based proteomics with a mathematical similarity analysis of response profiles. Proteomic responses to sublethal concentrations of Ptx and PsG were compared to a library of antibiotic stress response profiles revealing that both induce a stress response characteristic for agents targeting the bacterial cell envelope by interfering with membrane-bound steps of cell wall biosynthesis. Microscopy-based assays confirmed that both compounds compromise the integrity of the bacterial cell wall without disrupting the membrane potential. Furthermore, LC-MSE analysis showed that the greater potency of PsG against B.subtilis, reflected in a lower MIC and a more pronounced proteomic response, may be rooted in a more effective association with and penetration of B. subtilis cells. We conclude that Ptx and PsG target the integrity of the gram-positive cell wall.

Application of a Bacillus subtilis Whole-Cell Biosensor (PliaI-lux) for the Identification of Cell Wall Active Antibacterial Compounds.

Whole-cell biosensors, based on the visualization of a reporter strain's response to a particular stimulus, are a robust and cost-effective means to monitor defined environmental conditions or the presence of chemical compounds. One specific field in which such biosensors are frequently applied is drug discovery, that is, the screening of large numbers of bacterial or fungal strains for the production of antimicrobial compounds. Here, we describe the application of a luminescence-based Bacillus subtilis biosensor for the discovery of cell wall active substances; this article is an update to our previous chapter published in 2017. The system is based on the well-characterized promoter PliaI, which is induced in response to a wide range of conditions that cause cell envelope stress, particularly antibiotics that interfere with the membrane-anchored steps of cell wall biosynthesis. A simple "spot-on-lawn" assay, where colonies of potential producer strains are grown directly on a lawn of the reporter strain, allows for quantitative and time-resolved detection of antimicrobial compounds. Due to the very low technical demands of this procedure, we expect it to be easily applicable to a large variety of candidate producer strains and growth conditions.

Mutation-Based Antibiotic Resistance Mechanism in Methicillin-Resistant Staphylococcus aureus Clinical Isolates.

β-Lactam antibiotics target penicillin-binding proteins and inhibit the synthesis of peptidoglycan, a crucial step in cell wall biosynthesis. Staphylococcus aureus acquires resistance against β-lactam antibiotics by producing a penicillin-binding protein 2a (PBP2a), encoded by the mecA gene. PBP2a participates in peptidoglycan biosynthesis and exhibits a poor affinity towards β-lactam antibiotics. The current study was performed to determine the diversity and the role of missense mutations of PBP2a in the antibiotic resistance mechanism. The methicillin-resistant Staphylococcus aureus (MRSA) isolates from clinical samples were identified using phenotypic and genotypic techniques. The highest frequency (60%, 18 out of 30) of MRSA was observed in wound specimens. Sequence variation analysis of the mecA gene showed four amino acid substitutions (i.e., E239K, E239R, G246E, and E447K). The E239R mutation was found to be novel. The protein-ligand docking results showed that the E239R mutation in the allosteric site of PBP2a induces conformational changes in the active site and, thus, hinders its interaction with cefoxitin. Therefore, the present report indicates that mutation in the allosteric site of PBP2a provides a more closed active site conformation than wide-type PBP2a and then causes the high-level resistance to cefoxitin.

Suppressor Mutations Linking gpsB with the First Committed Step of Peptidoglycan Biosynthesis in Listeria monocytogenes.

The late cell division protein GpsB is important for cell wall biosynthesis in Gram-positive bacteria. GpsB of the human pathogen L. monocytogenes interacts with one of the key enzymes of this pathway, penicillin binding protein A1 (PBP A1), and influences its activity. PBP A1 catalyzes the last two steps of cell wall biosynthesis, but it is unknown how GpsB controls PBP A1. We observed that a L. monocytogenes gpsB mutant forms spontaneous suppressors and have mapped their mutations to genes mediating and influencing the first step of cell wall biosynthesis, likely stimulating the influx of metabolites into this pathway. We assume that GpsB is important to ensure productive incorporation of cell wall precursors into the peptidoglycan sacculus by PBP A1.

Application of a Bacillus subtilis Whole-Cell Biosensor (PliaI-lux) for the Identification of Cell Wall Active Antibacterial Compounds.

