Rv1218c, an ABC transporter of Mycobacterium tuberculosis with implications in drug discovery

Efflux systems are important in determining the efficacy of antibiotics used in the treatment of bacterial infections. In the last decade much attention has been paid to studying the efflux pumps of mycobacteria. New classes of compounds are under investigation for development into potential candidate drugs for the treatment of tuberculosis. Quite often, these have poor bactericidal activities but exhibit excellent target (biochemical) inhibition. Microarray studies conducted in our laboratories for deciphering the mode of action of experimental drugs revealed the presence of putative ABC transporters. Among these transporters, Rv1218c was chosen for studying its physiological relevance in mediating efflux in Mycobacterium tuberculosis. A ΔRv1218c mutant of M. tuberculosis displayed a 4- to 8-fold increase in the inhibitory and bactericidal potency for different classes of compounds. The MICs and MBCs were reversed to wild-type values when the full-length Rv1218c gene was reintroduced into the ΔRv1218c mutant on a multicopy plasmid. Most of the compound classes had significantly better bactericidal activity in the ΔRv1218c mutant than in the wild-type H37Rv, suggesting the involvement of Rv1218c gene product in effluxing these compounds from M. tuberculosis. The implication of these findings on tuberculosis drug discovery is discussed.     

MmpL3 is the cellular target of the antitubercular pyrrole derivative BM212

The 1,5-diarylpyrrole derivative BM212 was previously shown to be active against multidrug-resistant clinical isolates and Mycobacterium tuberculosis residing within macrophages as well as against Mycobacterium avium and other atypical mycobacteria. To determine its mechanism of action, we identified the cellular target. Spontaneous Mycobacterium smegmatis, Mycobacterium bovis BCG, and M. tuberculosis H37Rv mutants that were resistant to BM212 were isolated. By the screening of genomic libraries and by whole-genome sequencing, we found that all the characterized mutants showed mutations in the mmpL3 gene, allowing us to conclude that resistance to BM212 maps to the MmpL3 protein, a member of the MmpL (mycobacterial membrane protein, large) family. Susceptibility was unaffected by the efflux pump inhibitors reserpine, carbonylcyanide m-chlorophenylhydrazone, and verapamil. Uptake/efflux experiments with [(14)C]BM212 demonstrated that resistance is not driven by the efflux of BM212. Together, these data strongly suggest that the MmpL3 protein is the cellular target of BM212.     

Contribution of the Rv2333c efflux pump (the Stp protein) from Mycobacterium tuberculosis to intrinsic antibiotic resistance in Mycobacterium bovis BCG

OBJECTIVES: To characterize the efflux pump encoded by the gene Rv2333c from Mycobacterium tuberculosis, and assess its contribution to intrinsic antibiotic resistance using Mycobacterium bovis BCG as a model organism. METHODS: Firstly, the Rv2333c gene was expressed from a multicopy plasmid in M. bovis BCG. Secondly, the gene was inactivated in the chromosome of M. bovis BCG. Antibiotic susceptibility tests and tetracycline uptake/efflux experiments were carried out with the strains mentioned above. RESULTS: When the Rv2333c gene was inactivated in the M. bovis BCG chromosome, there was a decrease in the MIC values of spectinomycin and tetracycline, and an increase in [3H]tetracycline accumulation. When the Rv2333c gene was cloned into a multicopy plasmid, there was an increase in the MIC values of spectinomycin and tetracycline, and a decrease in [3H]tetracycline accumulation. These results indicate that both antibiotics are substrates of the Rv2333c efflux pump, which has been named Stp, for Spectinomycin Tetracycline efflux Pump. CONCLUSIONS: The Rv2333c efflux pump (Stp protein) of M. tuberculosis contributes to intrinsic spectinomycin and tetracycline resistance.     

