Direct interaction of a vancomycin derivative with bacterial enzymes involved in cell wall biosynthesis.

BACKGROUND: The glycopeptide antibiotic vancomycin complexes DAla-DAla termini of bacterial cell walls and peptidoglycan precursors and interferes with enzymes involved in murein biosynthesis. Semisynthetic vancomycins incorporating hydrophobic sugar substituents exhibit efficacy against DAla-DLac-containing vancomycin-resistant enterococci, albeit by an undetermined mechanism. Contrasting models that invoke either cooperative dimerization and membrane anchoring or direct inhibition of bacterial transglycosylases have been proposed to explain the bioactivity of these glycopeptides. RESULTS: Affinity chromatography has revealed direct interactions between a semisynthetic hydrophobic vancomycin (DCB-PV), and select Escherichia coli membrane proteins, including at least six enzymes involved in peptidoglycan assembly. The N(4)-vancosamine substituent is critical for protein binding. DCB-PV inhibits transglycosylation in permeabilized E. coli, consistent with the observed binding of the PBP-1B transglycosylase-transpeptidase. CONCLUSIONS: Hydrophobic vancomycins interact directly with a select subset of bacterial membrane proteins, suggesting the existence of discrete protein targets. Transglycosylase inhibition may play a role in the enhanced bioactivity of semisynthetic glycopeptides.

Design and synthesis of novel antibacterial agents with inhibitory activity against DNA polymerase III.

4-Substituted 2-amino-6-(anilino)pyrimidines have been found to be selective inhibitors of DNA polymerase III, a replicative enzyme known to be essential in the DNA synthesis of Gram-positive bacteria. Among the analogues, 18 displayed an IC(50) of 10 microM against DNA polymerase III from Staphylococcus aureus.

Inhibition of IMP-1 metallo-beta-lactamase and sensitization of IMP-1-producing bacteria by thioester derivatives.

IMP-1 metallo-beta-lactamase is a transferable carbapenem-hydrolyzing enzyme found in some clinical isolates of Pseudomonas aeruginosa, Serratia marcescens and Klebsiella pneumoniae. Bacteria that express IMP-1 show significantly reduced sensitivity to carbapenems and other beta-lactam antibiotics. A series of thioester derivatives has been shown to competitively inhibit purified IMP-1. As substrates for IMP-1, the thioesters yielded thiol hydrolysis products which themselves were reversible competitive inhibitors. The thioesters also increased sensitivity to the carbapenem L-742,728 in an IMP-1-producing laboratory stain of Escherichia coli, but will need further modification to improve their activity in less permeable organisms such as Pseudomonas and Serratia. Nonetheless, the thioester IMP-1 inhibitors offer an encouraging start to overcoming metallo-beta-lactamase-mediated resistance in bacteria.

In vitro activities of the potent, broad-spectrum carbapenem MK-0826 (L-749,345) against broad-spectrum beta-lactamase-and extended-spectrum beta-lactamase-producing Klebsiella pneumoniae and Escherichia coli clinical isolates.

An important mechanism of bacterial resistance to beta-lactam antibiotics is inactivation by beta-lactam-hydrolyzing enzymes (beta-lactamases). The evolution of the extended-spectrum beta-lactamases (ESBLs) is associated with extensive use of beta-lactam antibiotics, particularly cephalosporins, and is a serious threat to therapeutic efficacy. ESBLs and broad-spectrum beta-lactamases (BDSBLs) are plasmid-mediated class A enzymes produced by gram-negative pathogens, principally Escherichia coli and Klebsiella pneumoniae. MK-0826 was highly potent against all ESBL- and BDSBL-producing K. pneumoniae and E. coli clinical isolates tested (MIC range, 0.008 to 0.12 microgram/ml). In E. coli, this activity was associated with high-affinity binding to penicillin-binding proteins 2 and 3. When the inoculum level was increased 10-fold, increasing the amount of beta-lactamase present, the MK-0826 MIC range increased to 0.008 to 1 microgram/ml. By comparison, similar observations were made with meropenem while imipenem MICs were usually less affected. Not surprisingly, MIC increases with noncarbapenem beta-lactams were generally substantially greater, resulting in resistance in many cases. E. coli strains that produce chromosomal (Bush group 1) beta-lactamase served as controls. All three carbapenems were subject to an inoculum effect with the majority of the BDSBL- and ESBL-producers but not the Bush group 1 strains, implying some effect of the plasmid-borne enzymes on potency. Importantly, MK-0826 MICs remained at or below 1 microgram/ml under all test conditions.

