Tomofusa Tsuchiya

Publications of Tomofusa Tsuchiya

• Functional study of the novel multidrug efflux pump KexD from Klebsiella pneumoniae.

  Authors: Wakano Ogawa, Motoyasu Onishi, Ruiting Ni, Tomofusa Tsuchiya, Teruo Kuroda

  Gene. 02/2012; 498(2):177-82.

  We cloned a gene, kexD, that provides a multidrug-resistant phenotype from multidrug-resistant Klebsiella pneumoniae MGH78578. The deduced amino acid sequence of KexD is similar to that of the innerWe cloned a gene, kexD, that provides a multidrug-resistant phenotype from multidrug-resistant Klebsiella pneumoniae MGH78578. The deduced amino acid sequence of KexD is similar to that of the inner membrane protein, RND-type multidrug efflux pump. Introduction of the kexD gene into Escherichia coli KAM32 resulted in a MIC that was higher for erythromycin, novobiocin, rhodamine 6G, tetraphenylphosphonium chloride, and ethidium bromide than that of the control. Intracellular ethidium bromide levels in E. coli cells carrying the kexD gene were lower than that in the control cells under energized conditions, suggesting that KexD is a component of an energy-dependent efflux pump. RND-type pumps typically consist of three components: an inner membrane protein, a periplasmic protein, and an outer membrane protein. We discovered that KexD functions with a periplasmic protein, AcrA, from E. coli and K. pneumoniae, but not with the periplasmic proteins KexA and KexG from K. pneumoniae. KexD was able to utilize either TolC of E. coli or KocC of K. pneumoniae as an outer membrane component. kexD mRNA was not detected in K. pneumoniae MGH78578 or ATCC10031. We isolated erythromycin-resistant mutants from K. pneumoniae ATCC10031, and some showed a multidrug-resistant phenotype similar to the drug resistance pattern of KexD. Two strains of multidrug-resistant mutants were investigated for kexD expression; kexD mRNA levels were increased in these strains. We conclude that changing kexD expression can contribute to the occurrence of multidrug-resistant K. pneumoniae.

• Quercetin diacylglycoside analogues showing dual inhibition of DNA gyrase and topoisomerase IV as novel antibacterial agents.

  Authors: Abugafar M L Hossion, Yoshito Zamami, Rafiya K Kandahary, Tomofusa Tsuchiya, Wakano Ogawa, Akimasa Iwado, Kenji Sasaki

  Journal of medicinal chemistry. 06/2011; 54(11):3686-703.

  A structure-guided molecular design approach was used to optimize quercetin diacylglycoside analogues that inhibit bacterial DNA gyrase and topoisomerase IV and show potent antibacterial activityA structure-guided molecular design approach was used to optimize quercetin diacylglycoside analogues that inhibit bacterial DNA gyrase and topoisomerase IV and show potent antibacterial activity against a wide spectrum of relevant pathogens responsible for hospital- and community-acquired infections. In this paper, such novel 3,7-diacylquercetin, quercetin 6''-acylgalactoside, and quercetin 2'',6''-diacylgalactoside analogues of lead compound 1 were prepared to assess their target specificities and preferences in bacteria. The significant enzymatic inhibition of both Escherichia coli DNA gyrase and Staphylococcus aureus topoIV suggest that these compounds are dual inhibitors. Most of the investigated compounds exhibited pronounced inhibition with MIC values ranging from 0.13 to 128 μg/mL toward the growth of multidrug-resistant Gram-positive methicillin-resistant S. aureus, methicillin sensitive S. aureus, vancomycin-resistant enterococci (VRE), vancomycin intermediate S. aureus, and Streptococcus pneumoniae bacterial strains. Structure-activity relationship studies revealed that the acyl moiety was absolutely essential for activity against Gram-positive organisms. The most active compound 5i was 512-fold more potent than vancomycin and 16-32-fold more potent than 1 against VRE strains. It also has realistic in situ intestinal absorption in rats and showed very low acute toxicity in mice. So far, this compound can be regarded as a leading antibacterial agent.

• SugE, a new member of the SMR family of transporters, contributes to antimicrobial resistance in Enterobacter cloacae.

