Effects of moenomycin on Escherichia coli

The antibiotic moenomycin is a valuable biochemical tool for studying the metabolism of peptidoglycan and the autolytic system in Escherichia coli, since as a specific inhibitor of peptidoglycan polymerases it can efficiently promote cell lysis. In liquid media the bacteriolytic effect on E. coli K12 was dependent on the concentration of moenomycin, on growth phase and on growth rate. Before lysis cells underwent major morphological alterations. In sucrose-containing medium complete transformation to osmotically sensitive spheroplasts was easily achieved by addition of moenomycin. The minimum inhibitory concentration of the antibiotic varied with the strain of E. coli and was highly dependent on the growth medium. A tritiated derivative of moenomycin, [3H]decahydromoenomycin A, was prepared and found to have the same inhibiting efficiency. Its binding to E. coli membranes and membrane proteins was investigated. The absence of irreversible binding suggested that moenomycin might be a competitive inhibitor of the peptidoglycan polymerases. Spontaneous moenomycin resistant variants were isolated at a frequency of about 10(-9).     

Induction of vancomycin resistance in Enterococcus faecium by non-glycopeptide antibiotics

Abstract Bacitracin and other antibiotics that inhibit late stages in peptidoglycan biosynthesis induce vancomycin resistance in a high-level, inducibly vancomycin-resistant strain of Enterococcus faecium . Exposure to bacitracin led to synthesis of the lactate-containing UDP-MurNAc-pentadepsipeptide precursor required for vancomycin resistance. These findings indicate that inhibition of peptidoglycan biosynthesis can lead to induction of vancomycin resistance and raise the possibility that multiple signals may serve to induce resistance.     

The role of the novel Fem protein VanK in vancomycin resistance in Streptomyces coelicolor

The non-pathogenic, non-glycopeptide-producing actinomycete Streptomyces coelicolor carries a cluster of seven genes (vanSRJKHAX) that confers inducible, high level resistance to vancomycin. The vanK gene has no counterpart in previously characterized vancomycin resistance clusters, yet vanK is required for vancomycin resistance in S. coelicolor. VanK belongs to the Fem family of enzymes, which add the branch amino acid(s) to the stem pentapeptide of peptidoglycan precursors. Upon exposure to vancomycin, the VanRS two-component system switches on expression of all seven van genes, and the VanHAX enzymes reprogram the cell wall such that precursors terminate D-Ala-D-lactate (Lac) rather than D-Ala-D-Ala, thus conferring resistance to vancomycin, which only binds D-Ala-D-Ala-containing precursors. Here we provide biochemical and genetic evidence that VanK is required for vancomycin resistance because the constitutively expressed FemX enzyme, encoded elsewhere on the chromosome, cannot recognize D-Lac-containing precursors as a substrate, whereas VanK can. Consistent with this view, D-Lac-containing precursors carrying the Gly branch are present in the wild type transiently exposed to vancomycin but are undetectable in a vanK mutant treated in the same way. Further, femX null mutants are viable in the presence of vancomycin but die in its absence. Because only VanK can recognize D-Lac-containing precursors, vancomycin-induced expression of VanHAX in a vanK mutant is lethal, and so vanK is required for vancomycin resistance.     

Peptidoglycan precursor pools associated with MraY and FtsW deficiencies or antibiotic treatments

Blocking peptidoglycan synthesis in Escherichia coli with moenomycin or vancomycin led to the accumulation of UDP-MurNAc-pentapeptide and of its immediate upstream precursors, whereas with cephaloridine or penicillin G the pool of UDP-MurNAc-pentapeptide decreased. With MraY and FtsW deficiencies the decrease of UDP-MurNAc-pentapeptide was accompanied by an increase of the upstream nucleotide precursors and the appearance of UDP-MurNAc-tetrapeptide.     