Rv2686c-Rv2687c-Rv2688c, an ABC fluoroquinolone efflux pump in Mycobacterium tuberculosis

The Mycobacterium tuberculosis Rv2686c-Rv2687c-Rv2688c operon, encoding an ABC transporter, conferred resistance to ciprofloxacin and, to a lesser extent, norfloxacin, moxifloxacin, and sparfloxacin to Mycobacterium smegmatis. The resistance level decreased in the presence of the efflux pump inhibitors reserpine, carbonyl cyanide m-chlorophenylhydrazone, and verapamil. Energy-dependent efflux of ciprofloxacin from M. smegmatis cells containing the Rv2686c-Rv2687c-Rv2688c operon was observed.     

铜绿假单胞菌对碳青霉烯类抗生素的耐药表型与外排泵表达水平的关系

目的 了解铜绿假单胞菌(Pseudomonas aeruginosa,Pa)临床菌株外排泵抑制剂对碳青霉烯类抗生素活性的影响;探索铜绿假单胞菌对亚胺培南(IMP)和美罗培南(MEP)的耐药性与其外排泵表达水平关系.方法 对IMP耐药的Pa采用琼脂对倍稀释法进行外排泵抑制剂carbanyl cyanide m-chlorophenylhydrazone(CCCP,107株)与Pile-Arg-β-naphthylamide(PAβN,71株)的抑制试验,观察IMP和MEP的MIC变化;对32株对IMP和MEP不同耐药表型的Pa,采用实时荧光定量PCR法检测3种外排泵基因(mexA、mexD、mexF)的表达量.结果 联合外排泵抑制剂后,IMP、MEP耐药率与之前相比差异均无统计学意义,其中IMP联合CCCP、PApN前后耐药率X2值分别为0.338和0.086,P＞0.05;MEP联合CCCP、PABN前后耐药率X2值分别为1.065和1.458,P＞0.05.MIC值没有变化的菌株占50%以上;仅8株P且的MIC值降至原MIC值的1/4.在27株碳青霉烯类耐药Pa株中,24株(88.9%)存在外排泵高表达;其中3种外排泵(MexAB-OprM、MexCD-OprJ、MexEF-OprN)均高表达的菌株数量最多,13株,占54.2%.MexAB-OprM和MexCD-OprJ同时高表达以及MexAB-OprM和MexEF-OprN同时高表达菌株分别有3株,各占12.5%.仅MexEF-OprN高表达及仅MexAB-OprM高表达的菌株分别为2株,各占8.3%;未见仅MexCD-OprJ高表达者.3种外排泵基因mexA、mexD、mexF在对IMP及MEP均敏感的菌株中表达水平分别为0.48±0.48、0.48±0.53和0.30±0.41,与碳青霉烯类耐药组之间差异均有统计学意义(P＜0.05),碳青霉烯类耐药组的表达水平高于对IMP及MEP均敏感组的表达水平.MexA的表达水平在IMP、MEP均耐药组和IMP耐药、MEP敏感组间差别有统计学意义,前者高于后者.结论 当外排泵抑制剂CCCP和PAβN浓度分别为5μg/ml和20μg/ml时,对碳青霉烯类抗Pa活性影响较小,不能明显增强其对耐药菌的抗菌活性.MexAB-OprM的高表达与MEP耐药性有关,而MexCD-OprJ和MexEF-OprN的高表达与IMP耐药性有关,与MEP耐药性的关系尚有待进一步研究.     

Relative contributions of the AcrAB,MdfA and NorE efflux pumps to quinolone resistance in Escherichia coli

Quinolones are widely used, broad-spectrum antimicrobial agents. In screens for genes that, when overexpressed, allow Escherichia coli to grow on otherwise lethal concentrations of the fluoroquinolone norfloxacin, the ydhE gene was identified. We have shown that ydhE encodes a multidrug efflux pump with a narrower substrate range than that of its closest homologue, encoded by norM, and named the gene norE. The relative contributions to drug resistance of NorE compared with the two other known E. coli quinolone pumps, AcrAB and MdfA, have been defined. Overexpression of each of the three pumps separately resulted in roughly similar levels of quinolone resistance, whereas simultaneous overexpression of norE or mdfA in combination with acrAB gave synergic increases in quinolone resistance. The level of quinolone resistance mediated by efflux pumps seems to be constrained to an approximately 10-fold maximum, even with increased production of the pumps. We measured the drug resistance of an isogenic set of strains containing the various permutations of single, double and triple drug efflux pump mutants. The DeltanorE and DeltamdfA mutants were somewhat more susceptible to fluoroquinolones than the parent strain, and acrAB mutants were four- to six-fold more susceptible. Mutants lacking two or all three efflux pumps were not significantly more susceptible to fluoroquinolones than those lacking only one of the three pumps.     