  Authors: Gui-Xin He, Chu Zhang, Robert R Crow, Conner Thorpe, Huizhong Chen, Sanath Kumar, Tomofusa Tsuchiya, Manuel F Varela

  Antimicrobial agents and chemotherapy. 05/2011; 55(8):3954-7.

  We cloned a gene, sugE, from the chromosome of Enterobacter cloacae ATCC 13047. Analysis of the susceptibilities of the sugE-containing strain (Escherichia coli KAM32/pSUGE28) and sugE-deficient E.We cloned a gene, sugE, from the chromosome of Enterobacter cloacae ATCC 13047. Analysis of the susceptibilities of the sugE-containing strain (Escherichia coli KAM32/pSUGE28) and sugE-deficient E. cloacae (EcΔsugE) showed that SugE confers resistance to cetyltrimethylammonium bromide, cetylpyridinium chloride, tetraphenylphosphonium, benzalkonium chloride, ethidium bromide, and sodium dodecyl sulfate. We also investigated expression of sugE. We confirm here that SugE from E. cloacae is an SMR family transporter as determined by observing its energy-dependent drug efflux activity.

• Errata: Properties and Expression of a Multidrug Efflux Pump AcrAB-KocC from Klebsiella pneumoniae[Biol. Pharm. Bull. 31(4): 577-582 (2008)].

  Authors: Dai-Wei Li, Motoyasu Onishi, Takanori Kishino, Taira Matsuo, Wakano Ogawa, Teruo Kuroda, Tomofusa Tsuchiya

  Biological & pharmaceutical bulletin. 01/2011; 34(4):594.

• Patch clamp analysis of the respiratory chain in Bacillus subtilis.

  Authors: Koji Nakamura, Satoshi Ikeda, Taira Matsuo, Aiko Hirata, Masaya Takehara, Tetsuo Hiyama, Fujio Kawamura, Iwao Kusaka, Tomofusa Tsuchiya, Teruo Kuroda, Isamu Yabe

  Biochimica et biophysica acta. 01/2011; 1808(4):1103-7.

  Bacillus subtilis is a representative Gram-positive bacterium. In aerobic conditions, this bacterium can generate an electrochemical potential across the membrane with aerobic respiration. Here, weBacillus subtilis is a representative Gram-positive bacterium. In aerobic conditions, this bacterium can generate an electrochemical potential across the membrane with aerobic respiration. Here, we developed the patch clamp method to analyze the respiratory chain in B. subtilis. First, we prepared giant protoplasts (GPs) from B. subtilis cells. Electron micrographs and fluorescent micrographs revealed that GPs of B. subtilis had a vacuole-like structure and that the intravacuolar area was completely separated from the cytoplasmic area. Acidification of the interior of the isolated and purified vacuole-like structure, due to H(+) translocation after the addition of NADH, revealed that they consisted of everted cytoplasmic membranes. We called these giant provacuoles (GVs) and again applied the patch clamp technique. When NADH was added as an electron donor for the respiratory system, a significant NADH-induced current was observed. Inhibition of KCN and 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) demonstrated that this current is certainly due to aerobic respiration in B. subtilis. This is the first step for more detailed analyses of respiratory chain in B. subtilis, especially H(+) translocation mechanism.

• Design, synthesis, and biological evaluation of a novel series of quercetin diacylglucosides as potent anti-MRSA and anti-VRE agents.

  Authors: Abugafar M L Hossion, Nao Otsuka, Rafiya K Kandahary, Tomofusa Tsuchiya, Wakano Ogawa, Akimasa Iwado, Yoshito Zamami, Kenji Sasaki

  Bioorganic & medicinal chemistry letters. 09/2010; 20(17):5349-52.

  A series of novel quercetin diacylglucosides were designed and first synthesized by Steglich esterification on the basis of MRSA strains inhibiting natural compound A. The in vitro inhibition ofA series of novel quercetin diacylglucosides were designed and first synthesized by Steglich esterification on the basis of MRSA strains inhibiting natural compound A. The in vitro inhibition of different multi-drug resistant bacterial strains and Escherichia coli DNA gyrase B was investigated. In the series, compound 10h was up to 128-fold more potent against vancomycin-resistant enterococci and more effective than A, which represents a promising new candidate as a potent anti-MRSA and anti-VRE agent.