In vivo and in vitro action of new antibiotics interfering with the utilization of N-acetyl-glucosamine-N-acetyl-muramyl-pentapeptide

Recent literature on the antibiotics enduracidin, moenomycin, prasinomycin, and 11.837 RP suggested an interaction with murein synthesis. Incubation of sensitive strains from Bacillus cereus and Staphylococcus aureus in a "wall medium" containing labeled l-alanine showed that all four antibiotics inhibited the incorporation of alanine into murein and gave rise to accumulation of radioactive uridine diphosphate-N-acetyl-muramyl (UDP-MurNAc)-pentapeptide. Peptidoglycan was synthesized when the particulate enzyme of B. stearothermophilus was incubated with the murein precursors UDP-N-acetyl-glucosamine (UDP-GlcNAc) and UDP-MurNAc-pentapeptide. The newly formed polymer was less accessible for lysozyme and more strongly bound to the acceptor than the same product from the Escherichia coli particulate enzyme. After incubation in the presence of penicillin, a greater part of the peptidoglycan was lysozyme sensitive and more loosely bound to the acceptor. The antibiotics enduracidin, moenomycin, prasinomycin, and 11.837 RP inhibited peptidoglycan synthesis by the B. stearothermophilus particulate enzyme. The rate of synthesis of GlcNAc-MurNAc(-pentapeptide)-P-P-phospholipid was independent from the addition of these antibiotics, but its utilization was strongly inhibited. With the present results, it is not possible to distinguish the mechanisms of action of enduracidin, moenomycin, prasinomycin, and 11.837 RP from the mechanisms of action of vancomycin and ristocetin.     

Synthesis of peptidoglycan by high molecular weight penicillin-binding proteins of Bacillus subtilis and Bacillus stearothermophilus

The high molecular weight penicillin-binding proteins (PBP(s) ) Bacillus subtilis PBPs 1, 2, and 4 and Bacillus stearothermophilus PBPs 1-4 were shown to catalyze peptidoglycan synthesis from the undecaprenol-containing lipid intermediate substrate in two assay systems. In a filter paper assay system, high levels of substrate polymerization occurred when reaction mixtures were incubated on Whatman 3MM filter paper. The pH optimum for peptidoglycan synthesis was 7.5 for B. subtilis PBPs 1, 2, and 4 and 8.5 for B. stearothermophilus PBPs 1-4. Polymerization was Mg2+-independent and was unaffected by sulfhydryl reagents. Reconstitution with membrane lipids or addition of detergent (optimal concentration, 0.1%) was necessary for synthesis to occur. Bacitracin, penicillin, and cephalothin did not affect polymerization while vancomycin, ristocetin, moenomycin, and macarbomycin were strong inhibitors. In a test tube assay system, optimal synthesis occurred either in the presence of 10% ethylene glycol, 10% glycerol, and 8% methanol or in the presence of 10% N-acetylglucosamine. The products of lysozyme digestion of the synthesized peptidoglycan were analyzed by gel filtration and paper chromatography. B. stearothermophilus PBPs 1-4 synthesized a peptidoglycan product that was 5-7% cross-linked. No evidence for cross-linking was apparent in the peptidoglycan product of B. subtilis PBPs 1, 2, and 4.     

The vancomycin resistance VanRS two-component signal transduction system of Streptomyces coelicolor

We took advantage of the vancomycin-dependent phenotype of Streptomyces coelicolor femX null mutants to isolate a collection of spontaneous, drug-independent femX suppressor mutants that expressed the vancomycin-resistance (van) genes constitutively. All of the suppressor mutations were in vanS but, unexpectedly, many were predicted to be loss-of-function mutations. Confirming this interpretation, a constructed vanS deletion mutation also resulted in constitutive expression of the van genes, suggesting that VanS negatively regulated VanR function in the absence of drug. In contrast, a vanS pta ackA triple mutant, which should not be able synthesize acetyl phosphate, failed to express the van genes, whereas a pta ackA double mutant showed wild-type, regulated induction of the van genes. These results suggest that in the absence of vancomycin, acetyl phosphate phosphorylates VanR, and VanS acts as a phosphatase to suppress the levels of VanR approximately P. On exposure to vancomycin, VanS activity switches from a phosphatase to a kinase and vancomycin resistance is induced. In S. coelicolor, the van genes are induced by both vancomycin and the glycopeptide A47934, whereas in Streptomyces toyocaensis (the A47934 producer) resistance is induced by A47934 but not by vancomycin. We exploited this distinction to replace the S. coelicolor vanRS genes with the vanRS genes from S. toyocaensis. The resulting strain acquired the inducer profile of S. toyocaensis, providing circumstantial evidence that the VanS effector ligand is the drug itself, and not an intermediate in cell wall biosynthesis that accumulates as result of drug action. Consistent with this suggestion, we found that non-glycopeptide inhibitors of the late steps in cell wall biosynthesis such as moenomycin A, bacitracin and ramoplanin were not inducers of the S. coelicolor VanRS system, in contrast to results obtained in enterococcal VanRS systems.     

