Role of the membrane fusion protein in the assembly of resistance-nodulation-cell division multidrug efflux pump in Pseudomonas aeruginosa

The tripartite xenobiotic-antibiotic transporter of Pseudomonas aeruginosa consists of the inner membrane transporter (e.g., MexB, MexY), the periplasmic membrane-fusion-protein (e.g., MexA, MexX), and the outer membrane channel protein (e.g., OprM). These subunits were assumed to assemble into a transporter unit during export of the substrates. However, subunit interaction and their specificity in native form remained to be elucidated. To address these important questions, we analyzed the role of the individual subunits for the assembly of MexAB-OprM by pull-down assay tagging only one of the subunits. We found stable MexA-MexB-OprM complex without chemical cross-linking that withstand all purification procedures. Results of bi-partite interactions analysis showed tight association between MexA and OprM in the absence of MexB, whereas the expression systems lacking MexA failed to co-purify MexB or OprM. None of the heterologous subunit combinations such as MexA+MexY(his)+OprM and MexX+MexB(his)+OprM showed interaction. These results implied that the membrane fusion protein is central to the tripartite xenobiotic transporter assembly.     

Directed evolution of a bacterial efflux pump: adaptation of the E. coli TolC exit duct to the Pseudomonas MexAB translocase

Bacterial multidrug efflux pumps operate by periplasmic recruitment and opening of TolC family outer membrane exit ducts by cognate inner membrane translocases. Directed evolution of active hybrid pumps was achieved by challenging a library of mutated, shuffled TolC variants to adapt to the non-cognate Pseudomonas MexAB translocase, and confer resistance to the efflux substrate novobiocin. Amino acid substitutions in MexAB-adapted TolC variants that endowed high resistance were recreated independently, and revealed that MexAB-adaptation was conferred only by substitutions located in the lower alpha-helical barrel of TolC, specifically the periplasmic equatorial domain and entrance coiled coils. These changes converge to the native MexAB partner OprM, and indicate an interface key to the function and diversity of efflux pumps.     

Ligand-transporter interaction in the AcrB multidrug efflux pump determined by fluorescence polarization assay

The AcrB of Escherichia coli pumps out a wide range of compounds, including most of the currently available antibiotics, and contributes significantly to the serious problem of multidrug resistance of pathogenic bacteria. Quantitative analysis of drug efflux by this pump requires the measurement of the affinity of ligands. Yet there has been no success in determining these values. We introduce here an approach of steady-state fluorescence polarization to study the interactions between four different ligands and the purified AcrB transporter in a detergent environment. Our assays indicate that the transporter binds these drugs with K(D) values ranging from 5.5 to 74.1microM.     

The AcrAB RND efflux system from the live vaccine strain of Francisella tularensis is a multiple drug efflux system that is required for virulence in mice

The ability of bacterial pathogens to infect and cause disease is dependent upon their ability to resist antimicrobial components produced by their host, such as bile acids, fatty acids and other detergent-like molecules, and products of the innate immune system (e.g. cationic antimicrobial peptides). Bacterial resistance to the antimicrobial effects of such compounds is often mediated by active efflux systems belonging to the resistance-nodulation-division (RND) family of transporters. RND efflux systems have been implicated in antibiotic resistance and virulence extending their clinical relevance. In this report the hypothesis that the Francisella tularensis AcrAB RND efflux system contributes to antimicrobial resistance and pathogenesis has been tested. A null mutation was generated in the gene encoding the AcrB RND efflux pump protein of the live vaccine strain of F. tularensis. The resulting mutant exhibited increased sensitivity to multiple antibiotics and antimicrobial compounds. Murine challenge experiments revealed that the acrB mutant was attenuated. Collectively these results suggest that the F. tularensis AcrAB RND efflux system encodes a multiple drug efflux system that is important for virulence.     

Linkage of the efflux-pump expression level with substrate extrusion rate in the MexAB-OprM efflux pump of Pseudomonas aeruginosa

The amount of the subunit proteins of the MexAB-OprM efflux pump in Pseudomonas aeruginosa was quantified by the immunoblotting method. A single cell of the wild-type strain contained about 2500, 1000, and 1200 copies of MexA, MexB, and OprM, respectively, and their stoichiometry therefore was 2:1:1. The mexR mutant produced an eightfold higher level of these proteins than did wild-type cells. Assuming that MexB and OprM exist as a trimer in a pump assembly, the total number of MexAB-OprM per wild-type cell was calculated to be about 400 assemblies. The substrate efflux rate of MexAB-OprM was calculated from the fluorescent intensity of ethidium in intact cells that a single cell extruded ethidium at a maximum of about 3 x 10(-19) mol s(-1) and, therefore, the turnover rate of a single pump unit was predicted to be about 500 s(-1).     

