The MexJK efflux pump of Pseudomonas aeruginosa requires OprM for antibiotic efflux but not for efflux of triclosan

Using the biocide triclosan as a selective agent, several triclosan-resistant mutants of a susceptible Pseudomonas aeruginosa strain were isolated. Cloning and characterization of a DNA fragment conferring triclosan resistance from one of these mutants revealed a hitherto uncharacterized efflux system of the resistance nodulation cell division (RND) family, which was named MexJK and which is encoded by the mexJK operon. Expression of this operon is negatively regulated by the product of mexL, a gene located upstream of and transcribed divergently from mexJK. The triclosan-resistant mutant contained a single nucleotide change in mexL, which caused an amino acid change in the putative helix-turn-helix domain of MexL. The MexL protein belongs to the TetR family of repressor proteins. The MexJK system effluxed tetracycline and erythromycin but only in the presence of the outer membrane protein channel OprM; OprJ and OprN did not function with MexJK. Triclosan efflux required neither of the outer membrane protein channels tested but necessitated the MexJ membrane fusion protein and the MexK inner membrane RND transporter. The results presented in this study suggest that MexJK may function as a two-component RND pump for triclosan efflux but must associate with OprM to form a tripartite antibiotic efflux system. Furthermore, the results confirm that triclosan is an excellent tool for the study of RND multidrug efflux systems and that this popular biocide therefore readily selects mutants which are cross-resistant with antibiotics.     

Dication and trication which can increase the permeability of Escherichia coli outer membrane

Recent success in the preparation of the monomer, dimer and trimer in compound 48/80 prompted us to investigate the action of these compounds on Escherichia coli cells. It was found that compound 48/80 inhibited growth of E. coli cells, while the monomer, dimer and trimer in 48/80 did not. However, the following experiments showed that the dimer and trimer disrupted the permeability barrier of the outer membrane of E. coli. First, addition of the dimer or trimer in cell suspension stimulated the uptake of tetraphenylphosphonium cation. Second, the synergistic effect of the dimer on the action of gramicidin caused the efflux of K+. In experiments using isolated cytoplasmic membrane vesicles, addition of gramicidin alone caused the efflux of K+. Thus, it was speculated that, with whole cells, the dimer formed some defect structure in the outer membrane, through which gramicidin reached the cytoplasmic membrane and increased the K+ permeability. The temperature dependence of efflux K+ showed that the dimer in 48/80 rendered the outer membrane permeable to gramicidin at temperatures above the phase transition of the outer membrane.     

Identification and characterization of the emhABC efflux system for polycyclic aromatic hydrocarbons in Pseudomonas fluorescens cLP6a

The hydrocarbon-degrading environmental isolate Pseudomonas fluorescens LP6a possesses an active efflux mechanism for the polycyclic aromatic hydrocarbons phenanthrene, anthracene, and fluoranthene but not for naphthalene or toluene. PCR was used to detect efflux pump genes belonging to the resistance-nodulation-cell division (RND) superfamily in a plasmid-cured derivative, P. fluorescens cLP6a, which is unable to metabolize hydrocarbons. One RND pump, whose gene was identified in P. fluorescens cLP6a and was designated emhB, showed homology to the multidrug and solvent efflux pumps in Pseudomonas aeruginosa and Pseudomonas putida. The emhB gene is located in a gene cluster with the emhA and emhC genes, which encode the membrane fusion protein and outer membrane protein components of the efflux system, respectively. Disruption of emhB by insertion of an antibiotic resistance cassette demonstrated that the corresponding gene product was responsible for the efflux of polycyclic aromatic hydrocarbons. The emhB gene disruption did not affect the resistance of P. fluorescens cLP6a to tetracycline, erythromycin, trimethoprim, or streptomycin, but it did decrease resistance to chloramphenicol and nalidixic acid, indicating that the EmhABC system also functions in the efflux of these compounds and has an unusual selectivity. Phenanthrene efflux was observed in P. aeruginosa, P. putida, and Burkholderia cepacia but not in Azotobacter vinelandii. Polycyclic aromatic hydrocarbons represent a new class of nontoxic, highly hydrophobic compounds that are substrates of RND efflux systems, and the EmhABC system in P. fluorescens cLP6a has a narrow substrate range for these hydrocarbons and certain antibiotics.     