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.

Carbohydroxamido-oxazolidines: antibacterial agents that target lipid A biosynthesis.

A series of carbohydroxamido-oxazolidine inhibitors of UDP-3-O-[R-3-hydroxymyristoyl]-GlcNAc deacetylase, the enzyme responsible for the second step in lipid A biosynthesis, was identified. The most potent analog L-161,240 showed an IC50 = 30 nM in the DEACET assay and displayed an MIC of 1-3 microg/mL against wild-type E. coli.

Antibacterial agents that inhibit lipid A biosynthesis.

Lipid A constitutes the outer monolayer of the outer membrane of Gram-negative bacteria and is essential for bacterial growth. Synthetic antibacterials were identified that inhibit the second enzyme (a unique deacetylase) of lipid A biosynthesis. The inhibitors are chiral hydroxamic acids bearing certain hydrophobic aromatic moieties. They may bind to a metal in the active site of the deacetylase. The most potent analog (with an inhibition constant of about 50 nM) displayed a minimal inhibitory concentration of about 1 microgram per milliliter against Escherichia coli, caused three logs of bacterial killing in 4 hours, and cured mice infected with a lethal intraperitoneal dose of E. coli.

The envA permeability/cell division gene of Escherichia coli encodes the second enzyme of lipid A biosynthesis. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase.

The envA gene of Escherichia coli has been shown previously to be essential for cell viability (Beall, B. and Lutkenhaus, J. (1987) J. Bacteriol. 169, 5408-5415), yet it encodes a protein of unknown function. Extracts of strains harboring the mutant envA1 allele display 3.5-18-fold reductions in UDP-3-O-acyl-N-acetylglucosamine deacetylase specific activity. The deacetylase is the second enzymatic step of lipid A biosynthesis. The structural gene coding for the deacetylase has not been assigned. In order to determine if the envA gene encodes the deacetylase, envA was cloned into an isopropyl-1-thio-beta-D-galactopyranoside-inducible T7-based expression system. Upon induction, a protein of the size of envA was highly overproduced, as judged by SDS-PAGE. Direct deacetylase assays of cell lysates revealed a concomitant approximately 5,000-fold overproduction of activity. Assays of the purified, overproduced EnvA protein demonstrated a further approximately 5-fold increase in specific activity. N-terminal amino acid sequencing of the purified protein showed that the first 20 amino acids matched the predicted envA nucleotide sequence. Contaminating species were present at less than 1% of the level of the EnvA protein. Thus, envA is the structural gene for UDP-3-O-acyl-GlcNAc deacetylase. Based on its function in lipid A biosynthesis, we propose the new designation lpxC for this gene.

Leakage of periplasmic enzymes from envA1 strains of Escherichia coli.

Previous work ascribed antibiotic hypersensitivity of the envA1 mutant to lowered lipopolysaccharide levels and exposure of the lipid bilayer. In the detailed characterization of the EnvA permeability phenotype presented here, the envA1 mutation was shown to confer leakage of the periplasmic enzymes beta-lactamase and RNase I. Leakage was observed in three different genetic backgrounds, including the original envA1 strain and its parent. In contrast, no detectable leakage of the cytoplasmic enzyme beta-galactosidase was observed. Sensitivity of envA1 strains to a range of antibiotics not previously reported was tested, and lipophilicity (partition coefficient) of a number of antibiotics was determined. On the basis of observations of periplasmic leakage and sensitivity to large hydrophilic antibiotics and lysozyme, part of the permeability phenotype of the envA1 mutant is proposed to be due to transient rupture and resealing of the EDTA-sensitive outer membrane layer. In this regard, the EnvA permeability phenotype falls into a general class of permeability/leaky mutants of both Escherichia coli and Salmonella typhimurium.

Bacteriophage T4 DNA replication protein 41. Cloning of the gene and purification of the expressed protein.

