Changes in the expression of mexB, mexY, and oprD in clinical Pseudomonas aeruginosa isolates.

Changes in expression levels of drug efflux pump genes, mexB and mexY, and porin gene oprD in Pseudomonas aeruginosa were investigated in this study. Fifty-five multidrug-resistant P. aeruginosa (MDRP) strains were compared with 26 drug-sensitive strains and 21 strains resistant to a single antibiotic. The effect of the efflux inhibitor Phe-Arg-ß-naphthylamide on drug susceptibility was determined, and gene expression was quantified using real-time quantitative real-time reverse transcription polymerase chain reaction. In addition, the levels of metallo-ß-lactamase (MBL) and 6'-N-aminoglycoside acetyltransferase [AAC(6')-Iae] were investigated. Efflux pump inhibitor treatment increased the sensitivity to ciprofloxacin, aztreonam, and imipenem in 71%, 73%, and 29% of MDRPs, respectively. MBL and AAC(6')-Iae were detected in 38 (69%) and 34 (62%) MDRP strains, respectively. Meanwhile, 76% of MDRP strains exhibited more than 8-fold higher mexY expression than the reference strain PAO1. Furthermore, 69% of MDRP strains expressed oprD at levels less than 0.01-fold of those in PAO1. These findings indicated that efflux pump inhibitors in combination with ciprofloxacin or aztreonam might aid in treating MDRP infections.

Spatial Characteristics of the Efflux Pump MexB Determine Inhibitor Binding.

The multidrug efflux transporters MexB and MexY in Pseudomonas aeruginosa and AcrB in Escherichia coli contribute to these organisms' multidrug resistance. Efflux pump inhibitor (EPI) ABI-PP inhibits MexB and AcrB, but not MexY. We previously determined the structure of ABI-PP bound to the hydrophobic trap (the inhibitor-binding pit) of AcrB and MexB. The insensitivity of MexY to ABI-PP was attributed to a bulky tryptophan (Trp). AcrB(Phe178Trp) became uninhibited by ABI-PP, while MexY(Trp177Phe) resensitized MexY for ABI-PP. Interestingly, ABI-PP was able to inhibit MexB(Phe178Trp). Thus, it is not clear which bulky amino acid mutations are critical for inhibitor binding in MexB. Here, we investigated the pit of MexB in more detail, and elucidated which Trp mutation locations in the pit were hindering ABI-PP binding, but did not affect the function of the efflux pumps. Mutating positions 139, 277, 279, and 612 to tryptophan eliminated the inhibitory effect. However, the tryptophan mutation at position 571 did not cause any effect. These results show that the effectiveness of EPIs is greatly affected by mutations in different locations, and that binding of EPIs is partly attributed by spatial characteristics. These results should be taken into account for new inhibitor and drug discovery.

Structural basis for the inhibition of bacterial multidrug exporters.

The multidrug efflux transporter AcrB and its homologues are important in the multidrug resistance of Gram-negative pathogens. However, despite efforts to develop efflux inhibitors, clinically useful inhibitors are not available at present. Pyridopyrimidine derivatives are AcrB- and MexB-specific inhibitors that do not inhibit MexY; MexB and MexY are principal multidrug exporters in Pseudomonas aeruginosa. We have previously determined the crystal structure of AcrB in the absence and presence of antibiotics. Drugs were shown to be exported by a functionally rotating mechanism through tandem proximal and distal multisite drug-binding pockets. Here we describe the first inhibitor-bound structures of AcrB and MexB, in which these proteins are bound by a pyridopyrimidine derivative. The pyridopyrimidine derivative binds tightly to a narrow pit composed of a phenylalanine cluster located in the distal pocket and sterically hinders the functional rotation. This pit is a hydrophobic trap that branches off from the substrate-translocation channel. Phe 178 is located at the edge of this trap in AcrB and MexB and contributes to the tight binding of the inhibitor molecule through a π-π interaction with the pyridopyrimidine ring. The voluminous side chain of Trp 177 located at the corresponding position in MexY prevents inhibitor binding. The structure of the hydrophobic trap described in this study will contribute to the development of universal inhibitors of MexB and MexY in P. aeruginosa.

Structures of the multidrug exporter AcrB reveal a proximal multisite drug-binding pocket.