The hydrophobic uptake pathway across the outer membrane of the antibiotic supersusceptible Pseudomonas aeruginosa mutant Z61

Abstract The Pseudomonas aeruginosa antibiotic supersusceptible mutant Z61 was 50–400-fold more susceptible than its wild-type parent K799 to 5 hydrophobic antibiotics. The strain Z61 outer membrane also demonstrated enhanced permeability towards a hydrophobic fluorescent probe. Strain Z61 cells had an altered cell surface, as revealed by phase-partitioning experiments, a lower amount of Lipid A phosphate, and a reduction in the number of Mg²⁺ binding sites in Lipid A, as demonstrated by dansyl polymyxin competition experiments. An antibiotic permation pathway directly through the outer membrane bilayer, rather than through porin proteins, is proposed for strain Z61.     

Use of steroids to monitor alterations in the outer membrane of Pseudomonas aeruginosa.

Testosterone (a strongly hydrophobic steroid) and testosterone hemisuccinate (a negatively charged derivative) were used as probes to investigate alterations in the outer membrane of Pseudomonas aeruginosa. Diffusion rates of the steroids across the lipid bilayer were measured by coupling the influx of these compounds to their subsequent oxidation by an intracellular delta1-dehydrogenase enzyme. Wild-type cells of P. aeruginosa (strain PAO1) were found to be 25 times more permeable to testosterone than to testosterone hemisuccinate. The uptake of the latter compound appeared to be partially dependent on the external pH, thus suggesting a preferential diffusion of the uncharged protonated form across the cell envelope. Using various PAO mutants, we showed that the permeation of steroids was not affected by overexpression of active efflux systems but was increased up to 5.5-fold when the outer membrane contained defective lipopolysaccharides or lacked the major porin OprF. Such alterations in the hydrophobic uptake pathway were not, however, associated with an enhanced permeability of the mutants to the small hydrophilic molecule N,N,N',N'-tetramethyl-p-phenylene diamine. Thirty-six agents were also assayed for their ability to damage the cell surface of strain PAO1, using testosterone as a probe. Polymyxins, rBPI23, chlorhexidine, and dibromopropamidine demonstrated the strongest permeabilizing activities on a molar basis in the presence of 1 mM MgCl2. These amphiphilic polycations increased the transmembrane diffusion of testosterone up to 50-fold and sensitized the PAO1 cells to hydrophobic antibiotics. All together, these data indicated that the steroid uptake assay provides a direct and accurate measurement of the hydrophobic uptake pathway in P. aeruginosa.     

Non‐equivalent roles of two periplasmic subunits in the function and assembly of triclosan pump TriABC from Pseudomonas aeruginosa

In Gram‐negative bacteria, multidrug efflux transporters function in complexes with periplasmic membrane fusion proteins (MFPs) that enable antibiotic efflux across the outer membrane. In this study, we analyzed the function, composition and assembly of the triclosan efflux transporter TriABC–OpmH from Pseudomonas aeruginosa. We report that this transporter possesses a surprising substrate specificity that encompasses not only triclosan but the detergent SDS, which are often used together in antibacterial soaps. These two compounds interact antagonistically in a TriABC‐dependent manner and negate antibacterial properties of each other. Unlike other efflux pumps that rely on a single MFP for their activities, two different MFPs, TriA and TriB, are required for triclosan/SDS resistance mediated by TriABC–OpmH. We found that analogous mutations in the α‐helical hairpin and membrane proximal domains of TriA and TriB differentially affect triclosan efflux and assembly of the complex. Furthermore, our results show that TriA and TriB function as a dimer, in which TriA is primarily responsible for stabilizing interactions with the outer membrane channel, whereas TriB is important for the stimulation of the transporter. We conclude that MFPs are engaged into complexes as asymmetric dimers, in which each protomer plays a specific role.     