Whole-cell biosensors, based on the visualization of a reporter strain's response to a particular stimulus, are a robust and cost-effective means to monitor defined environmental conditions or the presence of chemical compounds. One specific field in which such biosensors are frequently applied is drug discovery, i.e., the screening of large numbers of bacterial or fungal strains for the production of antimicrobial compounds. We here describe the application of a luminescence-based Bacillus subtilis biosensor for the discovery of cell wall active substances. The system is based on the well-characterized promoter P liaI , which is induced in response to a wide range of conditions that cause cell envelope stress, particularly antibiotics that interfere with the membrane-anchored steps of cell wall biosynthesis. A simple "spot-on-lawn" assay, where colonies of potential producer strains are grown directly on a lawn of the reporter strain, allows for quantitative and time-resolved detection of antimicrobial compounds. Due to the very low technical demands of this procedure, we expect it to be easily applicable to a large variety of candidate producer strains and growth conditions.

Subcellular localization, interactions and dynamics of the phage‐shock protein‐like Lia response in Bacillus subtilis

The liaIH operon of Bacillus subtilis is the main target of the envelope stress-inducible two-component system LiaRS. Here, we studied the localization, interaction and cellular dynamics of Lia proteins to gain insights into the physiological role of the Lia response. We demonstrate that LiaI serves as the membrane anchor for the phage-shock protein A homologue LiaH. Under non-inducing conditions, LiaI locates in highly motile membrane-associated foci, while LiaH is dispersed throughout the cytoplasm. Under stress conditions, both proteins are strongly induced and colocalize in numerous distinct static spots at the cytoplasmic membrane. This behaviour is independent of MreB and does also not correlate with the stalling of the cell wall biosynthesis machinery upon antibiotic inhibition. It can be induced by antibiotics that interfere with the membrane-anchored steps of cell wall biosynthesis, while compounds that inhibit the cytoplasmic or extracytoplasmic steps do not trigger this response. Taken together, our data are consistent with a model in which the Lia system scans the cytoplasmic membrane for envelope perturbations. Upon their detection, LiaS activates the cognate response regulator LiaR, which in turn strongly induces the liaIH operon. Simultaneously, LiaI recruits LiaH to the membrane, presumably to protect the envelope and counteract the antibiotic-induced damage.

Cell Envelope Stress Response in Cell Wall-Deficient L-Forms of Bacillus subtilis

L-forms are cell wall-deficient bacteria that can grow and proliferate in osmotically stabilizing media. Recently, a strain of the Gram-positive model bacterium Bacillus subtilis was constructed that allowed controlled switching between rod-shaped wild-type cells and corresponding L-forms. Both states can be stably maintained under suitable culture conditions. Because of the absence of a cell wall, L-forms are known to be insensitive to β-lactam antibiotics, but reports on the susceptibility of L-forms to other antibiotics that interfere with membrane-anchored steps of cell wall biosynthesis are sparse, conflicting, and strongly influenced by strain background and method of L-form generation. Here we investigated the response of B. subtilis to the presence of cell envelope antibiotics, with regard to both antibiotic resistance and the induction of the known LiaRS- and BceRS-dependent cell envelope stress biosensors. Our results show that B. subtilis L-forms are resistant to antibiotics that interfere with the bactoprenol cycle, such as bacitracin, vancomycin, and mersacidin, but are hypersensitive to nisin and daptomycin, which both affect membrane integrity. Moreover, we established a lacZ-based reporter gene assay for L-forms and provide evidence that LiaRS senses its inducers indirectly (damage sensing), while the Bce module detects its inducers directly (drug sensing).

Proteomic response of Bacillus subtilis to lantibiotics reflects differences in interaction with the cytoplasmic membrane.

Mersacidin, gallidermin, and nisin are lantibiotics, antimicrobial peptides containing lanthionine. They show potent antibacterial activity. All three interfere with cell wall biosynthesis by binding lipid II, but they display different levels of interaction with the cytoplasmic membrane. On one end of the spectrum, mersacidin interferes with cell wall biosynthesis by binding lipid II without integrating into bacterial membranes. On the other end of the spectrum, nisin readily integrates into membranes, where it forms large pores. It destroys the membrane potential and causes leakage of nutrients and ions. Gallidermin, in an intermediate position, also readily integrates into membranes. However, pore formation occurs only in some bacteria and depends on membrane composition. In this study, we investigated the impact of nisin, gallidermin, and mersacidin on cell wall integrity, membrane pore formation, and membrane depolarization in Bacillus subtilis. The impact of the lantibiotics on the cell envelope was correlated to the proteomic response they elicit in B. subtilis. By drawing on a proteomic response library, including other envelope-targeting antibiotics such as bacitracin, vancomycin, gramicidin S, or valinomycin, YtrE could be identified as the most reliable marker protein for interfering with membrane-bound steps of cell wall biosynthesis. NadE and PspA were identified as markers for antibiotics interacting with the cytoplasmic membrane.

