Antitoxin ε Reverses Toxin ζ-Facilitated Ampicillin Dormants.

Toxin-antitoxin (TA) modules are ubiquitous in bacteria, but their biological importance in stress adaptation remains a matter of debate. The inactive ζ-ε2-ζ TA complex is composed of one labile ε2 antitoxin dimer flanked by two stable ζ toxin monomers. Free toxin ζ reduces the ATP and GTP levels, increases the (p)ppGpp and c-di-AMP pool, inactivates a fraction of uridine diphosphate-N-acetylglucosamine, and induces reversible dormancy. A small subpopulation, however, survives toxin action. Here, employing a genetic orthogonal control of ζ and ε levels, the fate of bacteriophage SPP1 infection was analyzed. Toxin ζ induces an active slow-growth state that halts SPP1 amplification, but it re-starts after antitoxin expression rather than promoting abortive infection. Toxin ζ-induced and toxin-facilitated ampicillin (Amp) dormants have been revisited. Transient toxin ζ expression causes a metabolic heterogeneity that induces toxin and Amp dormancy over a long window of time rather than cell persistence. Antitoxin ε expression, by reversing ζ activities, facilitates the exit of Amp-induced dormancy both in rec+ and recA cells. Our findings argue that an unexploited target to fight against antibiotic persistence is to disrupt toxin-antitoxin interactions.

Synthesis, in-vitro and in-silico study of novel thiazoles as potent antibacterial agents and MurB inhibitors.

Efficient procedures are herein reported for the synthesis of novel hybrid thiazoles via a one-pot three-component protocol. The protocol involves the reaction of novel aldehyde, thiosemicarbazide and halogen-containing reagents in solvent- and catalyst-free conditions. The structures of the new thiazoles were elucidated by elemental analyses and spectroscopic data. The in-vitro antibacterial screening and MurB enzyme inhibition assays were performed for the novel thiazoles. The thiazol-4(5H)-one derivative 6d, with p-MeO, exhibits the best antibacterial activities with minimum inhibitory concentration values of 3.9, 3.9, 7.8, and 15.6 µg/ml against Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus mutans, and Escherichia coli, respectively, as compared to the reference antibiotic drugs. It also exhibits the highest inhibition of the MurB enzyme with an IC50 of 8.1 µM. The structure-activity relationship was studied to determine the effect of the structures of the newly prepared molecules on the strength of the antibacterial activities. Molecular docking was also performed to predict the binding modes of the new thiazoles in the active sites of the E. coli MurB enzyme.

Structure guided design of an antibacterial peptide that targets UDP-N-acetylglucosamine acyltransferase.

UDP-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA) catalyzes the first step of lipid A biosynthesis, the transfer of an R-3-hydroxyacyl chain from its acyl carrier protein (ACP) to the 3-OH group of UDP-GlcNAc. Essential in the growth of Gram-negative bacteria, LpxA is a logical target for antibiotics design. A pentadecapeptide (Peptide 920) with high affinity towards LpxA was previously identified in a phage display library. Here we created a small library of systematically designed peptides with the length of four to thirteen amino acids using Peptide 920 as a scaffold. The concentrations of these peptides at which 50% of LpxA is inhibited (IC50) range from 50 nM to >100 µM. We determined the crystal structure of E. coli LpxA in a complex with a potent inhibitor. LpxA-inhibitor interaction, solvent model and all contributing factors to inhibitor efficacy were well resolved. The peptide primarily occludes the ACP binding site of LpxA. Interactions between LpxA and the inhibitor are different from those in the structure of Peptide 920. The inhibitory peptide library and the crystal structure of inhibitor-bound LpxA described here may further assist in the rational design of inhibitors with antimicrobial activity that target LpxA and potentially other acyltransferases.

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.

Regulation of the biosynthesis of N-acetylglucosaminylpyrophosphoryldolichol, feedback and product inhibition.