Insertion mutagenesis and membrane topology model of the Pseudomonas aeruginosa outer membrane protein OprM

Pseudomonas aeruginosa OprM is a protein involved in multiple-antibiotic resistance as the outer membrane component for the MexA-MexB-OprM efflux system. Planar lipid bilayer experiments showed that OprM had channel-forming activity with an average single-channel conductance of only about 80 pS in 1 M KCl. The gene encoding OprM was subjected to insertion mutagenesis by cloning of a foreign epitope from the circumsporozoite form of the malarial parasite Plasmodium falciparum into 11 sites. In Escherichia coli, 8 of the 11 insertion mutant genes expressed proteins at levels comparable to those obtained with the wild-type gene and the inserted malarial epitopes were surface accessible as assessed by indirect immunofluorescence. When moved to a P. aeruginosa OprM-deficient strain, seven of the insertion mutant genes expressed proteins at variable levels comparable to that of wild-type OprM and three of these reconstituted MIC profiles resembling those of the wild-type protein, while the other mutant forms showed variable MIC results. Utilizing the data from these experiments, in conjunction with multiple sequence alignments and structure predictions, an OprM topology model with 16 beta strands was proposed.     

Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes

Chloramphenicol has been reported to act as an inducer of the multidrug resistance in Escherichia coli. A resistant variant able to grow on plates containing 64 microg/ml chloramphenicol was obtained from the Enterobacter aerogenes ATCC 13048-type strain. Chloramphenicol resistance was due to an active efflux of this antibiotic and it was associated with resistance to fluoroquinolones and tetracycline, but not to aminoglycoside or beta-lactam antibiotics. MDR in the chloramphenicol-resistant variant is linked to the overexpression of the major AcrAB-TolC efflux system. This overexpression seems unrelated to the global Mar and the local AcrR regulatory pathways.     

Localization of the outer membrane subunit OprM of resistance-nodulation-cell division family multicomponent efflux pump in Pseudomonas aeruginosa

The outer membrane subunit OprM of the multicomponent efflux pump of Pseudomonas aeruginosa has been assumed to form a transmembrane xenobiotic exit channel across the outer membrane. We challenged this hypothesis to clarify the underlying ambiguity by manipulating the amino-terminal signal sequence of the OprM protein of the MexAB-OprM efflux pump in P. aeruginosa. [(3)H]Palmitate uptake experiments revealed that OprM is a lipoprotein. The following lines of evidence unequivocally established that the OprM protein functioned at the periplasmic space. (i) The OprM protein, in which a signal sequence including Cys-18 was replaced with that of periplasmic azurin, appeared in the periplasmic space but not in the outer membrane fraction, and the protein fully functioned as the pump subunit. (ii) The hybrid OprM containing the N-terminal transmembrane segment of the inner membrane protein, MexF, appeared exclusively in the inner membrane fraction. The hybrid protein containing 186 or 331 amino acid residues of MexF was fully active for the antibiotic extrusion, but a 42-residue protein was totally inactive. (iii) The mutant OprM, in which the N-terminal cysteine residue was replaced with another amino acid, appeared unmodified with fatty acid and was fractionated in both the periplasmic space and the inner membrane fraction but not in the outer membrane fraction. The Cys-18-modified OprM functioned for the antibiotic extrusion indistinguishably from that in the wild-type strain. We concluded, based on these results, that the OprM protein was anchored in the outer membrane via fatty acid(s) attached to the N-terminal cysteine residue and that the entire polypeptide moiety was exposed to the periplasmic space.     

