Peptidoglycan Synthesis Enzymes Research Articles

"One-Two Punch": Synergistic ß-Lactam Combinations for Mycobacterium abscessus and Target Redundancy in the Inhibition of Peptidoglycan Synthesis Enzymes.

Mycobacterium abscessus subsp. abscessus is one of the most difficult pathogens to treat and its incidence in disease is increasing. Dual β-lactam combinations act synergistically in vitro but are not widely employed in practice. A recent study shows that a combination of imipenem and ceftaroline significantly lowers the minimum inhibitory concentration of clinical isolates, despite both drugs targeting the same peptidoglycan synthesis enzymes. The underlying mechanism of this effect provides a basis for further investigations of dual β-lactam combinations in the treatment of M. abscessus subsp. abscessus, eventually leading to a clinical trial. Furthermore, dual β-lactam strategies may be explored for other difficult mycobacterial infections.

MreB and MreC act as the geometric moderators of the cell wall synthetic machinery in Thermus thermophiles

How cell morphology is maintained in thermophilic bacteria is unknown. In this study, the functions and mechanisms of the potential cell shape determinants (e.g. MreB, MreC, MreD and RodA homologues) of the model extremely thermophilic bacterium Thermus thermophilus were initially analyzed. Deletion of mreC, mreD or rodA only resulted in heterozygous mutants indicating that these genes are all essential. In the MreB-inhibited (by A22) strain and the heterozygous mreC, mreD or rodA mutant, cell morphologies were drastically changed, and enlarged spherical cells were eventually dead indicating that they are vital for cell shape maintenance. When fused to sGFP, MreB, MreC, MreD, RodA, and the enzymes involved in peptidoglycan synthesis (e.g. PBP2 and MurG) exhibited similar subcellular localization pattern, appearing as patches, or bands slightly angled to the cell length. The localizations and functions of all the 6 proteins required a natural peptidoglycan synthesis pattern, additionally those of MreD, RodA and MurG were dependent on MreB polymerization. Consistently, through comprehensive bacterial two-hybrid analyses, it was revealed that MreB could interact with itself, MreC, MreD, RodA and MurG, and MreC could associate with PBP2. In conclusion, in T. thermophilus, MreB, MreC, MreD, RodA and the peptidoglycan synthesis enzymes probably form a network of interactions centered with MreB and bridged with MreC, thereby maintaining cell morphology.

Allicin causes fragmentation of the peptidoglycan coat in Staphylococcus aureus by effecting synthesis and aiding hydrolysis: a determination by MALDI-TOF mass spectrometry on whole cells.

To determine the effect of allicin on Staphylococcus aureus cell wall peptidoglycans by the application of MALDI-TOF mass spectrometry on whole cells and to relate this to current knowledge of wall-processing enzymes. Two different S. aureus strains were grown for 48 h after which period each culture was split into two, one part was then treated with sub-inhibitory levels of allicin while the other part left untreated as a control. After a further 24 h whole cells were recovered and analysed by MALDI-TOF mass spectrometry. Changes in the mass spectra between the treated and untreated cells revealed fragmented peptidoglycans identified by mass calculation only in the treated cells. These peptidoglycan fragments where identified as the products of specific peptidoglycan hydrolases. Allicin is known to target cysteine thiol groups. These are absent in peptidoglycan hydrolases and we might have expected identical results in both of the treated and untreated cells. Peptidoglycan synthesis enzymes such as the Fem family of enzymes do contain cysteines. Fem enzymes A, B and X all have a conserved conformation of 99 % for over 100 S. aureus strains and are therefore potential targets for allicin. Examination of FemA structure showed that cysteine102 is accessible from the surface. We propose that allicin has an inhibitory mechanism alongside others of targeting FemA and possibly other Fem enzymes by curtailing glycine bridging and leading to fragmentation. This study provided an insight into yet another antimicrobial mechanism of allicin.

RodZ: a key-player in cell elongation and cell division in Escherichia coli.