• Gene Cloning and Characteristics of the RND-Type Multidrug Efflux Pumps MuxABC-OpmB possessing two RND components in Pseudomonas aeruginosa.

  Authors: Takehiko Mima, Naoki Kohira, Yang Li, Hiroshi Sekiya, Wakano Ogawa, Teruo Kuroda, Tomofusa Tsuchiya

  Microbiology (Reading, England). 09/2009;

  muxA-muxB-muxC-opmB (formerly PA2528-PA2527-PA2526-opmB), encoding a putative Resistance Nodulation cell Division (RND)-type multidrug efflux pump system was cloned from Pseudomonas aeruginosa PAO1.muxA-muxB-muxC-opmB (formerly PA2528-PA2527-PA2526-opmB), encoding a putative Resistance Nodulation cell Division (RND)-type multidrug efflux pump system was cloned from Pseudomonas aeruginosa PAO1. The P. aeruginosa YM64, a drug hypersusceptible strain, introduced with muxABC-opmB showed elevated MICs of aztreonam, macrolides, novobiocin and tetracycline. Since muxB and muxC, both of which encode RND components, were essential for function, MuxABC-OpmB is thought to be a drug efflux pump with four components. One novobiocin resistant mutant, PMX725, isolated from P. aeruginosa PMX7 showed elevated resistance not only to novobiocin but also to aztreonam, macrolides, and tetracycline. The increased mRNA expression of muxABC-opmB was observed in the mutant PMX725 compared with the parental strain. Sequencing analysis revealed that a single nucleotide insertion occurred in the deduced promoter region for muxABC-opmB in PMX725. In this report, we characterized the last RND-type multidrug efflux pump predicted from genome sequence in P. aeruginosa.

• Gene Cloning and Characterization of EfmA, a Multidrug Efflux Pump, from Enterococcus faecium.

  Authors: Toshihiro Nishioka, Wakano Ogawa, Teruo Kuroda, Takashi Katsu, Tomofusa Tsuchiya

  Biological & pharmaceutical bulletin. 04/2009; 32(3):483-8.

  A DNA fragment responsible for resistance to antimicrobial agents was cloned from chromosomal DNA of Enterococcus faecium FN-1, a clinically isolated strain. Escherichia coli KAM32, aA DNA fragment responsible for resistance to antimicrobial agents was cloned from chromosomal DNA of Enterococcus faecium FN-1, a clinically isolated strain. Escherichia coli KAM32, a drug-hypersusceptible mutant, was used as a host for gene cloning. Cells of E. coli KAM32 harboring a recombinant plasmid (pTFM8) carrying the DNA fragment became resistant to fluoroquinolones, macrolides, ethidium bromide, 4',6-diamidino-2-phenylindole (DAPI) and tetraphenylphosphonium chloride (TPPCl). Three complete open reading frames (ORFs) were found in the DNA insert of pTFM8, and the deduced amino acid sequences of one of the ORFs showed high similarity to Mdt(A) from Lactococcus lactis. Mdt(A) is a multidrug efflux pump belonging to a major facilitator superfamily. We designated the ORF efmA. E. coli KAM32 cells harboring the efmA showed energy-dependent efflux of DAPI and TPP(+). We also observed norfloxacin/H(+) antiport due to EfmA. The mRNA expression of efmA was observed in E. faecium FN-1 grown without any exogenously added antimicrobial agents. Thus, we conclude that efmA is constitutively expressed under laboratory growth conditions and would contribute to intrinsic resistance against multiple antimicrobial agents in E. faecium FN-1.

• Synergistic Effect of Kaempferol Glycosides Purified from Laurus nobilis and Fluoroquinolones on Methicillin-Resistant Staphylococcus aureus.

  Authors: Mei-Hua Liu, Nao Otsuka, Kumiko Noyori, Sumiko Shiota, Wakano Ogawa, Teruo Kuroda, Tsutomu Hatano, Tomofusa Tsuchiya

  Biological & pharmaceutical bulletin. 04/2009; 32(3):489-92.