Interplay between the efflux pump and the outer membrane permeability barrier in fluorescent dye accumulation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa encodes three types of xenobiotic efflux pumps, MexAB-OprM, MexCD-OprJ, and MexEF-OprN, which are regulated by the nalB, nfxB, and nfxC genes, respectively, and their high expression renders the cells resistant to multiple species of antibiotics. We evaluated the role of the outer membrane permeability barrier and the efflux pump in lowering the intracellular concentration of fluorescent probes. The wild-type, nalB, nfxB, and nfxC strains with an intact outer membrane showed equally high capability in draining out intracellular fluorescent dye, 2-(4-dimethylaminostyryl)-1-ethylpyridinium and ethidium bromide. When the outer membrane barrier was dismantled by the EDTA treatment, wild-type, nfxC, nfxB, and nalB strains showed significantly different levels of dye accumulation. The polymyxin B-treated cells showed an even more pronounced difference in dye accumulation among the nfxC, nfxB, and nalB mutants. We concluded from these results that the xenobiotic extrusion pumps interplay with the outer membrane permeability barrier in lowering the intracellular substrate concentration. Among three extrusion pumps in P. aeruginosa, MexAB-OprM was the most efficient, followed by MexCD-OprJ and MexEF-OprN pumps for the fluorescent dye extrusion.     

A Novel indole compound that inhibits Pseudomonas aeruginosa growth by targeting MreB is a substrate for MexAB-OprM

Drug efflux systems contribute to the intrinsic resistance of Pseudomonas aeruginosa to many antibiotics and biocides and hamper research focused on the discovery and development of new antimicrobial agents targeted against this important opportunistic pathogen. Using a P. aeruginosa PAO1 derivative bearing deletions of opmH, encoding an outer membrane channel for efflux substrates, and four efflux pumps belonging to the resistance nodulation/cell division class including mexAB-oprM, we identified a small-molecule indole-class compound (CBR-4830) that is inhibitory to growth of this efflux-compromised strain. Genetic studies established MexAB-OprM as the principal pump for CBR-4830 and revealed MreB, a prokaryotic actin homolog, as the proximal cellular target of CBR-4830. Additional studies establish MreB as an essential protein in P. aeruginosa, and efflux-compromised strains treated with CBR-4830 transition to coccoid shape, consistent with MreB inhibition or depletion. Resistance genetics further suggest that CBR-4830 interacts with the putative ATP-binding pocket in MreB and demonstrate significant cross-resistance with A22, a structurally unrelated compound that has been shown to promote rapid dispersion of MreB filaments in vivo. Interestingly, however, ATP-dependent polymerization of purified recombinant P. aeruginosa MreB is blocked in vitro in a dose-dependent manner by CBR-4830 but not by A22. Neither compound exhibits significant inhibitory activity against mutant forms of MreB protein that bear mutations identified in CBR-4830-resistant strains. Finally, employing the strains and reagents prepared and characterized during the course of these studies, we have begun to investigate the ability of analogues of CBR-4830 to inhibit the growth of both efflux-proficient and efflux-compromised P. aeruginosa through specific inhibition of MreB function.     

Essential residues in the chromate transporter ChrA of Pseudomonas aeruginosa

The chrA gene of Pseudomonas aeruginosa plasmid pUM505 encodes the hydrophobic protein ChrA, which confers resistance to chromate by the energy-dependent efflux of chromate ions. Chromate-sensitive mutants were isolated by in vivo random mutagenesis. Transport experiments with cell suspensions of selected mutants showed that 51CrO4(2-) extrusion was drastically lowered as compared to suspensions of the strain with the wild-type plasmid, confirming that the mutations affected a chromate efflux system. DNA sequence analysis showed that most point mutations affected amino acids clustered in the N-terminal half of ChrA, altering either cytoplasmic regions or transmembrane segments, and replaced residues moderately to highly conserved in ChrA homologs. PhoA and LacZ translational fusions were used to confirm the membrane topology at the N-terminal half of the ChrA protein.     