The assembly of the core oligosaccharide region of asparagine-linked glycoproteins proceeds by means of the dolichol pathway. The first step of this pathway, the reaction of dolichol phosphate with UDP-GlcNAc to form N-acetylglucosaminylpyrophosphoryldolichol (GlcNAc-P-P-dolichol), is under investigation as a possible site of metabolic regulation. This report describes feedback inhibition of this reaction by the second intermediate of the pathway, N-acetylglucosaminyl-N-acetylglucosaminylpyrophosphoryldolichol (GlcNAc-GlcNAc-P-P-dolichol), and product inhibition by GlcNAc-P-P-dolichol itself. These influences were revealed when the reactions were carried out in the presence of showdomycin, a nucleoside antibiotic, present at concentrations that block the de novo formation of GlcNAc-GlcNAc-P-P-dolichol but not that of GlcNAc-P-P-dolichol. The apparent K(i) values for GlcNAc-P-P-dolichol and GlcNAc-GlcNAc-P-P-dolichol under basal conditions were 4.4 and 2.8 microM, respectively. Inhibition was also observed under conditions where mannosyl-P-dolichol (Man-P-dol) stimulated the biosynthesis of GlcNAc-P-P-dolichol; the apparent K(i) values for GlcNAc-P-P-dolichol and GlcNAc-GlcNAc-P-P-dolichol were 2.2 and 11 microM, respectively. Kinetic analysis of the types of inhibition indicated competitive inhibition by GlcNAc-P-P-dolichol toward the substrate UDP-GlcNAc and non-competitive inhibition toward dolichol phosphate. Inhibition by GlcNAc-GlcNAc-P-P-dolichol was uncompetitive toward UDP-GlcNAc and competitive toward dolichol phosphate. A model is presented for the kinetic mechanism of the synthesis of GlcNAc-P-P-dolichol. GlcNAc-P-P-dolichol also exerts a stimulatory effect on the biosynthesis of Man-P-dol, i.e. a reciprocal relationship to that previously observed between these two intermediates of the dolichol pathway. This network of inhibitory and stimulatory influences may be aspects of metabolic control of the pathway and thus of glycoprotein biosynthesis in general.

Effect of phosphoenolpyruvate analogs on the activity of enoylpyruvate transferase and the effect of UDP-N-acetylglucosamine on the reactivity of the active site SH group.

Effect of several phosphoenolpyruvate analogs on the activity of enoylpyruvate (phosphoenolpyruvate: UDP-2-acetamido-2-deoxy-D-glucose 2-enoyl-1-carboxyethyltransferase, EC 2.5.1.7) transferase was examined. The results suggest that the phosphoenolpyruvate binding site of the transferase is very similar to that of pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40). Evidence is presented to show that the binding of UDP-GlcNAc to the transferase enhances the reactivity of the active site SH group.

Inhibition of UDP-N-acetylglucosamine pyrophosphorylase by uridine.

UDP-N-acetylglucosamine pyrophosphorylases (UTP: 2-acetamido-2-deoxy-alpha-D-glucose-1-phosphate uridylyltransferase, EC 2.7.7.23) from baker's yeast and Neurospora crassa IFO 6178 were inhibited by uridine which is the nucleoside moiety of UDP-GlcNAc. The inhibition was shown in both directions of pyrophosphorolysis and of synthesis of UDP-GlcNAc. Kinetic analysis revealed that uridine demonstrated a noncompetitive type of inhibition with UDP-GlcNAc and competitive inhibition with PPi. The Ki values for the baker's yeast enzyme were 1.8 mM for UDP-GlcNAc and 0.16 mM for PPi, and the values for the Neurospora enzyme were 1.1 mM for UDP-GlcNAc and 0.15 mM for PPi, respectively. Uridine did not bind irreversibly to the enzyme, as the activity was restored with dialysis. No other nucleosides caused inhibition of the enzyme activity except uridine. Some uridine derivatives, such as 5-hydroxyuridine, 5,6-dihydrouridine and pseudouridine, also inhibited the enzyme activity. But doexyuridine showed only slight inhibition, and 5'-UMP and orotidine caused no inhibition of the enzyme activity.