Yoshihiro Oyamada

Identification and molecular characterization of an N-acetylmuramyl-l-alanine amidase Sle1 involved in cell separation of Staphylococcus aureus

We purified a peptidoglycan hydrolase involved in cell separation from a Staphylococcus aureus atl null mutant and identified its gene. Characterization of the gene product shows a 32 kDa N‐acetylmuramyl‐l‐alanine amidase that we designated Sle1. Analysis of peptidoglycan digests showed Sle1 preferentially cleaved N‐acetylmuramyl‐l‐Ala bonds in dimeric cross‐bridges that interlink the two murein strands in the peptidoglycan. An insertion mutation of sle1 impaired cell separation and induced S. aureus to form clusters suggesting Sle1 is involved in cell separation of S. aureus. The Sle1 mutant revealed a significant decrease in pathogenesis using an acute infection mouse model. Atl is the major autolysin of S. aureus, which has been implicated in cell separation of S. aureus. Generation of an atl/sle1 double mutant revealed that the mutant cell separation was heavily impaired suggesting that S. aureus uses two peptidoglycan hydrolases, Atl and Sle1, for cell separation. Unlike Atl, Sle1 is not directly involved in autolysis of S. aureus.

Accumulation of Mutations in both gyrB and parE Genes Is Associated with High-Level Resistance to Novobiocin in Staphylococcus aureus

ABSTRACT Coumarin-resistant mutants of Staphylococcus aureus were isolated by three-step selection with novobiocin at different concentrations. Sequencing analysis of the gyrB and parE genes of the first-, second-, and third-step mutants revealed that successive point mutations first occurred specifically in the gyrB gene, followed by a point mutation in the parE gene and then an additional point mutation in the gyrB gene. These findings demonstrate that DNA gyrase is the primary target and that topoisomerase IV is the secondary target for novobiocin and that the accumulation of point mutations in both the gyrB and the parE genes is associated with high-level resistance to novobiocin in S. aureus. Moreover, our results show that the amino acid substitutions (Asp-89 to Gly and Ser-128 to Leu) found in GyrB are associated with resistance to novobiocin but not to coumermycin A1, suggesting that the interactions of novobiocin and coumermycin A1 with GyrB differ at the molecular level.

Cell-wall thickness : possible mechanism of acriflavine resistance in meticillin-resistant Staphylococcus aureus

Acriflavine resistance in the clinical meticillin-resistant Staphylococcus aureus isolate KT24 was found not to be mediated by multidrug efflux pumps encoded by qacA/B, smr, qacE, qacG, qacH, qacJ or norA. Early uptake and accumulation of ethidium bromide in MRSA KT24 was significantly lower than that in a susceptible strain, although the efflux rates were similar. Therefore, a permeability barrier in MRSA KT24 may be the conceivable mechanism of acriflavine resistance. Interestingly, it was found that MRSA KT24 had a significantly thickened cell wall, and that cell-wall thickness increased gradually during bacterial growth. In contrast, cell size and surface area in MRSA KT24 were not different from those in the susceptible strain. Moreover, MRSA KT24 exposure to sub-MIC concentrations of acriflavine resulted in a thicker cell wall. These results indicate that cell-wall thickness may be responsible for acriflavine resistance in S. aureus.

A 4-Aminofurazan Derivative—A189—Inhibits Assembly of Bacterial Cell Division Protein FtsZ In Vitro and In Vivo

Out of 95,000 commercially available chemical compounds screened by the anucleate cell blue assay, 138 selected hit compounds were further screened. As a result, A189, a 4‐aminofurazan derivative was found to inhibit FtsZ GTPase with an IC50 of 80 μg/ml and to exhibit antibacterial activity against Staphylococcus aureus and Escherichia coli. Light scattering demonstrated that A189 inhibited FtsZ assembly in vitro, and microscopic observation of A189‐treated E. coli indicated that A189 perturbed FtsZ ring formation and made bacterial cells filamentous. However, nucleoids staining with DAPI revealed that A189 did not affect DNA replication and chromosome segregation in bacterial filamentous cells. Furthermore, A189 made sulA‐deleted E. coli cells filamentous. Taken together, these findings suggest that A189 inhibits FtsZ GTPase activity, resulting in perturbation of FtsZ ring formation, which leads to bacterial cell death.

A new screening method to identify inhibitors of the Lol (localization of lipoproteins) system, a novel antibacterial target.