In situ assay for identifying inhibitors of bacterial transglycosylase

An in situ transglycosylase assay has been developed using endogenously synthesized lipid II. The assay involves the preferential synthesis and accumulation of lipid II in a reaction mixture containing the cell wall membrane material isolated from Escherichia coli, exogenously supplied UDP-MurNAc-pentapeptide, and radiolabeled UDP-GlcNAc. In the presence of Triton X-100, the radiolabeled product formed is almost exclusively lipid II, while the subsequent formation of peptidoglycan is inhibited. Removal of the detergent resulted in the synthesis of peptidoglycan (25% incorporation of radiolabeled material) from the accumulated lipid II. This reaction was inhibited by moenomycin, a known transglycosylase inhibitor. In addition, tunicamycin, which affects an earlier step of the pathway by inhibiting MraY, had no effect on the formation of peptidoglycan in this assay, as expected. Similarly, ampicillin and bacitracin did not inhibit the formation of peptidoglycan under the conditions established.     

Properties of cell wall peptidoglycan synthesized by amino acid deprived re1A mutants of Escherichia coli

Cell wall peptidoglycan synthesis in Escherichia coli is under stringent control. During amino acid deprivation, peptidoglycan synthesis is inhibited in re1A+ bacteria but not in re1A mutants. The relaxed synthesis of peptidoglycan by amino acid deprived re1A bacteria was inhibited by several beta-lactam antibiotics at concentrations which inhibited cell elongation in growing cultures suggesting that the transpeptidase activity of penicillin-binding protein (PBP-1B) was involved in this process. Structural studies on the peptidoglycan also indicated the involvement of transpeptidation in relaxed peptidoglycan synthesis. The peptidoglycan synthesized during amino acid deprivation was cross-linked to the existing cell wall peptidoglycan, and the degree of cross-linkage was the same as that of peptidoglycan synthesized by growing control cells. The relaxed synthesis of peptidoglycan was also inhibited by moenomycin, an inhibitor of the in vitro transglycosylase activities of PBPs, but the interpretation of this result depends on whether the transglycosylases are the sole targets of moenomycin in vivo. Most of the peptidoglycan lipoprotein synthesized by histidine-deprived re1A+ bacteria was in the free form as previously reported, possibly because of the restriction in peptidoglycan synthesis. In support of this proposal, most of the lipoprotein synthesized during histidine deprivation of re1A mutants was found to be covalently linked to peptidoglycan. Nevertheless, the peptidoglycan synthesized by amino acid deprived re1A bacteria was apparently deficient in bound lipoprotein as compared with peptidoglycan synthesized by normal growing control bacteria suggesting that the rate of lipoprotein synthesis during amino acid deprivation may be limiting.     

Anchor structure of staphylococcal surface proteins. IV. Inhibitors of the cell wall sorting reaction.

Surface proteins of Staphylococcus aureus are covalently linked to the bacterial cell wall by a mechanism requiring a COOH-terminal sorting signal with a conserved LPXTG motif. Cleavage between the threonine and the glycine of the LPXTG motif liberates the carboxyl of threonine to form an amide bond with the amino of the pentaglycine cross-bridge in the staphylococcal peptidoglycan. We asked whether antibiotic cell wall synthesis inhibitors interfere with the anchoring of surface proteins. Penicillin G, a transpeptidation inhibitor, had no effect on surface protein anchoring, whereas vancomycin and moenomycin, inhibitors of cell wall polymerization into peptidoglycan strands, slowed the sorting reaction. Cleavage of surface protein precursors did not require a mature assembled cell wall and was observed in staphylococcal protoplasts. A search for chemical inhibitors of the sorting reaction identified methanethiosulfonates and p-hydroxymercuribenzoic acid. Thus, sortase, the enzyme proposed to cleave surface proteins at the LPXTG motif, appears to be a sulfhydryl-containing enzyme that utilizes peptidoglycan precursors but not an assembled cell wall as a substrate for the anchoring of surface protein.     