RodZ is required for determination of cell shape in rod-shaped bacterium, such as Escherichia coli. RodZ is a transmembrane protein and forms a supramolecular complex called the Rod complex with other proteins, such as MreB-actin and peptidoglycan synthesis enzymes (for e.g., PBP2). Deletion of the rodZ gene changes the cell shape from rod to round or ovoid. Another supramolecular complex called divisome that controls cell division mainly consists of FtsZ-tubulin. MreB directly interacts with FtsZ and this interaction is critical to trigger a transition from cell elongation to cell division. Recently, we found that RodZ also directly interacts with FtsZ, and RodZ recruits MreB to the divisome. Formation of the division ring, called Z ring, is delayed if RodZ does not interact with FtsZ, indicating that RodZ might facilitate the formation of the Z ring during the cell division process. In this mini-review, we have summarized the roles of RodZ in cell elongation and cell division, especially based on our recent study.

Site-Specific Immobilization of the Peptidoglycan Synthase PBP1B on a Surface Plasmon Resonance Chip Surface.

Surface plasmon resonance (SPR) is one of the most powerful label‐free methods to determine the kinetic parameters of molecular interactions in real time and in a highly sensitive way. Penicillin‐binding proteins (PBPs) are peptidoglycan synthesis enzymes present in most bacteria. Established protocols to analyze interactions of PBPs by SPR involve immobilization to an ampicillin‐coated chip surface (a β‐lactam antibiotic mimicking its substrate), thereby forming a covalent complex with the PBPs transpeptidase (TP) active site. However, PBP interactions measured with a substrate‐bound TP domain potentially affect interactions near the TPase active site. Furthermore, in vivo PBPs are anchored in the inner membrane by an N‐terminal transmembrane helix, and hence immobilization at the C‐terminal TPase domain gives an orientation contrary to the in vivo situation. We designed a new procedure: immobilization of PBP by copper‐free click chemistry at an azide incorporated in the N terminus. In a proof‐of‐principle study, we immobilized Escherichia coli PBP1B on an SPR chip surface and used this for the analysis of the well‐characterized interaction of PBP1B with LpoB. The site‐specific incorporation of the azide affords control over protein orientation, thereby resulting in a homogeneous immobilization on the chip surface. This method can be used to study topology‐dependent interactions of any (membrane) protein.

Coarse-Grained Simulations Reveal Mechanisms of Bacterial Morphogenesis

How cells maintain their characteristic shapes throughout generations of growth is a fundamental question in bacterial cell biology. In most rod-shaped bacteria, the cell shape is determined by a peptidoglycan (PG) sacculus that surrounds the cell preventing lysis from turgor pressure. Despite decades of experiments, how the activities of PG synthesis enzymes are coordinated at the molecular and cellular levels to preserve the integrity and rod shape of the sacculus remains elusive. To explore different mechanistic models of sacculus growth, we have developed a coarse-grained simulation method that allows us to vary the properties and coordination of PG-remodeling enzymes while mechanical properties of the coarse-grained PG are derived from all-atom MD simulations of isolated PG. (Nguyen et al., PNAS. 112, E3689 (2015)). For the first time to our knowledge, individual enzymes, including transglycosylases, transpeptidases and endopeptidases, are explicitly represented. We have revealed the challenges a cell might face while remodeling its sacculus. For example, lysis can easily occur during bond cleavage because of turgor pressure; aggregation of new PG material can occur if enzymes are not processive; and the regular order and rod shape of the sacculus is easily lost if enzymatic activities are not coordinated. Exploring ways a cell might overcome these challenges, we find that local spatial and temporal coordination of the enzymes alone can be sufficient to maintain rod shape. Our results rationalize several experimental results, for instance explaining why the monofunctional transpeptidase PBP2 is essential and suggesting why different enzymes must exist in a complex that inserts new strands in pairs. Our approach also generates testable biological hypotheses. For instance, we predict a role for a housekeeping glycosidase; while our manuscript was in revision, just such a glycosidase was identified in cells (Cho et al., Cell 159, 1300 (2014)).

Continuous Fluorescence Assay for Peptidoglycan Glycosyltransferases.

Bacterial cell wall peptidoglycan is synthesized from its precursor lipid II by two enzymatic reactions. First, glycosyltransferases polymerize the glycan strands and second, DD-transpeptidases form cross-links between peptides of neighboring strands. Most bacteria possess bifunctional peptidoglycan synthesis enzymes capable of catalyzing both reactions. Here, we describe a continuous fluorescence glycosyltransferase assay using Dansyl-labeled lipid II as substrate. Progression of the reaction is monitored by the reduction in fluorescence over time. The assay is suitable to investigate the effect of protein interaction partners on the glycan strand synthesis activity of peptidoglycan polymerases.