Increases in permeability of Escherichia coli outer membrane induced by polycations

The action of polycations (such as polylysine and compound 48/80) on Escherichia coli was studied with use of Ca2+, K+ and TPP+ ion-selective electrodes. Rapid efflux of Ca2+ was observed when a polycation was added in cell suspension. The polycation treatment promoted a drug-inducing K+ release from the cytoplasmic membrane. TPP+ uptake was also increased by addition of a polycation. Without the polycation treatment, the uptake of TPP+ was largely suppressed due to a permeability barrier of the outer membrane. The results show that a polycation disrupted the permeability barrier of the outer membrane.     

mvaT mutation modifies the expression of the Pseudomonas aeruginosa multidrug efflux operon mexEF-oprN

Pseudomonas aeruginosa carries several multidrug efflux operons, including mexEF-oprN, that contribute to its resistance to multiple antibiotics. mvaT affects the expression of several P. aeruginosa genes. In this study, we show that the mvaT mutant PAODeltamvaT is more resistant than its parent PAO1 strain to chloramphenicol and norfloxacin but more sensitive to imipenem; yet both were less resistant to chloramphenicol, norfloxacin, and imipenem than 'typical'nfxC-type mutants. Neither strain carries the deletion described for nfxC-type mutants in mexT, the mexEF-oprN regulatory gene. Expression of mexEF-oprN is increased by five- to sixfold in PAODeltamvaT, while the expression of oprD is reduced by approximately twofold. mvaT mutation had no effect on the expression of other multidrug resistance operons, although it increased the expression of several ATP-binding cassette transporter genes. We show that mvaT mutation does not affect mexEF-oprN expression through mexT or mexS. We also explored several other potential mechanisms.     

Roles of MexXY- and MexAB-multidrug efflux pumps in intrinsic multidrug resistance of Pseudomonas aeruginosa PAO1

To envisage the roles of MexXY- and MexAB-multidrug efflux pumps in the intrinsic multidrug resistance of wild-type strain Pseudomonas aeruginosa PAO1, we constructed mutants lacking either individual or both efflux pumps. A mutant lacking MexXY showed increased susceptibility to aminoglycosides, erythromycin, and tetracycline, but not to beta-lactams, chloramphenicol, or quinolones. A mutant lacking MexAB showed increased susceptibility to beta-lactams, chloramphenicol, and nalidixic acid, but not to aminoglycosides, erythromycin, tetracycline, or fluoroquinolones. A mutant lacking both MexXY and MexAB showed an increased susceptibility to all antimicrobial agents tested compared with the wild type. Very similar results were obtained with a mutant lacking MexAB-OprM and a mutant lacking both MexXY and MexAB-OprM. Thus it is clear that OprM is essential not only for the function of MexAB, but also for the function of MexXY. Furthermore, we found that each pump compensated to some extent for the lack of another pump with respect to the common substrates (tetracycline, quinolones, and cefpirome). The introduction of a plasmid carrying the mexXY genes into P. aeruginosa PAO1 cells increased the resistance to fluoroquinolones. This suggests that the mexXY genes could be involved in acquired resistance to fluoroquinolones in P. aeruginosa PAO1.     

Evaluation of a series of 2-napthamide derivatives as inhibitors of the drug efflux pump AcrB for the reversal of antimicrobial resistance

Drug efflux pumps confer multidrug resistance to dangerous pathogens which makes these pumps important drug targets. We have synthesised a novel series of compounds based on a 2-naphthamide pharmacore aimed at inhibiting the efflux pumps from Gram-negative bacteria. The archeatypical transporter AcrB from Escherichia coli was used as model efflux pump as AcrB is widely conserved throughout Gram-negative organisms. The compounds were tested for their antibacterial action, ability to potentiate the action of antibiotics and for their ability to inhibit Nile Red efflux by AcrB. None of the compounds were antimicrobial against E. coli wild type cells. Most of the compounds were able to inhibit Nile Red efflux indicating that they are substrates of the AcrB efflux pump. Three compounds were able to synergise with antibiotics and reverse resistance in the resistant phenotype. Compound A3, 4-(isopentyloxy)-2-naphthamide, reduced the MICs of erythromycin and chloramphenicol to the MIC levels of the drug sensitive strain that lacks an efflux pump. A3 had no effect on the MIC of the non-substrate rifampicin indicating that this compound acts specifically through the AcrB efflux pump. A3 also does not act through non-specific mechanisms such as outer membrane or inner membrane permeabilisation and is not cytotoxic against mammalian cell lines. Therefore, we have designed and synthesised a novel chemical compound with great potential to further optimisation as inhibitor of drug efflux pumps.     