Phenothiazines and thioxanthenes inhibit multidrug efflux pump activity in Staphylococcus aureus

Efflux-related multidrug resistance (MDR) is a significant means by which bacteria can evade the effects of selected antimicrobial agents. Genome sequencing data suggest that Staphylococcus aureus may possess numerous chromosomally encoded MDR efflux pumps, most of which have not been characterized. Inhibition of these pumps, which may restore clinically relevant activity of antimicrobial agents that are substrates for them, may be an effective alternative to the search for new antimicrobial agents that are not substrates. The inhibitory effects of selected phenothiazines and two geometric stereoisomers of the thioxanthene flupentixol were studied using strains of S. aureus possessing unique efflux-related MDR phenotypes. These compounds had some intrinsic antimicrobial activity and, when combined with common MDR efflux pump substrates, resulted in additive or synergistic interactions. For S. aureus SA-1199B, which overexpresses the NorA MDR efflux pump, and for two additional strains of S. aureus having non-NorA-mediated MDR phenotypes, the 50% inhibitory concentration (IC(50)) for ethidium efflux for all tested compounds was between 4 and 15% of their respective MICs. Transport of other substrates was less susceptible to inhibition; the prochlorperazine IC(50) for acriflavine and pyronin Y efflux by SA-1199B was more than 60% of its MIC. Prochlorperazine and trans(E)-flupentixol were found to reduce the proton motive force (PMF) of S. aureus by way of a reduction in the transmembrane potential. We conclude that the mechanism by which phenothiazines and thioxanthenes inhibit efflux by PMF-dependent pumps is multifactorial and, because of the unbalanced effect of these compounds on the MICs and the efflux of different substrates, may involve an interaction with the pump itself and, to a lesser extent, a reduction in the transmembrane potential.     

Characterization of P55, a multidrug efflux pump in Mycobacterium bovis and Mycobacterium tuberculosis

The Mycobacterium bovis P55 gene, located downstream from the gene that encodes the immunogenic lipoprotein P27, has been characterized. The gene was identical to the open reading frame of the Rv1410c gene in the genome of Mycobacterium tuberculosis H37Rv, annotated as a probable drug efflux protein. Genes similar to P55 were present in all species of the M. tuberculosis complex and other mycobacteria such as Mycobacterium leprae and Mycobacterium avium. By Western blotting, P55 was located in the membrane fraction of M. bovis. When transformed into Mycobacterium smegmatis after cloning, P55 conferred aminoglycoside and tetracycline resistance. The levels of resistance to streptomycin and tetracycline conferred by P55 were decreased in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone and the pump inhibitors verapamil and reserpine. M. smegmatis cells expressing the plasmid-encoded P55 accumulated less tetracycline than the control cells. We conclude that P55 is a membrane protein implicated in aminoglycoside and tetracycline efflux in mycobacteria.     

Enhanced killing of intracellular multidrug-resistant Mycobacterium tuberculosis by compounds that affect the activity of efflux pumps