Mutation landscape of acquired cross-resistance to glycopeptide and β-lactam antibiotics in Enterococcus faecium.

Bypass of the d,d-transpeptidase activity of penicillin-binding proteins by an l,d-transpeptidase (Ldtfm) results in resistance to ampicillin and glycopeptides in Enterococcus faecium M9, a mutant obtained by nine consecutive selection steps. Resistance requires activation of a cryptic locus for production of the essential tetrapeptide-containing substrate of Ldtfm and impaired activity of protein phosphatase StpA. Here, whole-genome sequencing revealed a high mutation rate for the entire selection procedure (79 mutations in 900 generations). Acquisition of a mutation in the mismatch repair gene mutL had little impact on the frequency of rifampin-resistant mutants although the mutation spectrum of M9 was typical of impaired MutL with high transversion to transition (40/11) and substitution to deletion (51/28) ratios. M9 did not mainly accumulate neutral mutations since base substitutions occurred more frequently in coding sequences than expected (χ(2) = 5.0; P < 0.05) and silent mutations were underrepresented (χ(2) = 5.72; P < 0.02). None of the mutations directly affected recognition of the tetrapeptide substrate of Ldtfm by peptidoglycan synthesis enzymes. Instead, mutations appear to remodel regulatory circuits involving two-component regulatory systems and sugar metabolism. The high number of mutations required for activation of the l,d-transpeptidase pathway may strongly limit emergence of cross-resistance to ampicillin and glycopeptides by this mechanism.

Staphylococcus aureus Survives with a Minimal Peptidoglycan Synthesis Machine but Sacrifices Virulence and Antibiotic Resistance.

Many important cellular processes are performed by molecular machines, composed of multiple proteins that physically interact to execute biological functions. An example is the bacterial peptidoglycan (PG) synthesis machine, responsible for the synthesis of the main component of the cell wall and the target of many contemporary antibiotics. One approach for the identification of essential components of a cellular machine involves the determination of its minimal protein composition. Staphylococcus aureus is a Gram-positive pathogen, renowned for its resistance to many commonly used antibiotics and prevalence in hospitals. Its genome encodes a low number of proteins with PG synthesis activity (9 proteins), when compared to other model organisms, and is therefore a good model for the study of a minimal PG synthesis machine. We deleted seven of the nine genes encoding PG synthesis enzymes from the S. aureus genome without affecting normal growth or cell morphology, generating a strain capable of PG biosynthesis catalyzed only by two penicillin-binding proteins, PBP1 and the bi-functional PBP2. However, multiple PBPs are important in clinically relevant environments, as bacteria with a minimal PG synthesis machinery became highly susceptible to cell wall-targeting antibiotics, host lytic enzymes and displayed impaired virulence in a Drosophila infection model which is dependent on the presence of specific peptidoglycan receptor proteins, namely PGRP-SA. The fact that S. aureus can grow and divide with only two active PG synthesizing enzymes shows that most of these enzymes are redundant in vitro and identifies the minimal PG synthesis machinery of S. aureus. However a complex molecular machine is important in environments other than in vitro growth as the expendable PG synthesis enzymes play an important role in the pathogenicity and antibiotic resistance of S. aureus.

Serine/threonine protein phosphatase-mediated control of the peptidoglycan cross-linking L,D-transpeptidase pathway in Enterococcus faecium.