  In a previous study, we reported that two kaempferol glycosides isolated from Laurus nobilis L., kaempferol-3-O-alpha-L-(2'',4''-di-E-p-coumaroyl)-rhamnoside (C2) andIn a previous study, we reported that two kaempferol glycosides isolated from Laurus nobilis L., kaempferol-3-O-alpha-L-(2'',4''-di-E-p-coumaroyl)-rhamnoside (C2) and kaempferol-3-O-alpha-L-(2''-E-p-coumaroyl-4''-Z-p-coumaroyl)-rhamnoside (C3), showed strong antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci. Thereafter we found that these compounds greatly reduced the minimum inhibitory concentrations (MICs) of some fluoroquinolones in MRSA. In other words, C2 and C3 greatly potentiated anti-MRSA activity of fluoroquinolones. The effect of C2 and C3 with fluoroquinolones was found to be synergistic. The potentiation activity was observed with hydrophilic fluoroquinolones, such as norfloxacin and ciprofloxacin, but not with hydrophobic quinolones. We also found that norfloxacin reduced MICs of C2 and C3. The effect was synergistic. Possible mechanism of the synergistic effect was discussed.

• Modulation of Vibrio mimicus hemolysin through limited proteolysis by an endogenous metalloprotease.

  Authors: Tamaki Mizuno, Syed Z Sultan, Yoshimi Kaneko, Tomonaga Yoshimura, Yoko Maehara, Hiroshi Nakao, Tomofusa Tsuchiya, Sumio Shinoda, Shin-ichi Miyoshi

  The FEBS journal. 03/2009; 276(3):825-34.

  Vibrio mimicus is a causative agent of human gastroenteritis and food poisoning, and this species produces an enterotoxic hemolysin (V. mimicus hemolysin) as a virulence determinant. Vibrio mimicusVibrio mimicus is a causative agent of human gastroenteritis and food poisoning, and this species produces an enterotoxic hemolysin (V. mimicus hemolysin) as a virulence determinant. Vibrio mimicus hemolysin is secreted as an 80 kDa precursor, which is later converted to the 66 kDa mature toxin through removal of an N-terminal propeptide via cleavage of the Arg151-Ser152 bond. In this article, we investigate the role of the endogenous metalloprotease (V. mimicus protease) in the maturation of V. mimicus hemolysin. In vitro experiments using purified proteins showed that, although it activated the precursor at the early stage via cleavage of the Asn157-Val158 bond, V. mimicus protease finally converted the activated and physiologically maturated toxin to a 51 kDa protein through removal of the C-terminal polypeptide. This 51 kDa derivative was unable to lyse erythrocytes because of its inability to bind to the erythrocyte membrane. Vibrio mimicus protease-negative strains were found to produce high levels of V. mimicus hemolysin at the logarithmic phase of bacterial growth and maintained high hemolytic activity even at the stationary phase. These findings indicate that, although it is not directly related to toxin maturation in vivo, V. mimicus protease can modulate the activity of V. mimicus hemolysin and/or its precursor through limited proteolysis.

• Multidrug efflux transporters in the MATE family.

  Authors: Teruo Kuroda, Tomofusa Tsuchiya

  Biochimica et biophysica acta. 01/2009;

  The MATE (Multidrug And Toxic Compound Extrusion) family is the most recently categorized, one among five multidrug efflux transporter families. As far as we know, about twenty MATE transporters haveThe MATE (Multidrug And Toxic Compound Extrusion) family is the most recently categorized, one among five multidrug efflux transporter families. As far as we know, about twenty MATE transporters have been characterized so far. According to the information in sequence databases, huge numbers of MATE transporters seem to be present in various microorganisms. In this review, we would like to summarize the properties of the MATE-family transporters.

• Anti-methicillin Resistant Staphylococcus aureus (MRSA) Compounds Isolated from Laurus nobilis.

  Authors: Nao Otsuka, Mei-Hua Liu, Sumiko Shiota, Wakano Ogawa, Teruo Kuroda, Tsutomu Hatano, Tomofusa Tsuchiya

  Biological & pharmaceutical bulletin. 10/2008; 31(9):1794-7.