As the Lol system, which is involved in localization of lipoproteins, is essential for Escherichia coli growth and widely conserved among gram‐negative bacteria, it is considered to be a promising target for the development of anti‐gram‐negative bacterial agents. However, no high‐throughput screening method has so far been developed to screen for Lol system inhibitors. By combining three assay systems (anucleate cell blue assay, Lpp assay, and LolA‐dependent release inhibition assay) and a drug susceptibility test, we have successfully developed a new screening method for identification of compounds that inhibit the Lol system. Using this new screening method, we screened 23,600 in‐house chemical compounds and found 2 Lol system inhibitors. We therefore conclude that our new screening method can efficiently identify new antibacterial agents that target the Lol system.

Anucleate Cell Blue Assay: a Useful Tool for Identifying Novel Type II Topoisomerase Inhibitors

ABSTRACT About 95,000 compounds were screened by the anucleate cell blue assay. Fifty-one of the hit compounds had various structures and showed inhibitory activity against DNA gyrase and/or topoisomerase IV. Moreover, the compounds exhibited antibacterial activity against a fluoroquinolone- and novobiocin-resistant strain of Staphylococcus aureus. The anucleate cell blue assay is therefore a useful tool for finding novel type II topoisomerase inhibitors.

Cooperation between alteration of DNA gyrase genes and over-expression of MexB and MexX confers high-level fluoroquinolone resistance in Pseudomonas aeruginosa strains isolated from a patient who received a liver transplant followed by treatment with fluoroquinolones

Clinical isolates of highly fluoroquinolone‐resistant Pseudomonas aeruginosa had a mutation in either A or B subunit of DNA gyrase and over‐expressed MexB and MexX, the efflux system proteins. Introduction of wild‐type gyrase genes of Escherichia coli into the isolates made them as fluoroquinolone‐susceptible as the moderately fluoroquinolone‐resistant strains that only over‐expressed efflux system proteins. These findings demonstrate that high fluoroquinolone‐resistance in P. aeruginosa is attributed to cooperation between alteration in DNA gyrase genes and over‐expression of efflux systems proteins.

Mechanism of Inhibition of DNA Gyrase by ES-1273, a Novel DNA Gyrase Inhibitor

We investigated the mode of action of ES‐1273, a novel DNA gyrase inhibitor obtained by optimization of ES‐0615, which was found by screening our chemical library using anucleate cell blue assay. ES‐1273 exhibited the same antibacterial activity against S. aureus strains with amino acid change(s) conferring quinolone‐ and coumarin‐resistance as that against a susceptible strain. In addition, ES‐1273 inhibited DNA gyrase supercoiling activity, but not ATPase activity of the GyrB subunit of DNA gyrase. Moreover, ES‐1273 did not induce cleavable complex. These findings demonstrate that the mechanism by which ES‐1273 inhibits DNA gyrase is different from that of the quinolones or the coumarins. Preincubation of DNA gyrase and substrate DNA prevented inhibition of DNA gyrase supercoiling activity by ES‐1273. ES‐1273 antagonized quinolone‐induced cleavage. In electrophoretic mobility shift assay, no band representing DNA gyrase‐DNA complex was observed in the presence of ES‐1273. Taken together, these results indicate that ES‐1273 prevents DNA from binding to DNA gyrase. Furthermore, our results from surface plasmon resonance experiments strongly suggest that ES‐1273 interacts with DNA. Therefore, the interaction between ES‐1273 and DNA prevents DNA from binding to DNA gyrase, resulting in inhibition of DNA gyrase supercoiling. Interestingly, we also found that ES‐1273 inhibits topoisomerase IV and human topoisomerase IIoc, but not human topoisomerase I. These findings indicate that ES‐1273 is a type II topoisomerase specific inhibitor.

Cloning and expression of the major porin protein gene opcP of Burkholderia (formerly Pseudomonas) cepacia in Escherichia coli

The outer membrane protein OpcP1 of Burkholderia (formerly Pseudomonas) cepacia is one of the subunits forming the porin oligomer OpcPO by non-covalent association. OpcP1 was cleaved with lysyl endopeptidase and the N-terminal amino acid (aa) sequences of the polypeptide fragments were determined. Based on the sequence information, we cloned the opcP gene on a 10-kb EcoRI DNA fragment of the B. cepacia ATCC25416 chromosome. Nucleotide (nt) sequencing revealed a 1086-bp open reading frame (ORF), encoding a 361-aa polypeptide with a signal sequence of 20 residues. The predicted opcP gene encoded a mature protein of Mr 35,696, which agrees well with the value observed previously on SDS-PAGE. The opcP was sub-cloned into pTrc99A and introduced into Escherichia coli. Immunoblot analysis using murine antiserum specific to OpcP1 visualized the protein expressed in the E. coli cells after induction by isopropyl beta-D-thiogalactopyranoside (IPTG).