The substrate specificity of a neuronal glutamate transporter is determined by the nature of the coupling ion

Glutamate transporters are essential for terminating synaptic transmission. Glutamate is translocated together with three sodium ions. In the neuronal glutamate transporter EAAC1, lithium can replace sodium. To address the question of whether the coupling ion interacts with the 'driven' substrate during co-transport, the kinetic parameters of transport of the three substrates, L-glutamate and D- and L-aspartate by EAAC-1 in sodium- and lithium-containing media were compared. The major effect of the substitution of sodium by lithium was on Km. In the presence of sodium, the values for Km and Imax of these substrates were similar. In the presence of lithium, the Km for L-aspartate was increased around 13-fold. Remarkably, the corresponding increase for L-glutamate and D-aspartate was much larger, around 130-fold. In marked contrast, the Ki values for a non-transportable substrate analogue were similar in the presence of either sodium or lithium. The preference for L-aspartate in the presence of lithium was also observed when electrogenic transport of radioactive substrates was monitored in EAAC1-containing proteoliposomes. Our results indicate that, subsequent to substrate binding, the co-transported solutes interact functionally in the binding pocket of the transporter.     

Single-molecule detection of efflux pump machinery in Pseudomonas aeruginosa

Real-time single-molecule microscopy and spectroscopy were used to monitor single molecules moving in and out of live bacterial cells, Pseudomonas aeruginosa. Ethidium bromide (EtBr) was chosen as the fluorescence probe because it emitted a weak fluorescence in aqueous solution (outside of the cells) and became strongly fluorescent as it entered the cells and intercalated with DNA. Such changes in fluorescence intensity by individual EtBr molecules were measured to determine the influx and efflux rates of EtBr by the cells. The transport rates for EtBr through the energized extrusion pumps of these strains (WT, nalB-1, and DeltaABM) of P. aeruginosa were measured and showed stochastic behavior with the average being (2.86+/-0.12), (2.80+/-0.13), and (2.74+/-0.39) x s(-1), respectively. The transport rates of the three strains were independent of substrate concentration at the single-molecule level. In contrast to bulk (many molecules) measurements, single-molecule detection allowed the influx and efflux kinetics to be observed in low substrate concentrations at the molecular level.     

siRNA-mediated gene silencing of MexB from the MexA-MexB-OprM efflux pump in Pseudomonas aeruginosa

To gain insights into the effect of MexB gene under the short interfering RNA (siRNA), we synthesized 21 bp siRNA duplexes against the MexB gene. RT-PCR was performed to determine whether the siRNA inhibited the expression of MexB mRNA. Changes in antibiotic susceptibility in response to siRNA were measured by the E-test method. The efficacy of siRNAs was determined in a murine model of chronic P. aeruginosa lung infection. MexB-siRNAs inhibited both mRNA expression and the activity of P. aeruginosa in vitro. In vivo, siRNA was effective in reducing the bacterial load in the model of chronic lung infection and the P. aeruginosa-induced pathological changes. MexB-siRNA treatment enhanced the production of inflammatory cytokines in the early infection stage (P ＜ 0.05). Our results suggest that targeting of MexB with siRNA appears to be a novel strategy for treating P. aeruginosa infections. [BMB Reports 2014; 47(4): 203-208]     

Crystal structures of OprN and OprJ,outer membrane factors of multidrug tripartite efflux pumps of Pseudomonas aeruginosa

The genome of Pseudomonas aeruginosa encodes tripartite efflux pumps that extrude functionally and structurally dissimilar antibiotics from the bacterial cell. MexAB‐OprM, MexCD‐OprJ, MexEF‐OprN, and MexXY‐OprM are the main tripartite efflux pumps responsible for multidrug resistance in P. aeruginosa. The outer membrane factors OprN, OprJ, and OprM are essential components of functional tripartite efflux pumps. To elucidate the structural basis of multidrug resistance, we determined the crystal structures of OprN and OprJ. These structures revealed several features, including tri‐acylation of the N‐terminal cysteine, a small pore in the β‐barrel domain, and a tightly sealed gate in the α‐barrel domain. Despite the overall similarity of OprN, OprJ, and OprM, a comparison of their structures and electrostatic distributions revealed subtle differences at the periplasmic end of the α‐barrel domain. These results suggested that the overall structures of these outer membrane factors are specifically optimized for particular tripartite efflux pumps. Proteins 2016; 84:759–769. © 2016 Wiley Periodicals, Inc.     