  We found that an extract from Laurus nobilis L. (Lauraceae) leaves showed antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). We purified two flavonoids as theWe found that an extract from Laurus nobilis L. (Lauraceae) leaves showed antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). We purified two flavonoids as the effective compounds and identified them as kaempferol 3-O-alpha-L-(2'',4''-di-E-p-coumaroyl)-rhamnoside (C2) and kaempferol 3-O-alpha-L-(2''-Z-p-coumaroyl-4''-E-p-coumaroyl)-rhamnoside (C3). Both compounds showed strong antibacterial activity not only against MRSA but also against vancomycin-resistant enterococci (VRE). There was low or no antibacterial activity of C2 and C3 for Streptococcus pneumoniae, Pseudomonas aeruginosa and Serratia marcescens.

• Expression and function of TETRAN, a new type of membrane transporter.

  Authors: Hironori Ushijima, Miki Hiasa, Takushi Namba, Hyun-Jung Hwang, Tatsuya Hoshino, Shinji Mima, Tomofusa Tsuchiya, Yoshinori Moriyama, Tohru Mizushima

  Biochemical and biophysical research communications. 09/2008; 374(2):325-30.

  In contrast to transport across basolateral membranes, the mechanism governing transport of organic anions across the luminal membranes of proximal tubules has remained unclear. We recently foundIn contrast to transport across basolateral membranes, the mechanism governing transport of organic anions across the luminal membranes of proximal tubules has remained unclear. We recently found Tetracycline transporter-like protein (TETRAN), a human ortholog of yeast Tpo1p that can transport anionic Non-steroidal anti-inflammatory drugs (NSAIDs). In this study, we examine the expression and function of TETRAN. TETRAN mRNA is expressed in various human tissues, including kidney. When overexpressed in cultured cells, TETRAN was predominantly localized on cytoplasmic membranes. Immunohistochemical analysis of human and mouse kidney tissue showed that TETRAN was expressed at the luminal membranes of proximal tubules. Overexpression of TETRAN in cultured cells facilitated the uptake of organic anions such as indomethacin (a NSAID) and fluorescein. The results suggest that TETRAN is a novel human organic anion transporter, and that it serves as a transporter for some NSAIDs and various other organic anions at the final excretion step.

• Variation of extracellular proteases produced by Vibrio vulnificus clinical isolates: genetic diversity of the metalloprotease gene (vvp), and serine protease secretion by vvp-negative strains.

  Authors: Jiyou Wang, Tomoko Sasaki, Yoko Maehara, Hiroshi Nakao, Tomofusa Tsuchiya, Shin-ichi Miyoshi

  Microbial pathogenesis. 07/2008; 44(6):494-500.

  Vibrio vulnificus is a causative agent of septicemia or wound infection in human and eel; however, the genetic variation between human and eel isolates has been reported. In the present study, theVibrio vulnificus is a causative agent of septicemia or wound infection in human and eel; however, the genetic variation between human and eel isolates has been reported. In the present study, the difference in the vvp gene encoding a tissue-damaging metalloprotease was investigated. The gene of strain E86 from a diseased eel (type B vvp) was 95.2% identical with that of strain L-180 from human blood (type A vvp). PCR using oligonucleotide primers designed to differentiate two types of the gene showed that eel avirulent strains (9 isolates) commonly carry type A vvp, whereas eel virulent strains (18 isolates) revealed significant genetic variation. The vvp genes from 12 strains including strain E86 were placed on type B while those from 3 strains were on type A. Other strains were found to be vvp-negative, but PAGE and amino acid sequencing analysis showed that they secreted a serine protease (VVA0302) instead of the metalloprotease. This protease is an orthologue of a toxic protease from Vibrio parahaemolyticus, a human pathogen causing wound infection as well as gastroenteritis. These findings suggest that, in addition to metalloprotease, the extracellular serine protease may contribute to pathogenicity of V. vulnificus.

• Properties and expression of a multidrug efflux pump AcrAB-KocC from Klebsiella pneumoniae.

  Authors: Dai-Wei Li, Motoyasu Onishi, Takanori Kishino, Taira Matsuo, Wakano Ogawa, Teruo Kuroda, Tomofusa Tsuchiya

  Biological & pharmaceutical bulletin. 05/2008; 31(4):577-82.