A vancomycin-inducible lacZ reporter system in Bacillus subtilis: induction by antibiotics that inhibit cell wall synthesis and by lysozyme.

We have constructed a Bacillus subtilis strain in which expression of a vanH::lacZ gene fusion is regulated by VanR and VanS of Enterococcus faecium. This construct allows a nonpathogenic bacterial strain to be used as a model system for studying regulation of vancomycin resistance. Antibiotics and enzymes that affect cell wall biosynthesis and stability were tested for the ability to induce lacZ expression. As a result, fosfomycin and D-cycloserine were added to the group of peptidoglycan synthesis inhibitors shown to induce expression from the vanH promoter. Induction by cell wall hydrolytic enzymes, as well as by antibiotics whose actions may lead to the accumulation of chemically different peptidoglycan precursors, raises the possibility that models that postulate induction by peptidoglycan [correction of peptidodoglycan] precursors are wrong.     

The functional vanGCd cluster of Clostridium difficile does not confer vancomycin resistance

vanG_Cd, a cryptic gene cluster highly homologous to the vanG gene cluster of Enterococcus faecalis is largely spread in Clostridium difficile. Since emergence of vancomycin resistance would have dramatic clinical consequences, we have evaluated the capacity of the vanG_Cd cluster to confer resistance. We showed that expression of vanG_Cd is inducible by vancomycin and that VanG_Cd, VanXY_Cd and VanT_Cd are functional, exhibiting D‐Ala : D‐Ser ligase, D,D‐dipeptidase and D‐Ser racemase activities respectively. In other bacteria, these enzymes are sufficient to promote vancomycin resistance. Trans‐complementation of C. difficile with the vanC resistance operon of Enterococcus gallinarum faintly impacted the MIC of vancomycin, but did not promote vancomycin resistance in C. difficile. Sublethal concentration of vancomycin led to production of UDP‐MurNAc‐pentapeptide[D‐Ser], suggesting that the vanG_Cd gene cluster is able to modify the peptidoglycan precursors. Our results indicated amidation of UDP‐MurNAc‐tetrapeptide, UDP‐MurNAc‐pentapeptide[D‐Ala] and UDP‐MurNAc‐pentapeptide[D‐Ser]. This modification is passed on the mature peptidoglycan where a muropeptide Tetra‐Tetra is amidated on the meso‐diaminopimelic acid. Taken together, our results suggest that the vanG_Cd gene cluster is functional and is prevented from promoting vancomycin resistance in C. difficile.     

Functional characterization of the glycosyltransferase domain of penicillin-binding protein 1a from Thermotoga maritima

Class A penicillin-binding proteins (A-PBPs) are high-molecular weight membrane-bound bifunctional enzymes that catalyze the penicillin-sensitive transpeptidation and transglycosylation reaction steps involved in peptidoglycan assembling. We have over-expressed and characterized a soluble form of the glycosyltransferase domain of PBP1a (GT-PBP1a*) from the hyperthermophilic bacteria Thermotoga maritima. GT-PBP1a* efficiently catalyses peptidoglycan biosynthesis, as shown using an in vitro biosynthetized dansylated-lipid II substrate and a HPLC-coupled assay, and is specifically inhibited by moenomycin. GT-PBP1a* tends to spontaneously aggregate in detergent-free solution, a feature that supports existence of a secondary site for membrane association, distinct from the N-terminal transmembrane anchoring region. Overall, our preliminary data document the biochemical properties of GT-PBP1a* and should guide further studies aimed at deciphering the structural determinants involved into membrane binding by this class of enzymes.     

