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Biomedical subjects

Teruo Kuroda

Publications and source records attributed to Teruo Kuroda.

At least 19 recordsLinked to original sources
PubMed2006

Synergistic effect of [10]-gingerol and aminoglycosides against vancomycin-resistant enterococci (VRE).

An extract from ginger (root of Zingiber officinale) reduced the minimum inhibitory concentrations (MICs) of aminoglycosides in vancomycin-resistant enterococci (VRE). The effective compound was isolated and identified as [10]-gingerol. In the presence of [10]-gingerol at 1/10 concentration of its own MIC, the MIC of arbekacin was lowered by 1/32 to 1/16. [10]-Gingerol also reduced the MICs of other aminoglycosides, and of bacitracin and polymixin B, but not of other antimicrobial agents tested. Because [10]-gingerol reduced the MICs of several aminoglycosides both in strains possessing or lacking aminoglycoside-modification enzymes, it seems that the effect of [10]-gingerol is not related to these enzymes, which mainly confer bacterial resistance against aminoglycosides. It seemed that a detergent-like effect of [10]-gingerol potentiated the antimicrobial activity of the aminoglycosides. In fact, some detergents such as sodium dodecyl sulfate (SDS) and Triton X-100 reduced the MICs of aminoglycosides, bacitracin and polymixin B in VRE. Since the intrinsic resistance to aminoglycosides in enterococci is due to low level of entry of the drugs into the cells, increase in the membrane permeability caused by [10]-gingerol will enhance the influx of aminoglycosides into enterococcal cells.

Acetyltransferases↗
PubMed2006

KmrA multidrug efflux pump from Klebsiella pneumoniae.

We cloned a gene responsible for multidrug resistance from the chromosomal DNA of Klebsiella pneumoniae MGH78578 that showed multidrug resistance. We designated the gene kmrA. The deduced amino acid sequence of KmrA was similar to that of SmvA that is responsible for methyl viologen-resistance in Salmonella enterica sv. Typhi and Typhimurium. Introduction of the cloned kmrA gene into drug-hypersensitive Escherichia coli KAM32 cells made them resistant to acriflavine, 4',6-diamidino-2-phenylindole (DAPI), Hoechst 33342, tetraphenylphosphonium chloride (TPPCl), methyl viologen and ethidium bromide. We observed elevated energy-dependent efflux of ethidium in E. coli cells carrying the kmrA gene compared with control cells. We also cloned the smvA gene from S. enterica sv. Typhimurium LT2 and investigated the resistance pattern for several drugs. The pattern was similar between KmrA from K. pneumoniae and SmvA from S. enterica.

Anti-Bacterial Agents↗
PubMed2006

Gene cloning and characterization of SdrM, a chromosomally-encoded multidrug efflux pump, from Staphylococcus aureus.

There are more than 30 genes for putative multidrug efflux pumps in the chromosome of Staphylococcus aureus. Only a few of these have been analyzed so far. Here we cloned a new gene, SA1972, using a PCR method, from the chromosome of S. aureus N315. We found that the product SA1972 could lead to elevated resistance against several antimicrobial agents such as norfloxacin, acriflavine and ethidium bromide. We designated the gene as sdrM. We observed elevated energy-dependent efflux of acriflavine in S. aureus cells introduced with the sdrM gene. We conclude that SdrM is a multidrug efflux pump belonging to the major facilitator (MF) superfamily.

Acriflavine↗
PubMed2006

Functional gene cloning and characterization of MdeA, a multidrug efflux pump from Staphylococcus aureus.

A DNA fragment conferring drug resistance was cloned from the chromosomal DNA of Staphylococcus aureus N315 using a drug hypersensitive Escherichia coli KAM32 as the host. Although E. coli KAM32 cells were sensitive to many antimicrobial agents, transformed cells harboring a recombinant plasmid carrying the DNA region became resistant to several structurally unrelated antimicrobial agents, such as tetraphenylphosphonium chloride, Hoechst 33342 and norfloxacin. These results suggest that the cloned DNA fragment carries a gene(s) encoding a multidrug efflux pump. We partially determined the nucleotide sequence of the cloned DNA and found the mdeA gene within it. The E. coli cells transformed with the mdeA gene showed efflux activity of Hoechst 33342. On the other hand, S. aureus cells transformed with mdeA showed elevated resistance to doxorubicin, daunorubicin, tetraphenylphosphonium chloride, Hoechst 33342, ethidium bromide and rhodamine 6G. Elevated energy-dependent efflux of ethidium was observed with transformed S. aureus. We found that the mdeA gene was expressed under normal growth conditions in S. aureus N315.