Mechanisms of bacterial biocide and antibiotic resistance

Resistance to antibiotics is increasingly commonplace amongst important human pathogens. Although the mechanism(s) of resistance vary from agent to agent they typically involve one or more of: alteration of the drug target in the bacterial cell, enzymatic modification or destruction of the drug itself, or limitation of drug accumulation as a result of drug exclusion or active drug efflux. While most of these are agent specific, providing resistance to a single antimicrobial or class of antimicrobial, there are currently numerous examples of efflux systems that accommodate and, thus, provide resistance to a broad range of structurally unrelated antimicrobials--so-called multidrug efflux systems. Resistance to biocides is less common and likely reflects the multiplicity of targets within the cell as well as the general lack of known detoxifying enzymes. Resistance typically results from cellular changes that impact on biocide accumulation, including cell envelope changes that limit uptake, or expression of efflux mechanisms. Still, target site mutations leading to biocide resistance, though rare, are known. Intriguingly, many multidrug efflux systems also accommodate biocides (e.g. triclosan) such that strains expressing these are both antibiotic- and biocide-resistant. Indeed, concern has been expressed regarding the potential for agents such as triclosan to select for strains resistant to multiple clinically-relevant antibiotics. Some of the better characterized examples of such multidrug efflux systems can be found in the opportunistic pathogen Pseudomonas aeruginosa where they play an important role in the noted intrinsic and acquired resistance of this organism to antibiotics and triclosan. These tripartite pumps include an integral inner membrane drug-proton antiporter, an outer membrane- and periplasm-spanning channel-forming protein and a periplasmic link protein that joins these two. Expression of efflux genes is governed minimally by the product of a linked regulatory gene that is in most cases the target for mutation in multidrug resistant strains hyperexpressing these efflux systems. Issues for consideration include the natural function of these efflux systems and the therapeutic potential of targeting these systems in combating acquired multidrug resistance.     

Salt-inducible multidrug efflux pump protein in the moderately halophilic bacterium Chromohalobacter sp

It has been known that halophilic bacteria often show natural resistance to antibiotics, dyes, and toxic metal ions, but the mechanism and regulation of this resistance have remained unexplained. We have addressed this question by identifying the gene responsible for multidrug resistance. A spontaneous ofloxacin-resistant mutant derived from the moderately halophilic bacterium Chromohalobacter sp. strain 160 showed a two- to fourfold increased resistance to structurally diverse compounds, such as tetracycline, cefsulodin, chloramphenicol, and ethidium bromide (EtBr), and tolerance to organic solvents, e.g., hexane and heptane. The mutant produced an elevated level of the 58-kDa outer membrane protein. This mutant (160R) accumulated about one-third the level of EtBr that the parent cells did. An uncoupler, carbonyl cyanide m-chlorophenylhydrazone, caused a severalfold increase in the intracellular accumulation of EtBr, with the wild-type and mutant cells accumulating nearly equal amounts. The hrdC gene encoding the 58-kDa outer membrane protein has been cloned. Disruption of this gene rendered the cells hypersusceptible to antibiotics and EtBr and led to a high level of accumulation of intracellular EtBr. The primary structure of HrdC has a weak similarity to that of Escherichia coli TolC. Interestingly, both drug resistance and the expression of HrdC were markedly increased in the presence of a high salt concentration in the growth medium, but this was not observed in hrdC-disrupted cells. These results indicate that HrdC is the outer membrane component of the putative efflux pump assembly and that it plays a major role in the observed induction of drug resistance by salt in this bacterium.