Whereas human neutrophils are effective and efficient killers of bacteria, macrophages such as those derived from monocytes are almost devoid of killing activity. Nevertheless, monocytes can be transformed into effective killers of mycobacteria or staphylococci when exposed to clinical concentrations of a phenothiazine or to inhibitors of efflux pumps (reserpine and verapamil), or to ouabain, an inhibitor of K(+) transport. Because the rates of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) continue to escalate globally, and because no new effective drug has been made available for almost 40 years, compounds that enhance the killing activity of monocytes against MDR-TB are obviously needed. This review covers the specific characteristics of MDR-TB, identifies a variety of agents that address these characteristics and therefore have potential for managing MDR-TB. Because the mechanism by which these agents enhance the killing of intracellular bacteria is important for the intelligent design of new anti-tubercular agents, the review correlates the mechanisms by which these agents manifest their effects. Lastly, a model is presented which describes the mechanisms by which distinct efflux pumps of the phagosome-lysosome complex are inhibited by agents that are known to inhibit K(+) flux. The model also predicts the existence of a K(+) activated exchange (pump) that is probably located in the membrane that delineates the lysosome. This putative pump, which is immune to inhibitors of K+ flux, is identified as being the cause for the acidification of the lysosome thereby activating its hydrolytic enzymes. Because the non-killer macrophage can be transformed into an effective killer by a variety of compounds that inhibit K(+) transport, perhaps it would be wise to develop drugs that enhance the killing activity of these cells inasmuch as this approach would not be subject to any resistance, as is the eventual case for conventional antibiotics.     

多重耐药铜绿假单胞菌外排泵基因表达与耐药表型和耐药程度的关系

目的建立实时荧光定量PCR法检测多重耐药铜绿假单胞菌(Pa)的外排泵基因表达量,探讨不同类型外排泵基因表达与耐药表型和耐药程度的关系。方法收集临床分离的多重耐药Pa共80株,分析其耐药表型;用实时荧光定量PCR检测不同类型外排泵基因的表达量,用外排泵抑制剂CCCP及PAβN进行外排泵基因筛查,并比较其结果。结果 64株(80%)检出了不同类型外排泵基因,包括单独表达MexA 30株(37.5%),MexC 12株(15.0%),MexX 8株(10.0%),同时表达MexA和MexC者10株(12.5%),同时表达MexA和MexX者4株(5.0%)。外排泵抑制剂筛查试验总阳性率分别为CCCP抑制法73.8%,PAβN抑制法72.5%。MexA的表达主要增强对美罗培南、三代头孢菌素、氟喹诺酮类、大环内酯类的耐药,且随表达量的增加而增强。MexC型主要增强对氨基糖苷类、氟喹诺酮类、三代头孢菌素、哌拉西林/他唑巴坦类的耐药性,且随表达量的增加而增强。MexX型外排泵的表达主要增强对氨基糖苷类、大环内酯类、氟喹诺酮类和氨曲南的耐药性。MexA和MexC型同时表达与MexA型一致,并增强了对氟喹诺酮类的耐药。MexA和MexX型同时表达与MexA型一致,并增强了对庆大霉素的耐药而降低了对氟喹诺酮类的耐药性。结论 Pa的外排泵基因的表达量与特定种类的药物耐药程度有关,外排泵基因的高表达参与并加剧了Pa的耐药性。     

Role of the Mmr Efflux Pump in Drug Resistance in Mycobacterium tuberculosis

Efflux pumps are membrane proteins capable of actively transporting a broad range of substrates from the cytoplasm to the exterior of the cell. Increased efflux activity in response to drug treatment may be the first step in the development of bacterial drug resistance. Previous studies showed that the efflux pump Mmr was significantly overexpressed in strains exposed to isoniazid. In the work to be described, we constructed mutants lacking or overexpressing Mmr in order to clarify the role of this efflux pump in the development of resistance to isoniazid and other drugs in M. tuberculosis. The mmr knockout mutant showed an increased susceptibility to ethidium bromide, tetraphenylphosphonium, and cetyltrimethylammonium bromide (CTAB). Overexpression of mmr caused a decreased susceptibility to ethidium bromide, acriflavine, and safranin O that was obliterated in the presence of the efflux inhibitors verapamil and carbonyl cyanide m-chlorophenylhydrazone. Isoniazid susceptibility was not affected by the absence or overexpression of mmr. The fluorometric method allowed the detection of a decreased efflux of ethidium bromide in the knockout mutant, whereas the overexpressed strain showed increased efflux of this dye. This increased efflux activity was inhibited in the presence of efflux inhibitors. Under our experimental conditions, we have found that efflux pump Mmr is mainly involved in the susceptibility to quaternary compounds such as ethidium bromide and disinfectants such as CTAB. The contribution of this efflux pump to isoniazid resistance in Mycobacterium tuberculosis still needs to be further elucidated.     