Anti-Bacterial Agents↗
PubMed2006

Involvement of the Vibrio parahaemolyticus pvsC gene in export of the siderophore vibrioferrin.

The pvsC gene of unknown function has been found in the iron-regulated vibrioferrin biosynthesis operon of Vibrio parahaemolyticus (Tanabe, T. et al., J. Bacteriol. 185: 6938-6949, 2003). The amino acid sequence deduced from the gene showed significant similarity to 12-transmembrane segment efflux pumps belonging to the major facilitator superfamily. A nonpolar deletion of pvsC in V. parahaemolyticus resulted in a reduced release of vibrioferrin into the medium. Vibrioferrin release could be regained by introducing the intact pvsC gene on a complementing plasmid. These results indicate that the pvsC gene product functions as an inner membrane exporter of vibrioferrin.

Amino Acid Sequence↗
PubMed2005

Quantitative modeling of chloride conductance in yeast TRK potassium transporters.

So-called TRK proteins are responsible for active accumulation of potassium in plants, fungi, and bacteria. A pair of these proteins in the plasma membrane of Saccharomyces cerevisiae, ScTrk1p and ScTrk2p, also admit large, adventitious, chloride currents during patch-recording (Cl- efflux). Resulting steady-state current-voltage curves can be described by two simple kinetic models, most interestingly, voltage-driven channeling of ions through a pair of activation-energy barriers that lie within the membrane dielectric, near the inner (alpha) and outer (beta) surfaces. Two barrier heights (E(alpha) and E(beta)) and two relative distances (a1 and b2) from the surfaces specify the model. Measured current amplitude parallels intracellular chloride concentration and is strongly enhanced by acidic extracellular pH. The former implies an exponential variation of a1, between approximately 0.2 and approximately 0.4 of the membrane thickness, whereas the latter implies a linear variation of E(beta), by 0.69 Kcal mol(-1)/pH. The model requires membrane slope conductance to rise exponentially with increasingly large negative membrane voltage, as verified by data from a few yeast spheroplasts that tolerated voltage clamping at -200 to -300 mV. The behaviors of E(beta) and a1 accord qualitatively with a hypothetical structural model for fungal TRK proteins, suggesting that chloride ions flow through a central pore formed by symmetric aggregation of four TRK monomers.

Cation Transport Proteins↗
PubMed2005

Physiological roles of three Na+/H+ antiporters in the halophilic bacterium Vibrio parahaemolyticus.

Vibrio parahaemolyticus mutants lacking three Na+/H+ antiporters (NhaA, NhaB, NhaD) were constructed. The DeltanhaA strains showed significantly higher sensitivity to LiCl regarding their growth compared to the parental strain. The DeltanhaA and DeltanhaB strains exhibited higher sensitivities to LiCl. The mutant XACabd lacking all of the three antiporters could not grow in the presence of 500 mM LiCl at pH 7.0, or 50 mM at pH 8.5. The XACabd mutant was also sensitive to 1.0 M NaCl at pH 8.5. These results suggest that Na+/H+ antiporters, especially NhaA, are responsible for resistance to LiCl and to high concentrations of NaCl. Reduced Na+/H+ and Li+/H+ antiport activities were observed with everted membrane vesicles of DeltanhaB strains. However, Li+/H+ antiport activities of DeltanhaB strains were two times higher than those of DeltanhaA strains when cells were cultured at pH 8.5. It seems that expression of nhaA and nhaB is dependent on medium pH to some extent. In addition, HQNO (2-heptyl-4-hydroxyquinoline N-oxide), which is a potent inhibitor of the respiratory Na+ pump, inhibited growth of XACabd, but not of the wild type strain. Moreover, survival rate of XACabd under hypoosmotic stress was lower than that of wild type strain. It is likely that the Na+/H+ antiporters are involved in osmoregulation under hypoosmotic stress. Based on these findings, we propose that the Na+/H+ antiporters cooperate with the respiratory Na+ pump in ionic homeostasis in V. parahaemolyticus.