Human β-Defensin 3 Inhibits Cell Wall Biosynthesis in Staphylococci

Human beta-defensin 3 (hBD3) is a highly charged (+11) cationic host defense peptide, produced by epithelial cells and neutrophils. hBD3 retains antimicrobial activity against a broad range of pathogens, including multiresistant Staphylococcus aureus, even under high-salt conditions. Whereas antimicrobial host defense peptides are assumed to act by permeabilizing cell membranes, the transcriptional response pattern of hBD3-treated staphylococcal cells resembled that of vancomycin-treated cells (V. Sass, U. Pag, A. Tossi, G. Bierbaum, and H. G. Sahl, Int. J. Med. Microbiol. 298:619-633, 2008) and suggested that inhibition of cell wall biosynthesis is a major component of the killing process. hBD3-treated cells, inspected by transmission electron microscopy, showed localized protrusions of cytoplasmic contents, and analysis of the intracellular pool of nucleotide-activated cell wall precursors demonstrated accumulation of the final soluble precursor, UDP-MurNAc-pentapeptide. Accumulation is typically induced by antibiotics that inhibit membrane-bound steps of cell wall biosynthesis and also demonstrates that hBD3 does not impair the biosynthetic capacity of cells and does not cause gross leakage of small cytoplasmic compounds. In in vitro assays of individual membrane-associated cell wall biosynthesis reactions (MraY, MurG, FemX, and penicillin-binding protein 2 [PBP2]), hBD3 inhibited those enzymes which use the bactoprenol-bound cell wall building block lipid II as a substrate; quantitative analysis suggested that hBD3 may stoichiometrically bind to lipid II. We report that binding of hBD3 to defined, lipid II-rich sites of cell wall biosynthesis may lead to perturbation of the biosynthesis machinery, resulting in localized lesions in the cell wall as demonstrated by electron microscopy. The lesions may then allow for osmotic rupture of cells when defensins are tested under low-salt conditions.

Reconstruction of the Phenotypes of Methicillin-Resistant Staphylococcus aureu s by Replacement of the Staphylococcal Cassette Chromosome mec with a Plasmid-Borne Copy of Staphylococcus sciuri pbpD Gene

The mecA gene, the central determinant of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA), is not native to this bacterial species but may have originated in the animal commensal species Staphylococcus sciuri. All S. sciuri strains carry a close homologue of mecA in the form of pbpD, the genetic determinant of penicillin binding protein 4 (PBP 4) of S. sciuri. Here we describe an experimental system that could be used for additional tests for this proposition. The S. sciuri pbpD gene was cloned into a shuttle plasmid and introduced into methicillin-susceptible S. aureus strain COL-S derived from parental MRSA strain COL from which the resistance cassette staphylococcal cassette chromosome mec was excised. The S. sciuri pbpD determinant was transcribed and translated in the S. aureus transductants producing large amounts of the 84-kDa S. sciuri PBP 4 and was then deposited in the plasma membrane of the host bacterium. Transductants carrying the heterologous S. sciuri pbpD gene exhibited properties typical of those of parental MRSA strain COL, including broad-spectrum, high-level, and homogeneous resistance to structurally different beta-lactams. Antibiotic resistance was dependent on the functioning of S. aureus PBP 2 and was suppressed by the specific regulatory genes mecI and mecR and by inhibitors of an early step in cell wall biosynthesis. S. sciuri PBP 4 was also able to replace the essential physiological function(s) of the native PBP 2 of S. aureus and produce peptidoglycan typical of that of parental MRSA strain COL. Our results provide further support for the proposition that the resistance determinant mecA of MRSA strains has evolved from S. sciuri pbpD.

Use of Structure-Based Drug Design Approaches to Obtain Novel Anthranilic Acid Acyl Carrier Protein Synthase Inhibitors

Acyl carrier protein synthase (AcpS) catalyzes the transfer of the 4'-phosphopantetheinyl group from the coenzyme A to a serine residue in acyl carrier protein (ACP), thereby activating ACP, an important step in cell wall biosynthesis. The structure-based design of novel anthranilic acid inhibitors of AcpS, a potential antibacterial target, is presented. An initial high-throughput screening lead and numerous analogues were modeled into the available AcpS X-ray structure, opportunities for synthetic modification were identified, and an iterative process of synthetic modification, X-ray complex structure determination with AcpS, biological testing, and further modeling ultimately led to potent inhibitors of the enzyme. Four X-ray complex structures of representative anthranilic acid ligands bound to AcpS are described in detail.

Beta-lactams as versatile synthetic intermediates for the preparation of heterocycles of biological interest.