AcrB and its homologues are the principal multidrug transporters in Gram-negative bacteria and are important in antibiotic drug tolerance. AcrB is a homotrimer that acts as a tripartite complex with the outer membrane channel TolC and the membrane fusion protein AcrA. Minocycline and doxorubicin have been shown to bind to the phenylalanine cluster region of the binding monomer. Here we report the crystal structures of AcrB bound to the high-molecular-mass drugs rifampicin and erythromycin. These drugs bind to the access monomer, and the binding sites are located in the proximal multisite binding pocket, which is separated from the phenylalanine cluster region (distal pocket) by the Phe-617 loop. Our structures indicate that there are two discrete multisite binding pockets along the intramolecular channel. High-molecular-mass drugs first bind to the proximal pocket in the access state and are then forced into the distal pocket in the binding state by a peristaltic mechanism involving subdomain movements that include a shift of the Phe-617 loop. By contrast, low-molecular-mass drugs, such as minocycline and doxorubicin, travel through the proximal pocket without specific binding and immediately bind to the distal pocket. The presence of two discrete, high-volume multisite binding pockets contributes to the remarkably broad substrate recognition of AcrB.

AcrB-AcrA Fusion Proteins That Act as Multidrug Efflux Transporters.

UNLABELLED: The AcrAB-TolC system in Escherichia coli is an intrinsic RND-type multidrug efflux transporter that functions as a tripartite complex of the inner membrane transporter AcrB, the outer membrane channel TolC, and the adaptor protein AcrA. Although the crystal structures of each component of this system have been elucidated, the crystal structure of the whole complex has not been solved. The available crystal structures have shown that AcrB and TolC function as trimers, but the number of AcrA molecules in the complex is now under debate. Disulfide chemical cross-linking experiments have indicated that the stoichiometry of AcrB-AcrA-TolC is 1:1:1; on the other hand, recent cryo-electron microscopy images of AcrAB-TolC suggested a 1:2:1 stoichiometry. In this study, we constructed 1:1-fixed AcrB-AcrA fusion proteins using various linkers. Surprisingly, all the 1:1-fixed linker proteins showed drug export activity under both acrAB-deficient conditions and acrAB acrEF double-pump-knockout conditions regardless of the lengths of the linkers. Finally, we optimized a shorter linker lacking the conformational freedom imparted by the AcrB C terminus. These results suggest that a complex with equal amounts of AcrA and AcrB is sufficient for drug export function. IMPORTANCE: The structure and stoichiometry of the RND-type multidrug exporter AcrB-AcrA-TolC complex are still under debate. Recently, electron microscopic images of the AcrB-AcrA-TolC complex have been reported, suggesting a 1:2:1 stoichiometry. However, we report here that the AcrB-AcrA 1:1 fusion protein is active for drug export under acrAB-deficient conditions and also under acrAB acrEF double-deficient conditions, which eliminate the aid of free AcrA and its close homolog AcrE, indicating that the AcrB-AcrA 1:1 stoichiometry is enough for drug export function. In addition, the AcrB-AcrA fusion protein can function without the aid of free AcrA. We believe that these results are very important for considering the structure and mechanism of AcrAB-TolC-mediated multidrug export.

Fluorescence-based rapid measurement of sphingosine-1-phosphate transport activity in erythrocytes.

Sphingosine-1-phosphate (S1P) is present in the blood plasma and acts as a pivotal intercellular signal transmitter in the immune system by recruiting lymphocytes from the thymus and secondary lymphoid tissues. The plasma S1P concentration is maintained by the supply of S1P from erythrocytes. Previously, we showed that S1P release from erythrocytes is mediated by an ATP-dependent transporter. In this study, we attempted to establish a rapid and reliable method for measuring the S1P transport activity in erythrocytes by using a fluorescent S1P analog, 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled S1P. NBD-S1P was released from erythrocytes in a time-dependent manner. The NBD-S1P release was reduced after exposure to glyburide, which is an inhibitor of the S1P transporter in erythrocytes. Moreover, the release of NBD-S1P and S1P from erythrocytes was competitively inhibited by intracellular S1P and NBD-S1P, respectively. These results showed that the erythrocyte S1P transporter exports NBD-S1P. We optimized the sample-preparation conditions and lipid extraction to increase the sensitivity of the assay. Furthermore, we successfully measured NBD-S1P release without lipid extraction by decreasing the concentration of BSA in the assay buffer to 0.1%. This method will be useful for the high-throughput screening of S1P transporter inhibitors using conventional fluorometers.

ß-Lactam selectivity of multidrug transporters AcrB and AcrD resides in the proximal binding pocket.