ABSTRACTThe last step of peptidoglycan polymerization involves two families of unrelated transpeptidases that are the essential targets of β-lactam antibiotics. d,d-transpeptidases of the penicillin-binding protein (PBP) family are active-site serine enzymes that use pentapeptide precursors and are the main or exclusive cross-linking enzymes in nearly all bacteria. However, peptidoglycan cross-linking is performed mainly by active-site cysteine l,d-transpeptidases that use tetrapeptides in Mycobacterium tuberculosis, Clostridium difficile, and β-lactam-resistant mutants of Enterococcus faecium. We have investigated reprogramming of the E. faecium peptidoglycan assembly pathway by a switch from pentapeptide to tetrapeptide precursors and bypass of PBPs by l,d-transpeptidase Ldtfm. Mutational alterations of two signal transduction systems were necessary and sufficient for activation of the l,d-transpeptidation pathway, which is essentially cryptic in wild-type strains. The first one is a classical two-component regulatory system, DdcRS, that controls the activity of Ldtfm at the substrate level. As previously described, loss of DdcS phosphatase activity leads to production of the d,d-carboxypeptidase DdcY and conversion of the pentapeptide into the tetrapeptide substrate of Ldtfm. Here we show that full bypass of PBPs by Ldtfm also requires increased Ser/Thr protein phosphorylation resulting from impaired activity of phosphoprotein phosphatase StpA. This enzyme negatively controlled the level of protein phosphorylation both by direct dephosphorylation of target proteins and by dephosphorylation of its cognate kinase Stk. In combination with production of DdcY, increased protein phosphorylation by this eukaryotic-enzyme-like Ser/Thr protein kinase was sufficient for activation of the l,d-transpeptidation pathway in the absence of mutational alteration of peptidoglycan synthesis enzymes.

Dynamic FtsA and FtsZ localization and outer membrane alterations during polar growth and cell division in Agrobacterium tumefaciens

Growth and cell division in rod-shaped bacteria have been primarily studied in species that grow predominantly by peptidoglycan (PG) synthesis along the length of the cell. Rhizobiales species, however, predominantly grow by PG synthesis at a single pole. Here we characterize the dynamic localization of several Agrobacterium tumefaciens components during the cell cycle. First, the lipophilic dye FM 4-64 predominantly stains the outer membranes of old poles versus growing poles. In cells about to divide, however, both poles are equally labeled with FM 4-64, but the constriction site is not. Second, the cell-division protein FtsA alternates from unipolar foci in the shortest cells to unipolar and midcell localization in cells of intermediate length, to strictly midcell localization in the longest cells undergoing septation. Third, the cell division protein FtsZ localizes in a cell-cycle pattern similar to, but more complex than, FtsA. Finally, because PG synthesis is spatially and temporally regulated during the cell cycle, we treated cells with sublethal concentrations of carbenicillin (Cb) to assess the role of penicillin-binding proteins in growth and cell division. Cb-treated cells formed midcell circumferential bulges, suggesting that interrupted PG synthesis destabilizes the septum. Midcell bulges contained bands or foci of FtsA-GFP and FtsZ-GFP and no FM 4-64 label, as in untreated cells. There were no abnormal morphologies at the growth poles in Cb-treated cells, suggesting unipolar growth uses Cb-insensitive PG synthesis enzymes.

Scintillation proximity assay for inhibitors of Escherichia coli MurG and, optionally, MraY.

MurG and MraY, essential enzymes involved in the synthesis of bacterial peptidoglycan, are difficult to assay because the substrates are lipidic and hard to prepare in large quantities. Based on the use of Escherichia coli membranes lacking PBP1b, we report a high-throughput method to measure the activity of MurG and, optionally, MraY as well. In these membranes, incubation with the two peptidoglycan sugar precursors results in accumulation of lipid II rather than the peptidoglycan produced by wild-type membranes. MurG was assayed by addition of UDP-[3H]N-acetylglucosamine to membranes in which lipid I was preformed by incubation with UDP-N-acetyl-muramylpentapeptide, and the product was captured by wheat germ agglutinin scintillation proximity assay beads. In a modification of the assay, the activity of MraY was coupled to that of MurG by addition of both sugar precursors together in a single step. This allows simultaneous detection of inhibitors of either enzyme. Both assays could be performed using wild-type membranes by addition of the transglycosylase inhibitor moenomycin. Nisin and vancomycin inhibited the MurG reaction; the MraY-MurG assay was inhibited by tunicamycin as well. Inhibitors of other enzymes of peptidoglycan synthesis--penicillin G, moenomycin, and bacitracin--had no effect. Surprisingly, however, the beta-lactam cephalosporin C inhibited both the MurG and MraY-MurG assays, indicating a secondary mechanism by which this drug inhibits bacterial growth. In addition, it inhibited NADH dehydrogenase in membranes, a hitherto-unreported activity. These assays can be used to screen for novel antibacterial agents.