Since the advent of penicillin, the beta-lactam antibiotics have been the subject of much discussion and investigation, within both the scientific and public sectors. The primary biological targets of the beta-lactam antibacterial drugs are the penicillin binding proteins, a group of transpeptidases anchored within the bacterial cellular membrane, which mediate the final step of cell wall biosynthesis. The extensive use of common beta-lactam antibiotics such as penicillins and cephalosporins in medicine has resulted in an increasing number of resistant strains of bacteria through mutation and beta-lactamase gene transfer. Thus, a handful of nonconventional fused polycyclic beta-lactams have been described in the literature in order to overcome the defence mechanisms of the bacteria. In fact, tricyclic beta-lactam antibiotics, generally referred to as trinems, are a new class of synthetic antibacterial agents featuring good resistance to beta-lactamases and dehydropeptidases. In addition, recent discoveries have shown other biological properties of these compounds apart from their antibacterial action. In this sense, beta-lactams can serve as inhibitors of serine proteases, such as human leukocyte elastase (HLE) or thrombin, acyl-CoA cholesterol acyltransferase inhibitors and inhibitors of human cytomegalovirus. Additional impetus for research efforts on beta-lactam chemistry has been provided by the introduction of the beta-lactam synthon method, a term coined by Ojima 20 years ago, according to which 2-azetidinones can be employed as useful intermediates in organic synthesis. The usefulness of beta-lactams in the stereocontrolled synthesis of heterocycles of biological significance is based on the impressive variety of transformations, which can be derived from this system, due inter alia to a high chirality content that can be transferred into a variety of products. The cyclic 2-azetidinone skeleton has been extensively used as a template on which to build the heterocyclic structure fused to the four-membered ring, using the chirality and functionalisation of the beta-lactam nucleus as a stereocontrolling element. Alternatively, the direct one-pot generation of fused nitrogen heterocyclic systems from the nitrogen framework of 2-azetidinone derivatives has been achieved by selective bond breakage and rearrangement. It is our aim in this Review to highlight the state of the art in this endeavour, consisting either of the stereocontrolled synthesis of fused polycyclic beta-lactams of antibacterial interest, or stereoselective synthesis of different sized heterocycles of biological significance. Representative examples of the latter include indolizidines, pyrrolizidines, pirrolidines, pyrroles, taxoids and macrolide natural products.

Development of a whole-cell assay for peptidoglycan biosynthesis inhibitors.

Osmotically stabilized Escherichia coli cells subjected to freezing and thawing were utilized as the source of enzymes for a peptidoglycan pathway assay that can be used to simultaneously test all targets of the committed steps of cell wall biosynthesis. The use of (14)C-labeled UDP-N-acetylglucosamine (UDP-GlcNAc) as a substrate allows the direct detection of cross-linked peptidoglycan formed. The assay was validated with known antibiotics. Fosfomycin was the strongest inhibitor of the pathway assay, with a 50% inhibitory concentration of 1 microM. Flavomycin, bacitracin, vancomycin, D-cycloserine, penicillin G, and ampicillin also inhibited formation of radiolabeled peptidoglycan by the E. coli cells. Screening of compounds identified two inhibitors of the pathway, Cpd1 and Cpd2. Subsequent tests with a biochemical assay utilizing purified enzyme implicated UDP-GlcNAc enolpyruvyl transferase (MurA) as the target of Cpd1. This compound inhibits the first enzyme of the pathway in a time-dependent manner. Moreover, enzyme inactivation is dependent on preincubation in the presence of UDP-GlcNAc, which forms a complex with MurA, exposing its active site. Cpd1 also displayed antimicrobial activity against a panel of microorganisms. The pathway assay used in conjunction with assays for individual enzymes provides an efficient means of detecting and characterizing novel antimicrobial agents.

A 1.2-A snapshot of the final step of bacterial cell wall biosynthesis.

The cell wall imparts structural strength and shape to bacteria. It is made up of polymeric glycan chains with peptide branches that are cross-linked to form the cell wall. The cross-linking reaction, catalyzed by transpeptidases, is the last step in cell wall biosynthesis. These enzymes are members of the family of penicillin-binding proteins, the targets of beta-lactam antibiotics. We report herein the structure of a penicillin-binding protein complexed with a cephalosporin designed to probe the mechanism of the cross-linking reaction catalyzed by transpeptidases. The 1.2-A resolution x-ray structure of this cephalosporin bound to the active site of the bifunctional serine type D-alanyl-D-alanine carboxypeptidase/transpeptidase (EC ) from Streptomyces sp. strain R61 reveals how the two peptide strands from the polymeric substrates are sequestered in the active site of a transpeptidase. The structure of this complex provides a snapshot of the enzyme and the bound cell wall components poised for the final and critical cross-linking step of cell wall biosynthesis.