The bacteriophage T4 primase, composed of the T4 proteins 41 and 61, synthesizes pentaribonucleotides used to prime DNA synthesis on single-stranded DNA in vitro. 41 protein is also a DNA helicase that opens DNA in the same direction as the growing replication fork. Previously, Mattson et al. (Mattson, T., Van Houwe, G., Bolle, A., Selzer, G., and Epstein, R. (1977) Mol. Gen. Genet. 154, 319-326) located part of gene 41 on a 3400-base pair EcoRI fragment of T4 DNA (map units 24.3 to 21.15). In this paper, we report the cloning of T4 DNA representing map units 24.3 to 20.06 in a multicopy plasmid vector. Extracts of cells containing this plasmid complement gene 41- extracts in a DNA synthesis assay, indicating that this region contains all the information necessary for the expression of active 41 protein. We located gene 41 more precisely between T4 map units 22.01 to 20.06 since our cloning of this region downstream of the strong lambda promoter PL results in the production of active 41 protein at a level 100-fold greater than after T4 infection. We have purified 133 mg of homogeneous 41 protein from 27 g of these cells. Like the 41 protein from T4 infected cells, the purified 41 protein in conjunction with the T4 gene 61 priming protein catalyzes primer formation (assayed by RNA primer-dependent DNA synthesis with T4 polymerase, the genes 44/62 and 45 polymerase accessory proteins, and the gene 32 helix-destabilizing protein) and is a helicase whose activity is stimulated by T4 61 protein.

Purification of bacteriophage T4 gene 61 protein. A protein essential for synthesis of RNA primers in the T4 in vitro DNA replication system.

An activity dependent on an intact bacteriophage T4 gene 61 is required, along with the T4 gene 41 protein, for the synthesis of ribonucleotide primers in an in vitro T4 DNA replication system. In this paper, we present a method for purification of the protein catalyzing this gene 61-dependent activity based on an assay for primer-dependent DNA synthesis by the T4 DNA replication proteins. The T4 gene 32 helix-destabilizing protein influences the chromatographic behavior of 61 protein. The purification of 61 protein to near homogeneity by the scheme presented requires the presence of 32 protein in crude extracts. The isolated 61 protein is basic, with a molecular weight of 44,000, and is active as a monomer. Ribonucleotide primer synthesis shows a linear dependence on 61 protein concentration, but a sigmoidal dependence on 41 protein concentration. The dependence on 41 protein concentration is linear, however, if the 41 protein is first "activated" by incubation at high concentration in the presence of rGTP. Using this "activated" 41 protein and purified 61 protein, we show a stoichiometric relationship between the two proteins in the priming reaction consistent with the existence of a priming complex comprising an oligomer of 41 protein and a 61 protein monomer.

Bacteriophage T4 gene 41 protein, required for the synthesis of RNA primers, is also a DNA helicase.

Bacteriophage T4 gene 41 protein is one of the two phage proteins previously shown to be required for the synthesis of the pentaribonucleotide primers which initiate the synthesis of new chains in the T4 DNA replication system. We now show that a DNA helicase activity which can unwind short fragments annealed to complementary single-stranded DNA copurifies with the gene 41 priming protein. T4 gene 41 is essential for both the priming and helicase activities, since both are absent after infection by T4 phage with an amber mutation in gene 41. A complete gene 41 product is also required for two other activities previously found in purified preparations of the priming activity: a single-stranded DNA-dependent GTPase (ATPase) and an activity which stimulates strand displacement synthesis catalyzed by T4 DNA polymerase, the T4 gene 44/62 and 45 polymerase accessory proteins, and the T4 gene 32 helix-destabilizing protein (five-protein reaction). The 41 protein helicase requires a single-stranded DNA region adjoining the duplex region and begins unwinding at the 3' terminus of the fragment. There is a sigmoidal dependence on both nucleotide (rGTP, rATP) and protein concentration for this reaction. 41 Protein helicase activity is stimulated by our purest preparation of the T4 gene 61 priming protein, and by the T4 gene 44/62 and 45 polymerase accessory proteins. The direction of unwinding is consistent with the idea that 41 protein facilitates DNA synthesis on duplex templates by destabilizing the helix as it moves 5' to 3' on the displaced strand.