Tannic acid affects the phenotype of Staphylococcus aureus resistant to tetracycline and erythromycin by inhibition of efflux pumps

The widespread use of antibiotics created selective pressure for the emergence of strains that would persist despite antibiotic toxicity. The bacterial resistance mechanisms are several, with efflux pumps being one of the main ones. These pumps are membrane proteins with the function of removing antibiotics from the cell cytoplasm. Due to this importance, the aim of this work was to evaluate the inhibitory effect of tannic acid against efflux pumps expressed by the Staphylococcus aureus RN4220 and IS-58 strains. The efflux pump inhibition was assayed using a sub-inhibitory concentration of efflux pump standard inhibitors and tannic acid (MIC/8), observing their capacity to decrease the MIC of Ethidium bromide (EtBr) and antibiotics due the possible inhibitory effect of these substances. The MICs of EtBr and antibiotics were significantly different in the presence of tannic acid, indicating the inhibitory effect of this product against efflux pumps of both strains. These results indicate the possible usage of tannic acid as an inhibitor and an adjuvant in the antibiotic therapy against multidrug resistant bacteria (MDR).     

CIP耐药的铜绿假单胞菌两种分子耐药机制关系的研究

目的探讨环丙沙星（CIP）耐药的铜绿假单胞菌临床分离株主动外排药物与gyrA、parC基因突变的关系。方法联合碳酰氰基-对-氯苯腙（CCCP）和CIP对CIP耐药的铜绿假单胞菌株进行主动外排阳性株和阴性株的筛选,并对这些菌株的gyrA,parC基因进行聚合酶链式反应-限制性片段长度多态性分析（PCR—RFLP）。结果57％（55／97）的CIP耐药菌株最小抑菌浓度（MIC）可被逆转,gyrA单基因突变率为65％,gyrA和pa-C双基因突变率为35％,未发现parC单基因突变的菌株。主动外排阳性组与阴性组gyrA、parC基因突变情况差异无显著性。结论在本地区铜绿假单胞菌对CIP的耐药机制中,主动外排系统表达上调与抗菌药物作用靶位的改变均占有重要的地位,两者可能是并存的两种相对独立的机制。     

The Outer Membrane TolC-like Channel HgdD Is Part of Tripartite Resistance-Nodulation-Cell Division (RND) Efflux Systems Conferring Multiple-drug Resistance in the Cyanobacterium Anabaena sp. PCC7120

The TolC-like protein HgdD of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 is part of multiple three-component “AB-D” systems spanning the inner and outer membranes and is involved in secretion of various compounds, including lipids, metabolites, antibiotics, and proteins. Several components of HgdD-dependent tripartite transport systems have been identified, but the diversity of inner membrane energizing systems is still unknown. Here we identified six putative resistance-nodulation-cell division (RND) type factors. Four of them are expressed during late exponential and stationary growth phase under normal growth conditions, whereas the other two are induced upon incubation with erythromycin or ethidium bromide. The constitutively expressed RND component Alr4267 has an atypical predicted topology, and a mutant strain (I-alr4267) shows a reduction in the content of monogalactosyldiacylglycerol as well as an altered filament shape. An insertion mutant of the ethidium bromide-induced all7631 did not show any significant phenotypic alteration under the conditions tested. Mutants of the constitutively expressed all3143 and alr1656 exhibited a Fox⁻ phenotype. The phenotype of the insertion mutant I-all3143 parallels that of the I-hgdD mutant with respect to antibiotic sensitivity, lipid profile, and ethidium efflux. In addition, expression of the RND genes all3143 and all3144 partially complements the capability of Escherichia coli ΔacrAB to transport ethidium. We postulate that the RND transporter All3143 and the predicted membrane fusion protein All3144, as homologs of E. coli AcrB and AcrA, respectively, are major players for antibiotic resistance in Anabaena sp. PCC 7120.     