Lithium↗
PubMed2005

Gene cloning and characterization of four MATE family multidrug efflux pumps from Vibrio cholerae non-O1.

There are six putative genes for multidrug and toxic compound extrusion (MATE) family multidrug efflux pumps in the chromosome of Vibrio cholerae. We have so far analyzed two MATE family pumps in V. cholerae non-O1 NCTC4716. Here we cloned four remaining genes for putative MATE family efflux pumps by the PCR method from this microorganism and designated them as vcmB, vcmD, vcmH and vcmN. Each one of the four genes was introduced and expressed in the drug hypersusceptible host Escherichia coli KAM32 cells. We observed elevated MICs of multiple antimicrobial agents, such as fluoroquinolones, aminoglycosides, ethidium bromide and Hoechst 33342 in the transformants. Energydependent efflux of substrate was observed with the transformed cells. We found that efflux activities of VcmB, VcmD and VcmH were Na+-dependent, but that of VcmN was Na+-independent. Thus, all six of the MATE family multidrug efflux pumps of V. cholerae non-O1 have been characterized. We also found that all six genes were expressed in cells of V. cholerae non-O1.

Bacterial Proteins↗
PubMed2005

Gene cloning and properties of the RND-type multidrug efflux pumps MexPQ-OpmE and MexMN-OprM from Pseudomonas aeruginosa.

We cloned two operons for putative RND-type multidrug efflux pumps from Pseudomonas aeruginosa by a PCR method. We designated the genes in one operon mexPQ(-opmE) and in another operon mexMN. Introduction of the mexPQ-opmE into drug hypersensitive cells resulted in elevated MICs of macrolides, fluoroquinolones and some other drugs. Introduction of the mexMN into the hypersensitive cells possessing oprM, but not into cells not possessing oprM, resulted in elevated MICs of chloramphenicol and thiamphenicol. Thus, we conclude that MexPQ-OpmE and MexMN-OprM are functional multidrug efflux pumps when expressed in P. aeruginosa.

Bacterial Outer Membrane Proteins↗
PubMed2005

Remarkable synergies between baicalein and tetracycline, and baicalein and beta-lactams against methicillin-resistant Staphylococcus aureus.

During the screening of compounds that potentiate the effect of antimicrobial agents against methicillin-resistant Staphylococcus aureus(MRSA), we found that an extract of thyme (Thymus vulgaris L) leaves greatly reduced the minimum inhibitory concentration (MIC) of tetracycline against MRSA. We isolated the effective compound and identified it as baicalein (5, 6, 7-trihydroxyflavone). One of the clinically isolated MRSA strains possessed tetK, a gene encoding active efflux pump for tetracycline. We examined the effect of baicalein on the efflux of tetracycline, using Escherichia coli KAM32/pTZ1252 carrying the tetK. The E. coli KAM32/pTZ1252 showed 8 to 16 times higher MIC than E. coli KAM32. We observed strong inhibition of transport of tetracycline by baicalein with membrane vesicles prepared from E. coli KAM32/pTZ1252. Baicalein also showed synergy with tetracycline in a MRSA strain that doesn't possess tetK, or with beta-lactams. Thus, mechanisms of the synergies seem to be versatile.

Anti-Bacterial Agents↗
PubMed2005

Multidrug resistance in Klebsiella pneumoniae MGH78578 and cloning of genes responsible for the resistance.

Klebsiella pneumoniae MGH78578, a clinical isolate, showed high level of resistance to many antimicrobial agents. We cloned genes responsible for drug resistance from chromosomal DNA of K. pneumoniae MGH78578 by shotgun method using Escherichia coli KAM32, a drug hypersensitive strain, as host. We obtained 43 hybrid plasmids that made host cells resistant to several antimicrobial agents. We classified them into 17 groups based on growth properties in the presence of each one of 9 antimicrobial agents and on restriction patterns of each hybrid plasmid. Analysis of the cloned genes must be very useful for investigation of major parts of multidrug resistance systems including multidrug efflux pumps in K. pneumoniae MGH78578 in which genome sequence is available.