Evaluation of efflux pump inhibitors of MexAB- or MexXY-OprM in Pseudomonas aeruginosa using nucleic acid dyes

BackgroundIncreased expression of efflux pumps is an important mechanism of antibiotic resistance in Pseudomonas aeruginosa, and treatment with inhibitors of active efflux pumps seems an attractive strategy to combat with multidrug resistance. Assays using ethidium bromide (EtBr), which accumulates by binding to nucleic acids, are often employed to assess the efficacy of efflux pump inhibitors (EPIs). However, few studies have reported on assays using other nucleic acid dyes.ObjectiveWe used different classes of EPIs for MexAB- or MexXY-OprM to measure the accumulation of various fluorescent dyes, including SYBR Safe, AtlasSight, and GelGreen.MethodsEscherichia coli MG1655ΔacrBΔtolC strain harboring plasmids carrying the mexAB-oprM (pABM) or mexXY-oprM (pXYM) genes of P. aeruginosa were constructed. Then, the accumulation of the above-mentioned nucleic acid dyes and EtBr was measured to assess the efflux ability in the presence and absence of EPIs (MexAB-OprM-specific inhibitor of pyridopyrimidine derivative [ABI-PP], berberine, non-specific inhibitor of phenylalanine-arginine β-naphthylamide [PAβN], and protonophore of carbonyl cyanide m-chlorophenyl hydrazone [CCCP]).ResultsDecreased accumulations of nucleic acid dyes were observed in strains with pABM or pXYM compared with the parental strain. ABI-PP or berberine addition significantly increased the accumulation of any nucleic acids in the strains with the specific pumps. PAβN or CCCP addition showed increased accumulation of almost all dye in strains with pABM or pXYM. However, the inhibition patterns of EPIs differed according to the nucleic acid dyes used.ConclusionsAccumulation assays for EPIs were suitable to evaluate EPI candidates using various nucleic acid dyes.     

Involvement of the AcrAB-TolC efflux pump in the resistance, fitness, and virulence of Enterobacter cloacae

Multidrug efflux pumps have emerged as important mechanisms of antimicrobial resistance in bacterial pathogens. In order to cause infection, pathogenic bacteria require mechanisms to avoid the effects of host-produced compounds, and express efflux pumps may accomplish this task. In this study, we evaluated the effect of the inactivation of AcrAB-TolC on antimicrobial resistance, fitness, and virulence in Enterobacter cloacae, an opportunistic pathogen usually involved in nosocomial infections. Two different clinical isolates of E. cloacae were used, EcDC64 (multidrug resistance overexpressing the AcrAB-TolC efflux pump) and Jc194 (basal AcrAB-TolC expression). The acrA and tolC genes were deleted in strains EcDC64 and Jc194 to produce, respectively, EcΔacrA and EcΔtolC and JcΔacrA and JcΔtolC knockout (KO) derivatives. Antibiotic susceptibility testing was performed with all isolates, and we discovered that these mechanisms are involved in the resistance of E. cloacae to several antibiotics. Competition experiments were also performed with wild-type and isogenic KO strains. The competition index (CI), defined as the mutant/wild-type ratio, revealed that the acrA and tolC genes both affect the fitness of E. cloacae, as fitness was clearly reduced in the acrA and tolC KO strains. The median CI values obtained in vitro and in vivo were, respectively, 0.42 and 0.3 for EcDC64/EcΔacrA, 0.24 and 0.38 for EcDC64/EcΔtolC, 0.15 and 0.11 for Jc194/JcΔacrA, and 0.38 and 0.39 for Jc194/JcΔtolC. Use of an intraperitoneal mouse model of systemic infection revealed reduced virulence in both E. cloacae clinical strains when either the acrA or tolC gene was inactivated. In conclusion, the structural components of the AcrAB-TolC efflux pump appear to play a role in antibiotic resistance as well as environmental adaptation and host virulence in clinical isolates of E. cloacae.