Assignment of the outer-membrane-subunit-selective domain of the membrane fusion protein in the tripartite xenobiotic efflux pump of Pseudomonas aeruginosa

Early in vivo experiments revealed that the MexA-MexB dipartite pump unit of Pseudomonas aeruginosa conferred drug resistance to the cells, which expressed OprM, but not to the OprN-bearing cells. While the MexE-MexF unit interplayed with either the outer membrane subunits. Taking advantage of this subunit selectivity, we selected the MexA mutant that gained the ability to interplay with OprN. Four mutants have been isolated and all showed an amino acid substitution (Q116R) in the coiled-coil domain of MexA. The hybrid protein bearing the coiled-coil domain of MexA and the remainder domains from MexE retained the ability to interplay with OprM, but lost the functional interplay with OprN. These results established that the coiled-coil domain of the membrane fusion protein is responsible for selecting the compatible outer membrane subunit.     

Construction of a series of mutants lacking all of the four major mex operons for multidrug efflux pumps or possessing each one of the operons from Pseudomonas aeruginosa PAO1: MexCD-OprJ is an inducible pump

We constructed a series of deletion mutants lacking all of the four major mex operons for Mex multidrug efflux pumps or possessing each one of the operons from Pseudomonas aeruginosa PAO1. The drug specificity of MexAB-OprM, MexXY-OprM and MexCD-OprJ was investigated. Surprisingly, we found that the MexCD-OprJ was an inducible pump, inducers of which were tetraphenylphosphonium chloride, ethidium bromide, rhodamine 6G and acriflavine. Fluoroquinolones, chloramphenicol, erythromycin and tetracycline were not inducers although they were substrates of MexCD-OprJ.     

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.     

Factors that influence the permeability assay of the outer membrane of Pseudomonas aeruginosa

Brief exposure of Pseudomonas aeruginosa to a temperature of 10 degrees C or lower caused a significant leakage of the periplasmic beta-lactamase into the medium. The extent of leakage increased as the incubation temperature was lowered to 4 degrees C and reached a maximum at 0 degrees C. Cells grown in the presence of beta-lactamase inducers were unsuitable for the permeability assay. It was found that the diffusion rates of beta-lactams through the outer membrane of P. aeruginosa were much lower than those previously reported, as assayed under refined conditions. The diffusion rates of beta-lactams in one of the mutants tested were an order of magnitude lower than those of the other strains, despite the fact that the outer membrane protein profile of the strain appeared to be indistinguishable from those of the others. These results suggest that beta-lactam antibiotics diffuse through the outer membrane of P. aeruginosa, at least partly, through a non-porin pathway.     

The unusual substrate specificity of a virulence associated serine hydrolase from the highly toxic bacterium,Francisella tularensis

Francisella tularensis is the causative agent of the highly, infectious disease, tularemia. Amongst the genes identified as essential to the virulence of F. tularensis was the proposed serine hydrolase FTT0941c. Herein, we purified FTT0941c to homogeneity and then characterized the folded stability, enzymatic activity, and substrate specificity of FTT0941c. Based on phylogenetic analysis, FTT0941c was classified within a divergent Francisella subbranch of the bacterial hormone sensitive lipase (HSL) superfamily, but with the conserved sequence motifs of a bacterial serine hydrolase. FTT0941c showed broad hydrolase activity against diverse libraries of ester substrates, including significant hydrolytic activity across alkyl ester substrates from 2 to 8 carbons in length. Among a diverse library of fluorogenic substrates, FTT0941c preferred α-cyclohexyl ester substrates, matching with the substrate specificity of structural homologues and the broad open architecture of its modeled binding pocket. By substitutional analysis, FTT0941c was confirmed to have a classic catalytic triad of Ser115, His278, and Asp248 and to remain thermally stable even after substitution. Its overall substrate specificity profile, divergent phylogenetic homology, and preliminary pathway analysis suggested potential biological functions for FTT0941c in diverse metabolic degradation pathways in F. tularensis.     

Organic solvent-selective domain of the resistance-nodulation-division-type xenobiotic-antibiotic transporters of Pseudomonas aeruginosa

The hydrophobic substrate-selective domain of the resistance-nodulation-division-type xenobiotic transporter of Pseudomonas aeruginosa was assigned based on the different organic-solvent selectivities of MexB and MexY. The MexB-MexY hybrid protein consisting of two large periplasmic domains of MexB and the transmembrane domains of MexY showed MexB-type organic solvent selectivity. The results imply that the resistance-nodulation-division-type xenobiotic transporters recognize hydrophobic substrates such as organic solvents by their periplasmic domains and expel them to the external milieu. This is an elegant way to protect the cytoplasmic membrane from membrane-deteriorating agents.