Wakano Ogawa

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. Energy‐dependent 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.

Gene cloning and characteristics of the RND-type multidrug efflux pump MuxABC-OpmB possessing two RND components in Pseudomonas aeruginosa

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. Introduction of muxABC-opmB into P. aeruginosa YM64, a drug-hypersusceptible strain, led to 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. 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 had occurred in the deduced promoter region for muxABC-opmB in PMX725. In this study, we have characterized the last RND-type multidrug efflux pump predicted from the genome sequence in P. aeruginosa.

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

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 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.

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

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 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.

Molecular cloning and characterization of all RND-type efflux transporters in Vibrio cholerae non-O1.

Resistance Nodulation cell Division (RND) efflux transporters are thought to be involved in mediating multidrug resistance in Gram‐negative bacteria, including Vibrio cholerae non‐O1. There are six operons for putative RND‐type efflux transporters present in the chromosome of V. cholerae O1 including two operons, vexAB and vexCD, which had already been identified. All of the six operons were cloned from V. cholerae non‐O1, NCTC4716 by the PCR method, introduced, and expressed in cells of drug hypersusceptible Escherichia coli KAM33 (AacrAB, AydhE). Only vexEF conferred elevated minimum inhibitory concentrations (MICs) of some antimicrobial agents in the E. coli cells. However, VexEF did not confer increased MIC of any drug tested in tolC‐deficient E. coli KAM43 cells. On the other hand, when E. coli KAM43 was transformed with vexAB, vexCD or vexEF together with tolCVc of V. cholerae NCTC4716, we observed elevated MICs of various antimicrobial agents. Among them, E. coli KAM43 expressing both VexEF and TolCVc showed much higher MICs and much broader substrate specificity than the other two. We also observed ethidium efflux activity via VexEF‐TolCVc, and the activity required Na+. Thus, VexEF‐TolCVc is either a Na+‐activated or a Na+‐coupled transporter. To our knowledge, this is the first report on the requirement of Na+ for an RND‐type efflux transporter.

Characterization of all RND-type multidrug efflux transporters in Vibrio parahaemolyticus.

Resistance nodulation cell division (RND)‐type efflux transporters play the main role in intrinsic resistance to various antimicrobial agents in many gram‐negative bacteria. Here, we estimated 12 RND‐type efflux transporter genes in Vibrio parahaemolyticus. Because VmeAB has already been characterized, we cloned the other 11 RND‐type efflux transporter genes and characterized them in Escherichia coli KAM33 cells, a drug hypersusceptible strain. KAM33 expressing either VmeCD, VmeEF, or VmeYZ showed increased minimum inhibitory concentrations (MICs) for several antimicrobial agents. Additional four RND‐type transporters were functional as efflux pumps only when co‐expressed with VpoC, an outer membrane component in V. parahaemolyticus. Furthermore, VmeCD, VmeEF, and VmeYZ co‐expressed with VpoC exhibited a broader substrate specificity and conferred higher resistance than that with TolC of E. coli. Deletion mutants of these transporter genes were constructed in V. parahaemolyticus. TM32 (ΔvmeAB and ΔvmeCD) had significantly decreased MICs for many antimicrobial agents and the number of viable cells after exposure to deoxycholate were markedly reduced. Strains in which 12 operons were all disrupted had very low MICs and much lower fluid accumulation in rabbit ileal loops. These results indicate that resistance nodulation cell division‐type efflux transporters contribute not only to intrinsic resistance but also to exerting the virulence of V. parahaemolyticus.

Functionally cloned pdrM from Streptococcus pneumoniae encodes a Na(+) coupled multidrug efflux pump.

Multidrug efflux pumps play an important role as a self-defense system in bacteria. Bacterial multidrug efflux pumps are classified into five families based on structure and coupling energy: resistance−nodulation−cell division (RND), small multidrug resistance (SMR), major facilitator (MF), ATP binding cassette (ABC), and multidrug and toxic compounds extrusion (MATE). We cloned a gene encoding a MATE-type multidrug efflux pump from Streptococcus pneumoniae R6, and designated it pdrM. PdrM showed sequence similarity with NorM from Vibrio parahaemolyticus, YdhE from Escherichia coli, and other bacterial MATE-type multidrug efflux pumps. Heterologous expression of PdrM let to elevated resistance to several antibacterial agents, norfloxacin, acriflavine, and 4′,6-diamidino-2-phenylindole (DAPI) in E. coli KAM32 cells. PdrM effluxes acriflavine and DAPI in a Na+- or Li+-dependent manner. Moreover, Na+ efflux via PdrM was observed when acriflavine was added to Na+-loaded cells expressing pdrM. Therefore, we conclude that PdrM is a Na+/drug antiporter in S. pneumoniae. In addition to pdrM, we found another two genes, spr1756 and spr1877,that met the criteria of MATE-type by searching the S. pneumoniae genome database. However, cloned spr1756 and spr1877 did not elevate the MIC of any of the investigated drugs. mRNA expression of spr1756, spr1877, and pdrM was detected in S. pneumoniae R6 under laboratory growth conditions. Therefore, spr1756 and spr1877 are supposed to play physiological roles in this growth condition, but they may be unrelated to drug resistance.