ß-Lactams are mainstream antibiotics that are indicated for the prophylaxis and treatment of bacterial infections. The AcrA-AcrD-TolC multidrug efflux system confers much stronger resistance on Escherichia coli to clinically relevant anionic ß-lactam antibiotics than the homologous AcrA-AcrB-TolC system. Using an extensive combination of chimeric analysis and site-directed mutagenesis, we searched for residues that determine the difference in ß-lactam specificity between AcrB and AcrD. We identified three crucial residues at the "proximal" (or access) substrate binding pocket. The simultaneous replacement of these residues in AcrB by those in AcrD (Q569R, I626R, and E673G) transferred the ß-lactam specificity of AcrD to AcrB. Our findings indicate for the first time that the difference in ß-lactam specificity between AcrB and AcrD relates to interactions of the antibiotic with residues in the proximal binding pocket.

Molecular and physiological functions of sphingosine 1-phosphate transporters.

Sphingosine 1-phosphate (S1P) is a lipid mediator that plays important roles in diverse cellular functions such as cell proliferation, differentiation and migration. S1P is synthesized inside the cells and subsequently released to the extracellular space, where it binds to specific receptors that are located on the plasma membranes of target cells. Accumulating recent evidence suggests that S1P transporters including SPNS2 mediate S1P release from the cells and are involved in the physiological functions of S1P. In this review, we discuss recent advances in our understanding of the mechanism and physiological functions of S1P transporters. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.

Autoimmune disorder phenotypes in Hvcn1-deficient mice.

H(v) channels (voltage-gated proton channels) are expressed in blood cells, microglia and some types of epithelial cells. In neutrophils H(v) channels regulate the production of reactive oxygen species through regulation of membrane potential and intracellular pH. H(v) channels have also been suggested to play a role in sperm physiology in the human. However, the functions of the Hv channel at the whole-body level are not fully understood. In the present paper we show that Hvcn1 (voltage-gated hydrogen channel 1)-knockout mice show splenomegaly, autoantibodies and nephritis, that are reminiscent of human autoimmune diseases phenotypes. The number of activated T-cells was larger in Hvcn1-deficient mice than in the wild-type mice. Upon viral infection this was remarkably enhanced in Hvcn1-deficient mice. The production of superoxide anion in T-cells upon stimulation with PMA was significantly attenuated in the Hvcn1-deficient mice. These results suggest that H(v) channels regulate T-cell homoeostasis in vivo.

The sphingosine 1-phosphate transporter, SPNS2, functions as a transporter of the phosphorylated form of the immunomodulating agent FTY720.

FTY720 is a novel immunomodulating drug that can be phosphorylated inside cells; its phosphorylated form, FTY720-P, binds to a sphingosine 1-phosphate (S1P) receptor, S1P(1), and inhibits lymphocyte egress into the circulating blood. Although the importance of its pharmacological action has been well recognized, little is known about how FTY720-P is released from cells after its phosphorylation inside cells. Previously, we showed that zebrafish Spns2 can act as an S1P exporter from cells and is essential for zebrafish heart formation. Here, we demonstrate that human SPNS2 can transport several S1P analogues, including FTY720-P. Moreover, FTY720-P is transported by SPNS2 through the same pathway as S1P. This is the first identification of an FTY720-P transporter in cells; this finding is important for understanding FTY720 metabolism.

Crystal structures of a multidrug transporter reveal a functionally rotating mechanism.

AcrB is a principal multidrug efflux transporter in Escherichia coli that cooperates with an outer-membrane channel, TolC, and a membrane-fusion protein, AcrA. Here we describe crystal structures of AcrB with and without substrates. The AcrB-drug complex consists of three protomers, each of which has a different conformation corresponding to one of the three functional states of the transport cycle. Bound substrate was found in the periplasmic domain of one of the three protomers. The voluminous binding pocket is aromatic and allows multi-site binding. The structures indicate that drugs are exported by a three-step functionally rotating mechanism in which substrates undergo ordered binding change.

Regulation of the AcrAB multidrug efflux pump in Salmonella enterica serovar Typhimurium in response to indole and paraquat.

Salmonella enterica serovar Typhimurium has at least nine multidrug efflux pumps. Among these, AcrAB is constitutively expressed and is the most efficient, playing a role in both drug resistance and virulence. The acrAB locus is induced by indole, Escherichia coli-conditioned medium, and bile salts. This induction is dependent on RamA through the binding sequence in the upstream region of acrA that binds RamA. In the present study, we made a detailed investigation of the ramA and acrAB induction mechanisms in Salmonella in response to indole, a biological oxidant for bacteria. We found that acrAB and ramA induction in response to indole is dependent on RamR. However, the cysteine residues of RamR do not play a role in the induction of ramA in response to indole, and the oxidative effect of indole is therefore not related to ramA induction via RamR. Furthermore, we showed that paraquat, a superoxide generator, induces acrAB but not ramA. We further discovered that the mechanism of acrAB induction in response to paraquat is dependent on SoxS. The data indicate that there are at least two independent induction pathways for acrAB in response to extracellular signals such as indole and paraquat. We propose that Salmonella utilizes these regulators for acrAB induction in response to extracellular signals in order to adapt itself to environmental conditions.