Peptidoglycan synthesis in the absence of class A penicillin-binding proteins in Bacillus subtilis

Penicillin-binding proteins (PBPs) catalyze the final, essential reactions of peptidoglycan synthesis. Three classes of PBPs catalyze either trans-, endo-, or carboxypeptidase activities on the peptidoglycan peptide side chains. Only the class A high-molecular-weight PBPs have clearly demonstrated glycosyltransferase activities that polymerize the glycan strands, and in some species these proteins have been shown to be essential. The Bacillus subtilis genome sequence contains four genes encoding class A PBPs and no other genes with similarity to their glycosyltransferase domain. A strain lacking all four class A PBPs has been constructed and produces a peptidoglycan wall with only small structural differences from that of the wild type. The growth rate of the quadruple mutant is much lower than those of strains lacking only three of the class A PBPs, and increases in cell length and frequencies of wall abnormalities were noticeable. The viability and wall production of the quadruple-mutant strain indicate that a novel enzyme can perform the glycosyltransferase activity required for peptidoglycan synthesis. This activity was demonstrated in vitro and shown to be sensitive to the glycosyltransferase inhibitor moenomycin. In contrast, the quadruple-mutant strain was resistant to moenomycin in vivo. Exposure of the wild-type strain to moenomycin resulted in production of a phenotype similar to that of the quadruple mutant.     

Moenomycin-resistance is associated with vancomycin-intermediate susceptibility in Staphylococcus aureus

We have previously isolated a vancomycin-intermediate susceptibility mutant from methicillinresistant Staphylococcus aureus(MRSA) strain COL, and demonstrated the increased glycan-chain length and the decreased moenomycin-susceptibility. To further investigate the relationship between the resistance to vancomycin and to moenomycin, we isolated moenomycin-resistant mutants (4-16 fold higher compared to the parent) from 5 MRSA and 2 methicillin-sensitive S. aureus(MSSA) strains. The MRSA mutants showed a decreased susceptibility to vancomycin (2-4 fold), teicoplanin (2-4 fold) and an increased susceptibility to methicillin (2-8 fold). MSSA strains also showed similar results with those of MRSA strains except that there was no alteration of methicillin susceptibility. Among the mutants, three mutants including two MRSA mutants and one MSSA mutant were analyzed by electron microscopy, and they showed thickened cell walls compared to those of the parents. The glycan-chain length of the peptidoglycan of the mutant was shown to be slightly longer than that of the parent, but the muropeptide profile was very similar. The expression levels of all PBPs were similar to those of the parent. Furthermore, the nucleotide sequences of sgtA, sgtB and pbp2 in the mutant were identical to those of the parent. These results indicate that the moenomycin-resistance is closely associated with vancomycin-intermediate susceptibility in S. aureus.     

Molecular mechanisms of vancomycin resistance

Vancomycin and related glycopeptides are drugs of last resort for the treatment of severe infections caused by Gram‐positive bacteria such as Enterococcus species, Staphylococcus aureus, and Clostridium difficile. Vancomycin was long considered immune to resistance due to its bactericidal activity based on binding to the bacterial cell envelope rather than to a protein target as is the case for most antibiotics. However, two types of complex resistance mechanisms, each comprised of a multi‐enzyme pathway, emerged and are now widely disseminated in pathogenic species, thus threatening the clinical efficiency of vancomycin. Vancomycin forms an intricate network of hydrogen bonds with the d ‐Ala‐d ‐Ala region of Lipid II, interfering with the peptidoglycan layer maturation process. Resistance to vancomycin involves degradation of this natural precursor and its replacement with d ‐Ala‐d ‐lac or d ‐Ala‐d ‐Ser alternatives to which vancomycin has low affinity. Through extensive research over 30 years after the initial discovery of vancomycin resistance, remarkable progress has been made in molecular understanding of the enzymatic cascades responsible. Progress has been driven by structural studies of the key components of the resistance mechanisms which provided important molecular understanding such as, for example, the ability of this cascade to discriminate between vancomycin sensitive and resistant peptidoglycan precursors. Important structural insights have been also made into the molecular evolution of vancomycin resistance enzymes. Altogether this molecular data can accelerate inhibitor discovery and optimization efforts to reverse vancomycin resistance. Here, we overview our current understanding of this complex resistance mechanism with a focus on the structural and molecular aspects.