  We previously reported that we had cloned genes responsible for multidrug resistance from the chromosomal DNA of Klebsiella pneumoniae MGH78578 using a drug-hypersusceptible Escherichia coli strainWe previously reported that we had cloned genes responsible for multidrug resistance from the chromosomal DNA of Klebsiella pneumoniae MGH78578 using a drug-hypersusceptible Escherichia coli strain as a host. One of the recombinant plasmids pETV6 conferred resistance to host cells against a wide range of antimicrobial agents, dyes and detergents. It was revealed that this plasmid carried the acrBKp gene and a part of the acrAKp gene coding for a multidrug efflux pump belonging to the RND family. We cloned the whole acrAKpBKp operon of K. pneumoniae and characterized the pump. The AcrAB pump utilized TolC as an outer membrane component in cells of E. coli. Elevated energy-dependent efflux of ethidium was observed with cells possessing AcrAKp BKp-TolC. We cloned a gene coding for an ortholog of TolC from chromosomal DNA of K. pneumoniae, and designated it kocC. It seems that the AcrAKpBKp-KocC complex functions as a potent multidrug efflux pump in K. pneumoniae. We observed a higher level of expression of acrAKp in K. pneumoniae MGH78578, a multidrug resistant strain, compared with ATCC10031, a drug susceptible strain.

• Functional gene cloning and characterization of the SsmE multidrug efflux pump from Serratia marcescens.

  Authors: Yusuke Minato, Fereshteh Shahcheraghi, Wakano Ogawa, Teruo Kuroda, Tomofusa Tsuchiya

  Biological & pharmaceutical bulletin. 04/2008; 31(3):516-9.

  We cloned a gene responsible for multidrug resistance from chromosomal DNA of Serratia marcescens, and determined the nucleotide sequence. We designated the gene as ssmE. The deduced amino acidWe cloned a gene responsible for multidrug resistance from chromosomal DNA of Serratia marcescens, and determined the nucleotide sequence. We designated the gene as ssmE. The deduced amino acid sequence of SsmE showed high similarity with the small multidrug resistance (SMR)-type multidrug efflux pumps. Cells of Escherichia coli KAM32, a drug hyper-susceptible mutant, transformed with a plasmid pESM437 carrying the ssmE gene showed elevated minimum inhibitory concentrations of structurally unrelated antimicrobial agents. E. coli KAM32/pESM437 showed elevated energy dependent efflux of ethidium. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that ssmE was expressed in cells of S. marcescens.

• SmdAB, a heterodimeric ABC-Type multidrug efflux pump, in Serratia marcescens.

  Authors: Taira Matsuo, Jing Chen, Yusuke Minato, Wakano Ogawa, Tohru Mizushima, Teruo Kuroda, Tomofusa Tsuchiya

  Journal of bacteriology. 02/2008; 190(2):648-54.

  We cloned genes, designated smdAB, that encode a multidrug efflux pump from the chromosomal DNA of clinically isolated Serratia marcescens NUSM8906. For cells of the drug-hypersensitive strainWe cloned genes, designated smdAB, that encode a multidrug efflux pump from the chromosomal DNA of clinically isolated Serratia marcescens NUSM8906. For cells of the drug-hypersensitive strain Escherichia coli KAM32 harboring a recombinant plasmid carrying smdAB, structurally unrelated antimicrobial agents such as norfloxacin, tetracycline, 4',6-diamidino-2-phenylindole (DAPI), and Hoechst 33342 showed elevated MICs. The deduced amino acid sequences of both SmdA and SmdB exhibited similarities to the sequences of ATP-binding cassette (ABC)-type multidrug efflux pumps. The efflux of DAPI and Hoechst 33342 from E. coli cells expressing SmdAB was observed, and the efflux activities were inhibited by sodium o-vanadate, which is a well-known ATPase inhibitor. The introduction of smdA or smdB alone into E. coli KAM32 did not elevate the MIC of DAPI; thus, both SmdA and SmdB were required for function. These results indicate that SmdAB is probably a heterodimeric multidrug efflux pump of the ABC family in S. marcescens.

• VmeAB, an RND-type multidrug efflux transporter in Vibrio parahaemolyticus.

  Authors: Taira Matsuo, Katsuhiko Hayashi, Yuji Morita, Motohiro Koterasawa, Wakano Ogawa, Tohru Mizushima, Tomofusa Tsuchiya, Teruo Kuroda

  Microbiology (Reading, England). 01/2008; 153(Pt 12):4129-37.