Biological Transport↗
PubMed2004

A major Li(+) extrusion system NhaB of Pseudomonas aeruginosa : comparison with the major Na(+) extrusion system NhaP.

A gene encoding a Li(+) extrusion system was cloned from the chromosomal DNA of Pseudomonas aeruginosa and expressed in Escherichia coli cells. The gene enabled growth of E. coli KNabc cells, which were unable to grow in the presence of 10 mM LiCl or 0.1 M NaCl because of the lack of major Na(+) (Li(+))/H(+) antiporters. We detected Li(+)/H(+) and Na(+)/H(+) antiport activities in membrane vesicles prepared from E. coli KNabc cells that harbored a plasmid carrying the cloned gene. Activity of this antiporter was pH-dependent with an optimal pH activity between pH 7.5 and 8.5. These properties indicate that this antiporter is different from NhaP, an Na(+)/H(+) antiporter from P. aeruginosa that we reported previously, and that is rather specific to Na(+) but it cannot extrude Li(+) effectively. The gene was sequenced and an open reading frame (ORF) was identified. The amino acid sequence deduced from the ORF showed homology (about 60% identity and 90% similarity) with that of the NhaB Na(+)/H(+) antiporters of E. coli and Vibrio parahaemolyticus. Thus, we designated the antiporter as NhaB of P. aeruginosa. E. coli KNabc carrying the nhaB gene from P. aeruginosa was able to grow in the presence of 10 to 50 mM LiCl, although KNabc carrying nhaP was unable to grow in these conditions. The antiport activity of NhaB from P. aeruginosa was produced in E. coli and showed apparent Km values for Li(+) and Na(+) of 2.0 mM and 1.3 mM, respectively. The antiport activity was inhibited by amiloride with a Ki value for Li(+) and Na(+) of 0.03 mM and 0.04 mM, respectively.

Bacterial Proteins↗
PubMed2004

An H(+)-coupled multidrug efflux pump, PmpM, a member of the MATE family of transporters, from Pseudomonas aeruginosa.

We cloned the gene PA1361 (we designated the gene pmpM), which seemed to encode a multidrug efflux pump belonging to the MATE family, of Pseudomonas aeruginosa by the PCR method using the drug-hypersensitive Escherichia coli KAM32 strain as a host. Cells of E. coli possessing the pmpM gene showed elevated resistance to several antimicrobial agents. We observed energy-dependent efflux of ethidium from cells possessing the pmpM gene. We found that PmpM is an H(+)-drug antiporter, and this finding is the first reported case of an H(+)-coupled efflux pump in the MATE family. Disruption and reintroduction of the pmpM gene in P. aeruginosa revealed that PmpM is functional and that benzalkonium chloride, fluoroquinolones, ethidium bromide, acriflavine, and tetraphenylphosphonium chloride are substrates for PmpM in this microorganism.

ATP-Binding Cassette Transporters↗
PubMed2003

A new member of the tripartite multidrug efflux pumps, MexVW-OprM, in Pseudomonas aeruginosa.

OBJECTIVES: Multidrug efflux pumps are thought to be involved in mediating multidrug resistance in Pseudomonas aeruginosa. Here we aim to characterize hitherto uncharacterized multidrug efflux pumps from P. aeruginosa. MATERIALS AND METHODS: We isolated a mutant, YM442, which showed elevated resistance to several antimicrobial agents from P. aeruginosa YM44 lacking four major multidrug efflux pumps, MexAB, MexCD-OprJ, MexEF-OprN and MexXY. We cloned genes responsible for the resistance from chromosomal DNA of YM442 using YM44 as host. RESULTS: We designated the genes mexVW. Introduction of a recombinant plasmid pTAJ2 carrying the mexVW into YM44 cells conferred resistance to fluoroquinolones, tetracycline, chloramphenicol, erythromycin, ethidium bromide and acriflavine. Elevated ethidium bromide extrusion was observed with cells of YM442 and of YM44/pTAJ2. An outer membrane protein OprM was able to cooperate with MexVW. Elevated expression of the mexV gene was observed with YM442 compared with YM44. CONCLUSIONS: MexV (membrane fusion protein)-MexW (RND-type membrane protein)-OprM is a tripartite multidrug efflux pump. It is suggested that other outer membrane component(s) could cooperate with MexVW.