Properties of a Na+/galactose(glucose) symport system in Vibrio parahaemolyticus

We have investigated galactose transport in a mutant strain of Vibrio parahaemolyticus that lacks a glucose-PTS (phosphoenolpyruvate:carbohydrate phosphotransferase system) and a trehalose-PTS. Cells of the V. parahaemolyticus actively transported D-galactose and Na+ greatly stimulated the transport. Maximum stimulation of D-galactose transport activity was observed at 10mM NaCl, and Na+ could be replaced with Li+. Addition of galactose to the cell suspension under anaerobic conditions elicited Na+ uptake. Therefore, we conclude that this organism accomplishes galactose transport by a Na+/solute symport mechanism. Judging from inhibition results, D-galactose, D-glucose and to a lesser extent alpha-D-fucose are substrates of this transport system. The Na+/galactose symport system exhibited a high affinity for D-galactose (Km: 40 microM) and showed a relatively lower affinity for D-glucose (Km: 420 microM), but the maximum velocities for galactose and glucose transport were almost same (about 52 nmol/min per mg protein). The Na+/D-galactose symport system was induced by either D-galactose or alpha-D-fucose, and repressed by D-glucose.

Riccardin C derivatives cause cell leakage in Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major problem in clinical settings, and because it is resistant to most antimicrobial agents, MRSA infections are difficult to treat. We previously reported that synthetic macrocyclic bis(bibenzyl) derivatives, which were originally discovered in liverworts, had anti-MRSA activity. However, the action mechanism responsible was unclear. In the present study, we elucidated the action mechanism of macrocyclic bis(bibenzyl) RC-112 and its partial structure, IDPO-9 (2-phenoxyphenol). Survival experiments demonstrated that RC-112 had a bactericidal effect on MRSA, whereas IDPO-9 had bacteriostatic effects. IDPO-9-resistant mutants exhibited cross-resistance to triclosan, but not to RC-112. The mutation was identified in the fabI, enoyl-acyl carrier protein reductase gene, a target of triclosan. We have not yet isolated the RC-112-resistant mutant. On the other hand, the addition of RC-112, unlike IDPO-9, caused the inflow of ethidium and propidium into S. aureus cells. RC-112-dependent ethidium outflow was observed in ethidium-loaded S. aureus cells. Transmission electron microscopy also revealed that S. aureus cells treated with RC-112 had intracellular lamellar mesosomal-like structures. Intracellular Na+ and K+ concentrations were significantly changed by the RC-112 treatment. These results indicated that RC-112 increased membrane permeability to ethidium, propidium, Na+, and K+, and also that the action mechanism of IDPO-9 was different from those of the other compounds.

S-nitrosated α-1-acid glycoprotein kills drug-resistant bacteria and aids survival in sepsis

Treating infections with exogenous NO, which shows broad‐spectrum antimicrobial activity, appears to be effective. Similar to NO biosynthesis, biosynthesis of α‐1‐acid glycoprotein variant A (AGPa), with a reduced cysteine (Cys149), increases markedly during inflammation and infection. We hypothesized that AGPa is an S‐nitrosation target in acute‐phase proteins. This study aimed to determine whether S‐nitrosated AGPa (SNO‐AGPa) may be the first compound of this novel antibacterial class against multidrug‐resistant bacteria. AGPa was incubated with RAW264.7 cells activated by lipopolysaccharide and interferon‐γ. The antimicrobial effects of SNO‐AGPa were determined by measuring the turbidity of the bacterial suspensions in vitro and survival in a murine sepsis model in vivo, respectively. Results indicated that endogenous NO generated by activated RAW264.7 cells caused S‐nitrosation of AGPa at Cys149. SNO‐AGPa strongly inhibited growth of gram‐positive, gram‐negative, and multidrug‐resistant bacteria and was an extremely potent bacteriostatic compound (IC50: 10–9 to 10–6 M). The antibacterial mechanism of SNO‐AGPa involves S‐transnitrosation from SNO‐AGPa to bacterial cells. Treatment with SNO‐AGPa, but not with AGPa, markedly reduced bacterial counts in blood and liver in a mouse sepsis model. The sialyl residues of AGPa seem to suppress the antibacterial activity, since SNO‐asialo AGPa was more potent than SNO‐AGPa.—Watanabe, K., Ishima, Y., Akaike, T., Sawa, T., Kuroda, T., Ogawa, W., Watanabe, H., Suenaga, A., Kai, T., Otagiri, M., Maruyama, T. S‐nitrosated α‐1‐acid glycoprotein kills drug‐resistant bacteria and aids survival in sepsis. FASEB J. 27, 391–398 (2013). www.fasebj.org