Inhibition Of Peptidoglycan Biosynthesis Research Articles

Inhibition of peptidoglycan biosynthesis in gram-positive bacteria by LY146032.

LY146032, a cyclic lipopeptide antibiotic, is an inhibitor of cell wall peptidoglycan biosynthesis in gram-positive bacteria. Although LY146032 at relatively high concentrations inhibited the in vitro polymerization of UDP-linked sugar precursors, inhibition of cell wall formation in intact Staphylococcus aureus and Bacillus megaterium cells did not lead to the accumulation of UDP-N-acetyl-muramyl (MurNAc)-peptide(s). Experiments that measured formation of UDP-MurNAc-peptides revealed that LY146032 inhibited the formation of these nucleotide-linked intermediates. This antibiotic had a disruptive effect on membrane permeability as evidenced by the loss of intracellular potassium immediately after exposure to the drug. The lack of any major disruption of the phosphoenolpyruvate:sugar phosphotransferase system indicated that the membrane is not likely a lethal target for this antibiotic. The findings are consistent with a mechanism by which LY146032 inhibits the formation of precursor molecules utilized in peptidoglycan biosynthesis. The observed membrane effects likely result from transit of the inhibitor to its lethal target site.

Affinity chromatography of glycopeptide antibiotics

An affinity support was designed to facilitate the isolation and purification of glycopepetide antibiotics by mimicking their known affinity for the bacterial cell wall. Members of th is class of antibiotics inhibit peptidoglycan biosynthesis by specifically binding to pentapeptide precursors terminating with l-Lys— d-Ala— d-Ala. A series of ligands (Gly, d-Ala, d-Ala— d-Ala and α-N-Ac— l-Lys— d— d-Ala) were immobilized on a N-hydroxysuccinimide-activated agarose support and evaluated using the glycopeptides vacomycin and the aridicin complex. Conditions were developed to enable complete adsorption and efficient elution of both antibiotics. Of the four ligands, the readily available dipeptide offered the best compromise between high binding specificity and recovery on elution. Binding and subsequent high recovery of biologically active products were observed for eight other glycopeptide antibiotics. Column performance was shown by purification of vancomycin directly from a low titer fermentation broth. The applicability of this technique to large scale isolation was demonstrated by the preparative affinity chromatography of 36 g of the aridicins.

Involvement of the relA gene in the autolysis of Escherichia coli induced by inhibitors of peptidoglycan biosynthesis.

It is generally assumed that inhibitors of peptidoglycan biosynthesis do not kill nongrowing bacteria. An exceptional case is reported here. The addition of chloramphenicol to amino acid-deprived cultures of relA+ strains of Escherichia coli which were treated with beta-lactam antibiotics, D-cycloserine, or moenomycin resulted in lysis. This phenomenon is termed chloramphenicol-dependent lysis. To be effective, chloramphenicol had to be present at its minimum growth-inhibitory concentration (or higher). Analogs of chloramphenicol which did not bind to ribosomes were completely ineffective. Amino acid deprivation was actually not required to demonstrate chloramphenicol-dependent lysis, and cultures treated with growth-inhibitory levels of chloramphenicol alone were lysed when challenged with inhibitors of peptidoglycan synthesis. Peptidoglycan synthesis has been shown previously to be under stringent (relA+) control, and chloramphenicol is known to be an antagonist of stringent control. Thus, it is proposed that the mechanism of chloramphenicol-dependent lysis is based on the ability of chloramphenicol to relax peptidoglycan synthesis in nongrowing relA+ bacteria. This is also consistent with the observation that treatment of amino acid-deprived relA mutants with inhibitors of peptidoglycan synthesis resulted in lysis, i.e., without the mediation of chloramphenicol.

Chlorocardicin, a monocyclic .BETA.-lactam from a Streptomyces sp. I. Discovery, production and biological activities.