Development of a Microplate-Based Scintillation Proximity Assay for MraY Using a Modified Substrate

MraY is an established target for the discovery of antibacterial agents. The conventional assay for MraY uses radioactive substrate and analysis of products after paper chromatography or butanol extraction. Synthesis of radiolabeled substrate has been done in vitro using purified enzymes or by growing cells on radiolabeled precursors. The authors report a simple and rapid method to chemically radiolabel MraY substrate, UDP-MurNAc-pentapeptide. Specific activity obtained by this method was more than 100 times higher than the conventionally labeled substrate, and yields are high enough to support the requirements of high-throughput screening (HTS). The authors have developed a microplate-based homogeneous assay for MraY in which the product is captured on wheat germ agglutinin (WGA) scintillation proximity assay (SPA) beads. The assay was validated by showing inhibition by specific inhibitors of MraY but not by inhibitors of other enzymes of peptidoglycan synthesis. The assay uses wild-type membranes of Escherichia coli, giving it an advantage over recently described assays that need the protein to be overexpressed. In addition, it has an advantage over the high-throughput MraY-MurG coupled assay reported in the literature because it is MraY specific, and therefore hits obtained in this assay do not need further deconvolution. It has potential for use in HTS approaches to find novel inhibitors of MraY.

Modification of Peptidoglycan Structure by Penicillin Action in Cell Walls of Proteus mirabilis

Composition of cell wall peptidoglycan was studied in the gram‐negative bacterium Proteus mirabilis and in different wall‐defective growth forms of this organism which survive and multiply in the presence of penicillins. The results show that action of the classical benzylpenicillin does not proceed by a uniform mechanism of inhibition of peptide crosslinkage in peptidoglycan.Peptidoglycan of the unstable spheroplast L‐form of P. mirabilis grown in medium with 120 μg/ml benzylpenicillin contained monomers, and peptide‐crosslinked dimers and trimers of disaccharide‐peptide building blocks in a similar ratio as peptidoglycan of the normal bacterial form. Moreover, peptide side‐chains in peptidoglycan from penicillin‐grown L‐form spheroplasts were predominantly tetrapeptides l‐alanyl‐d‐γ‐glutamyl‐meso‐diaminopimelyl‐d‐alanine. Thus, specific penicillin‐sensitive enzymes of peptidoglycan synthesis, peptidoglycan transpeptidase and dd‐carboxypeptidase, must have remained uninhibited by the antibiotic in the L‐form. L‐form peptidoglycan, however, differed characteristically from peptidoglycan of normal Proteus bacteria by having a much lower content of O‐acetyl groups linked to muramic acid residues of the polymer. P. mirabilis grew actively in the form of osmotically stable spheres in medium with up to 100 μg/ml of the amidino penicillin mecillinam. Neither the peptide crosslinkage nor the O‐acetyl content of peptidoglycan were influenced to any great extent by mecillinam.Only the combined action of benzylpenicillin and mecillinam caused complete inhibition of growth in P. mirabilis, concomitant with deformation of cells to very large spheres. It is hypothesized that benzylpenicillin and mecillinam each inhibit different parts of a multiple, isofunctional enzyme system whose total inhibition requires the presence of both antibiotics.

New antipseudomonal penicillin, PC-904: affinity to penicillin-binding proteins and inhibition of the enzyme cross-linking peptidoglycan.

The mechanism of action of a new antipseudomonal penicillin, PC-904, was studied with respect to its binding affinities to penicillin-binding proteins (PBPs) and its inhibitory activities on cross-linking enzymes of peptidoglycan synthesis in vitro. PC-904 showed especially high affinity (compared with that of penicillin G) to Escherichia coli PBP-3. It also had high affinities to PBP-2 and -1Bs and low affinities to PBP-1A, -4, -5, and -6. Similar results were obtained with Pseudomonas aeruginosa, in which this antibiotic showed very high affinity (compared with that of penicillin G) to PBP-3, -1A (presumably corresponding to E. coli PBP-1Bs), and -2; there was especially high affinity to PBP-3 and much less affinity to PBP-1B (presumably corresponding to E. coli PBP-1A). These results are compatible with morphological observations that at concentrations near its minimal inhibitory concentration or less, this antibiotic induced the formation of filamentous cells of E. coli and P. aeruginosa. At higher concentrations or after prolonged incubation, it induced lysis of the cells. The remarkably high affinity of PC-904 to pseudomonal PBP-3, -1A, and -2 may partly explain the potent antipseudomonal activity of this antibiotic. In E. coli, the concentration of PC-904 required to inhibit the cross-linking reaction in enzymatic peptidoglycan synthesis, presumably carried out by PBP-1Bs, was as low as the inhibitory concentrations of penicillin G, ampicillin, and carbenicillin.