  Genes vmeA and vmeB, encoding a multidrug efflux transporter in the halophilic bacterium Vibrio parahaemolyticus, have been cloned using a drug-hypersusceptible Escherichia coli strain as the host.Genes vmeA and vmeB, encoding a multidrug efflux transporter in the halophilic bacterium Vibrio parahaemolyticus, have been cloned using a drug-hypersusceptible Escherichia coli strain as the host. Cells of E. coli KAM33 (DeltaacrAB DeltaydhE) carrying the vmeAB region from V. parahaemolyticus conferred much higher MICs for a variety of antimicrobial agents than did control cells. Cells possessing VmeAB under energized conditions maintained very low intracellular concentrations of ethidium. This was as expected for an energy-dependent efflux system, and supports the notion--based on sequence homology--that VmeAB belongs to the resistance nodulation cell division (RND) family of multidrug efflux transporters. It is likely that VmeAB forms functional complexes with the outer-membrane protein TolC in E. coli, because introduction of vmeAB into cells of E. coli KAM43, which lacks the tolC gene, failed to elevate the MICs for any of the antimicrobial agents tested. Therefore, a V. parahaemolyticus homologue of tolC was also cloned, designated vpoC, and was introduced together with vmeAB into cells of E. coli KAM43. The MICs of all agents tested were raised and were comparable to the values observed in E. coli KAM33 harbouring a plasmid carrying vmeAB. Finally, a vmeAB-deficient mutant of V. parahaemolyticus was constructed (designated TM3). TM3 showed slightly higher susceptibility than the parental V. parahaemolyticus to some antimicrobial agents. Survival rate of the TM3 when exposed to deoxycholate decreased compared with that of the parent.

• Gene cloning and characterization of KdeA, a multidrug efflux pump from Klebsiella pneumoniae.

  Authors: Yu Ping, Wakano Ogawa, Teruo Kuroda, Tomofusa Tsuchiya

  Biological & pharmaceutical bulletin. 11/2007; 30(10):1962-4.

  We cloned a gene related to multidrug resistance from a drug-resistant Klebsiella pneumoniae strain MGH78578. We designated the gene kdeA, which encodes a protein possessing 12 hydrophobic regions.We cloned a gene related to multidrug resistance from a drug-resistant Klebsiella pneumoniae strain MGH78578. We designated the gene kdeA, which encodes a protein possessing 12 hydrophobic regions. The deduced amino acid sequence of KdeA is similar to that of MdfA, a well-characterized multidrug efflux pump from Escherichia coli. Introduction of the kdeA gene into cells of the drug-hypersusceptible E. coli strain KAM32 resulted in elevated minimum inhibitory concentrations of chloramphenicol, norfloxacin, acriflavine, and ethidium bromide. We observed elevated energy-dependent ethidium efflux activity with cells carrying kdeA compared with control cells. We also observed expression of kdeA in cells of K. pneumoniae under normal growth conditions.

• Enhancement of antibacterial effects of epigallocatechin gallate, using ascorbic acid.

  Authors: Tsutomu Hatano, Mayumi Tsugawa, Miwako Kusuda, Shoko Taniguchi, Takashi Yoshida, Sumiko Shiota, Tomofusa Tsuchiya

  Phytochemistry. 10/2007;

  Although plant polyphenols such as (-)-epigallocatechin gallate (EGCG) have antibacterial activity towards methicillin-resistant Staphylococcus aureus (MRSA), such polyphenols are unstable inAlthough plant polyphenols such as (-)-epigallocatechin gallate (EGCG) have antibacterial activity towards methicillin-resistant Staphylococcus aureus (MRSA), such polyphenols are unstable in solution. Because the instability of polyphenols is attributable to their oxidation, we examined the effects of antioxidants and inhibitors of polyphenol oxidation on the maintenance of polyphenol antibacterial activity. The antibacterial activity of EGCG was enhanced in the presence of ascorbic acid, and ascorbic acid was the most effective for retaining the concentration of stable EGCG. On the other hand, the antibacterial activity of EGCG was lowered in the presence of casein in spite of its suppressing effect on the EGCG decrease. The effect of EGCG on the antibiotic resistance of MRSA was also enhanced in the presence of ascorbic acid. The addition of an antioxidant may affect other pharmacological effects of polyphenols in analogous ways, although this does not mean the clinical usefulness of the addition directly.