Vancomycin derivatives that inhibit peptidoglycan biosynthesis without binding D-Ala-D-Ala.

Vancomycin is an important drug for the treatment of Gram-positive bacterial infections. Resistance to vancomycin has begun to appear, posing a serious public health threat. Vancomycin analogs containing modified carbohydrates are very active against resistant microorganisms. Results presented here show that these carbohydrate derivatives operate by a different mechanism than vancomycin; moreover, peptide binding is not required for activity. It is proposed that carbohydrate-modified vancomycin compounds are effective against resistant bacteria because they interact directly with bacterial proteins involved in the transglycosylation step of cell wall biosynthesis. These results suggest new strategies for designing glycopeptide antibiotics that overcome bacterial resistance.

Kinetic and crystallographic studies of Escherichia coli UDP-N-acetylmuramate:L-alanine ligase.

Uridine diphosphate-N-acetylmuramate:L-alanine ligase (EC 6.3.2.8, UNAM:L-Ala ligase or MurC gene product) catalyzes the ATP-dependent ligation of the first amino acid to the sugar moiety of the peptidoglycan precursor. This is an essential step in cell wall biosynthesis for both gram-positive and gram-negative bacteria. Optimal assay conditions for initial velocity studies have been established. Steady-state assays were carried out to determine the effect of various parameters on enzyme activity. Factors studies included: cation specificity, ionic strength, buffer composition and pH. At 37 degrees C and pH 8.0, kcat was equal to 980 +/- 40 min-1, while K(m) values for ATP, UNAM, and L-alanine were, 130 +/- 10, 44 +/- 3, and 48 +/- 6 microM, respectively. Of the metals tested only Mn, Mg, and Co were able to support activity. Sodium chloride, potassium chloride, ammonium chloride, and ammonium sulfate had no effect on activity up to 75 mM levels. The enzyme, in appropriate buffer, was stable enough to be assayed over the pH range of 5.6 to 10.1. pH profiles of Vmax/K(m) for the three substrates and of Vmax were obtained. Crystallization experiments with the enzyme produced two crystal forms. One of these has been characterized by X-ray diffraction as monoclinic, space group C2, with cell dimensions a = 189.6, b = 92.1, c = 75.2 A, beta = 105 degrees, and two 54 kDa molecules per asymmetric unit. It was discovered that the enzyme will hydrolyze ATP in the absence of L-alanine. This L-alanine independent activity is dependent upon the concentrations of both ATP and UNAM; kcat for this activity is less than 4% of the biosynthetic activity measured in the presence of saturating levels of L-alanine. Numerous L-alanine analogs tested were shown to stimulate ATP hydrolysis. A number of these L-alanine analogs produced novel products as accessed by HPLC and mass spectral analysis. All of the L-alanine analogs tested as inhibitors were competitive versus L-alanine.

The mechanism of action of penicillin. Penicillin acylates the active site of Bacillus stearothermophilus D-alanine carboxypeptidase.

Penicillin kills susceptible bacteria by specifically inhibiting the transpeptidase that catalyzes the final step in cell wall biosynthesis, the cross-linking of peptidoglycan. It was hypothesized (Tipper, D., and Strominger, J. (1965) Proc. Natl. Acad. Sci. U.S.A. 54, 1133-1141) that 1) penicillin is a structural analog of the acyl-D-alanyl-D-alanine terminus of the pentapeptide side chains of nascent peptidoglycan, and that 2) penicillin, by virtue of its highly reactive beta-lactam structure, irreversibly acylates the active site of the cell wall transpeptidase. Although the cell wall transpeptidase has proven elusive, a closely related penicillin-sensitive cell wall enzyme, D-alanine carboxypeptidase, has been purified from membranes of Bacillus stearothermophilus by penicillin affinity chromatography. By amino acid sequence analysis of 14C-labeled cyanogen bromide peptides generated and purified from this carboxypeptidase covalently labeled with either [14C]penicillin G or the substrate, [14C]diacetyl-L-lysyl-D-alanyl-D-lactate, it was shown that the penicillin and substrate were both bound as esters to a serine at residue 36. Therefore, the second hypothesis stated above was proven to be correct for D-alanine carboxypeptidase. Several new methods were developed in the course of this work, including 1) a rapid penicillin-binding assay, 2) use of hydroxylamine to protect peptides against carbamylation during ion exchange chromatography in concentrated urea solutions, and 3) gel filtration chromatography in 70% formic acid, a universal solvent for peptides.