Peptidoglycan synthesis in L-phase variants of Bacillus licheniformis and Bacillus subtilis

Stable L-phase variants isolated from Bacillus licheniformis and Bacillus subtilis, when grown in osmotically stabilized media, do not synthesize peptidoglycan but have been found to accumulate the nucleotide precursors of this polymer. The enzymes involved in the synthesis of these precursors and the later membrane-bound stages of peptidoglycan synthesis have been investigated, and the L-phase variants have been shown to contain lesions, which provide a rational explanation for the absence of peptidoglycan and for the nature of the precursor accumulated. The majority of the L-phase variants contained a single enzymic defect, but two strains were isolated with double lesions. Five out of seven strains examined accumulated uridine 5'-diphosphate (UDP)-MurAc-L-ala-D-glu and were unable to synthesize diaminopimelic acid as a consequence of a defect in aspartate-beta-semialdehyde dehydrogenase activity. Two strains were deficient in UDP-MurAc: L-alanine ligase and accumulated UDP-MurAc. One strain accumulated the complete nucleotide precursor UDP-MurAc-L-ala-D-glu-mA2pm-D-ala-D-ala and was deficient in phospho-N-acetylmuramyl pentapeptide translocase. A second strain also had this lesion, together with defective aspartate-beta-semialdehyde dehydrogenase activity. The other enzymes of peptidoglycan synthesis were present in the L-phase variants, with activities similar to those found in the parent bacilli grown under identical conditions. Membrane preparations from certain of the L-phase variants were also capable of synthesizing the secondary polymers poly(glycerol phosphate) teichoic acid and teichuronic acid and also a polymer of N-acetylglucosamine.

Cell wall peptidoglycan mutants of Escherichia coli K-12: existence of two clusters of genes, mra and mrb, for cell wall peptidoglycan biosynthesis.

Temperature-sensitive mutants of Escherichia coli K-12 which cannot form cell wall peptidoglycan at 42 C were isolated. The thermosensitive steps were characterized biochemically, and the genes coding the enzymes of peptidoglycan synthesis were mapped. These genes were in two clusters: one group, located at about 1.5 min between leu and azi, was designated as mra (murein a); the second group, located at about 77.5 min close to argH and metB, was designated as mrb (murein b, with the order metB-argH-mrb). No simple relations were found between the gene location and the order or localization of enzymes involved in the sequence of reactions of cell wall peptidoglycan biosynthesis.

The cell membrane of mycoplasma.

Most mycoplasmologists support the proposal of Gibbons and Murray (1978) endowing the mycoplasmas with the lofty status of one of the four major divisions in the kindgom Procaryotae, the division named Mollicutes (mollis soft + cutes skin) to denote the lack of cell walls in these organisms. The recent report that penicillin-binding proteins and enzymes of peptidoglycan synthesis are absent from mycoplasmas as against their presence in the plasma membrane of the wall-less bacterial L-forms (Martin et al., 1980) supports the idea of a separate division for mycoplasmas. A different view has recently been expressed by Fox et al. (1980) who, on the basis of nucleotide sequences in 16 S rRNA, argue that mycoplasmas are genealogically wall-less descendents of clostridia, and consequently should not be given a separate high taxonomic status. Notwithstanding this somewhat philosophical controversy, the fact is that mycoplasmas differ from other prokaryotes in several unique properties, most useful in membrane studies. Thus, the mycoplasmas are unique in being the only self-replicating organisms with a single membranous structure—the plasma membrane. This is, perhaps, their greatest advantage in membrane studies, for it facilitates the isolation of pure plasma membranes uncontaminated by other membrane types. Moreover, the mycoplasmas’ lack of cell walls enables the application of gentle and simple techniques, such as osmotic lysis for membrane isolation (Razin, 1978, 1981).