Chlorocardicin is a new monocyclic beta-lactam produced by a Streptomyces sp. It is structurally related to nocardicin A but differs in having a m-chloro substituent on the p-hydroxyphenylglycine unit. The biological activity of chlorocardicin was similar to nocardicin A but the former showed less antagonism in complex media. Moderate in vitro activity was observed against Enterobacteriaceae and Pseudomonas aeruginosa. Chlorocardicin showed low activity against Staphylococcus aureus whereas nocardicin A was inactive. Both compounds were shown to be strongly potentiated by antibiotics that inhibit peptidoglycan biosynthesis and were antagonized by selected L- and D-amino acids.

A diazaborine derivative inhibits lipopolysaccharide biosynthesis.

Inhibitors of the biosynthesis of peptidoglycan, the rigid layer of the bacterial cell wall which is responsible for the shape of the cell, are among the most effective antibacterial agents known. Both Gram-positive and Gram-negative organisms are susceptible to the action of such compounds. As lipopolysaccharide (LPS) is an integral part of the outer membrane of Gram-negative bacteria, it is possible that a blockage of its biosynthesis might result in the arrest of cellular growth and eventually cell death, analogous to the effects seen with inhibitors of peptidoglycan biosynthesis. However, no antibacterial agent has been described which inhibits growth in this way. We present here experimental evidence which indicates that 1,2-dihydro-1-hydroxy-6-methyl-2-(propanesulphonyl)-thieno(3,2-D) (1,2,3)-diazaborine (code no. 84474), a heterocyclic, boron-containing substance, prevents bacterial proliferation by inhibiting LPS biosynthesis. There was a reduction in galactose incorporation into the LPS of whole cells, and no newly formed LPS chains appeared in whole bacteria in the presence of the compound, as demonstrated by electron microscopy. The 2-keto-3-deoxy-octonate metabolism seems to be the target of 84474 as arabinose 5-phosphate incorporation into macro-molecular material was affected.

Inhibition of peptidoglycan biosynthesis at a postcytoplasmic reaction in a stable L-phase variant of Streptococcus faecium.

Cultures of a stable L-phase variant of Streptococcus faecium F24 produced and retained peptidoglycan precursors intracellularly over the entire growth cycle in a chemically defined medium. The identity of the most abundant precursor, UDP N-acetylmuramyl-L-alanyl-D-glutamyl-L-lysyl-D-alanyl-D-alanine (UDP-MurNAc-pentapeptide), was confirmed by demonstrating in vitro the presence of enzymes required for the cytoplasmic stage of peptidoglycan biosynthesis. The initial membrane-bound reaction in peptidoglycan biosynthesis involving phospho-MurNAc-pentapeptide translocase and undecaprenyl-phosphate membrane carrier was catalyzed by protoplast membrane preparations but not by L-phase membrane preparations. However, both protoplast and L-phase membranes incorporated radioactivity from dTDP-L-[14C]rhamnose, the presumed precursor to a non-peptidoglycan cell surface component, into high-molecular-weight material. dTDP-L-rhamnose did not accumulate in growing cultures but was synthesized from D-glucose-1-phosphate and dTTP by cell-free extracts of the streptococcus and L-phase variant. Neither rhamnose- nor muramic acid-containing compounds were detected in culture fluids. It is suggested that continued inhibition of cell wall biosynthesis in this stable L-phase variant is the result of a defect expressed at the membrane stage of peptidoglycan biosynthesis specifically involving the translocation step.

Isolation and some properties of cell envelope altered mutants of Escherichia coli.

Mutants of Escherichia coli which have a defect in their permeability barrier were selected. The technique used was to employ a strain of E. coli having a deletion in the gene for lactose permease and to select for mutants which can grow on lactose at 40 C. Twenty such mutants were isolated and six of these were found to be more sensitive to actinomycin D, sodium deoxycholate, and sodium dodecyl sulfate than was the parental strain. They were also more sensitive to the antibiotics vancomycin and bacitracin, which inhibit peptidoglycan biosynthesis. These mutants were no more sensitive to several different colicins or phages than was the wild-type strain. One of the mutants selected by this technique has an abnormal morphology when grown on certain carbon sources in minimal medium, and this mutant is more extensively studied in the accompanying paper.