Bacterial Outer Membrane Proteins↗
PubMed2003

An RND-type multidrug efflux pump SdeXY from Serratia marcescens.

OBJECTIVES: Serratia marcescens, an important cause of nosocomial infections, shows intrinsic resistance to a wide variety of antimicrobial agents (multidrug resistance). Multidrug efflux pumps are often involved in the multidrug resistance in many bacteria. A study was undertaken to characterize the multidrug efflux pumps in S. marcescens. METHODS: The genes responsible for the multidrug resistance phenotype in S. marcescens were cloned into Escherichia coli KAM32, a drug-hypersusceptible strain, for further analysis. RESULTS: We cloned sdeXY genes and determined the nucleotide sequence. Clones that carried the sdeXY genes displayed reduced susceptibility to several antimicrobial agents including erythromycin, tetracycline, norfloxacin, benzalkonium chloride, ethidium bromide, acriflavine and rhodamine 6G. A protein similarity search using GenBank revealed that SdeY is a member of the resistance nodulation cell-division (RND) family of multidrug efflux proteins and SdeX is a member of the membrane fusion proteins. Introduction of sdeXY into E. coli cells possessing tolC, but not in cells lacking tolC, resulted in multidrug resistance. We observed energy-dependent ethidium efflux in cells of E. coli KAM32 possessing sdeXY and tolC. CONCLUSIONS: SdeXY is the first RND-type multidrug efflux pump to be characterized in multidrug-resistant S. marcescens.

Anti-Bacterial Agents↗
PubMed2003

Induction of mexCD-oprJ operon for a multidrug efflux pump by disinfectants in wild-type Pseudomonas aeruginosa PAO1.

Induction of the MexCD-OprJ multidrug efflux pump was investigated in wild-type Pseudomonas aeruginosa PAO1. MexCD-OprJ was induced by clinically important disinfectants such as benzalkonium chloride and chlorhexidine gluconate, and by some cytotoxic agents such as tetraphenylphosphonium chloride, ethidium bromide and rhodamine 6G. MexCD-OprJ was not induced by norfloxacin, tetracycline, chloramphenicol, streptomycin, erythromycin or carbenicillin, although they are substrates for the pump. Cells of PAO1 showed increased resistance to norfloxacin when grown in the presence of the inducers of the mexCD-oprJ operon mentioned above. These results indicate that MexCD-OprJ plays an important role in intrinsic multidrug resistance in wild-type P. aeruginosa in hospitals where disinfectants are used frequently.

Bacterial Proteins↗
PubMed2003

Gene cloning and characterization of VcrM, a Na+-coupled multidrug efflux pump, from Vibrio cholerae non-O1.

We cloned a DNA fragment responsible for drug resistance from chromosome of Vibrio cholerae non-O1. Nucleotide sequence analysis of this fragment revealed the presence of a single open reading frame encoding a protein consisting of 445 amino acid residues. We designated the gene as vcrM. Hydropathy analysis of the deduced amino acid sequence of VcrM suggests the presence of 12 trans-membrane segments. A dendrogram showed that VcrM is a member of the DinF-subfamily within the MATE family of multidrug efflux pumps. Expression of the cloned vcrM gene in drug-hypersensitive Escherichia coli KAM32 cells made them resistant to acriflavine, 4', 6-diamidino-2-phenylindole, Hoechst 33342, rhodamine 6G, tetraphenylphosphonium chloride (TPPCl) and ethidium bromide. Efflux of acriflavine due to VcrM was dependent on Na+ or Li+. Moreover, Na+ efflux was observed with VcrM when TPPCl was added to Na+-loaded cells. Therefore, we conclude that VcrM is a Na+/drug antiporter-type multidrug efflux pump.

Acriflavine↗