Roles of Salmonella multidrug efflux pumps in tigecycline resistance.

OBJECTIVES: salmonella enterica strains exhibiting decreased susceptibility to tigecycline have been reported. In this study, we sought to elucidate the roles of Salmonella multidrug efflux pumps and AcrAB regulators in tigecycline resistance. METHODS: we examined the involvement of multidrug efflux pumps and AcrAB regulators in resistance to tigecycline and other glycylcyclines by determining the MICs of the drugs for Salmonella multidrug efflux pump and AcrAB regulator-overproducing or -deleted strains. Strains of S. enterica serovar Typhimurium derived from the wild-type strain ATCC 14028s were used in this study. RESULTS: a plasmid carrying the tet gene conferred resistance to 9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline ('DMG-DMDOT') minocycline, doxycycline and tetracycline, but does not affect tigecycline resistance. Deletion of acrAB resulted in strains with significantly increased susceptibility to tigecycline and other glycylcyclines. Plasmids carrying the acrAB or acrEF gene restored increased susceptibility of the acrAB-deleted mutant to all tested compounds. Deletion of ramA, a positive regulator of acrAB, slightly increased susceptibility to tigecycline. Overexpression of ramA and deletion of ramR, a repressor of ramA, resulted in decreased susceptibility to all tested compounds. This phenotype, modulated by ramA or ramR, was not observed in the acrB-deleted background. CONCLUSIONS: AcrAB and AcrEF confer resistance to tigecycline and tetracycline derivatives in Salmonella. RamA and RamR are also involved in resistance to tigecycline in an AcrAB-dependent manner.

Effect of overexpression of small non-coding DsrA RNA on multidrug efflux in Escherichia coli.

OBJECTIVES: Several putative and proven drug efflux pumps are present in Escherichia coli. Because many such efflux pumps have overlapping substrate spectra, it is intriguing that bacteria, with their economically organized genomes, harbour such large sets of multidrug efflux genes. To understand how bacteria utilize these multiple efflux pumps, it is important to elucidate the process of pump expression regulation. The aim of this study was to determine a regulator of the multidrug efflux pump in this organism. METHODS: We screened a genomic library of E. coli for genes that decreased drug susceptibility in this organism. The library was developed from the chromosomal DNA of the MG1655 strain, and then the recombinant plasmids were transformed into an acrB-deleted strain. Transformants were screened for resistance to various antibiotics including oxacillin. RESULTS: We found that the multidrug susceptibilities of the acrB-deleted strain were decreased by the overexpression of small non-coding DsrA RNA as well as by the overexpression of known regulators of multidrug efflux pumps. Plasmids carrying the dsrA gene conferred resistance to oxacillin, cloxacillin, erythromycin, rhodamine 6G and novobiocin. DsrA decreased the accumulation of ethidium bromide in E. coli cells. Furthermore, expression of mdtE was significantly increased by dsrA overexpression, and the decreased multidrug susceptibilities modulated by DsrA were dependent on the MdtEF efflux pump. CONCLUSIONS: These results indicate that DsrA modulates multidrug efflux through activation of genes encoding the MdtEF pump in E. coli.

Regulation and physiological function of multidrug efflux pumps in Escherichia coli and Salmonella.

Multidrug efflux is an obstacle to the successful treatment of infectious diseases, and it is mediated by multidrug efflux pumps that recognize and export a broad spectrum of chemically dissimilar toxic compounds. Many bacterial genome sequences have been determined, allowing us to identify drug efflux genes encoded in the bacterial genome. Here, we present an approach to identifying drug efflux genes and their regulatory networks in Escherichia coli and Salmonella. Multidrug efflux pumps are often regulated by environmental signals and they are required for bacterial virulence in addition to multidrug resistance. It is now understood that these efflux pumps also have physiological roles. In this article, we investigate the physiological roles of drug efflux pumps in virulence. Because multidrug efflux pumps have roles in bacterial drug resistance and virulence, we propose that drug efflux pumps have greater clinical relevance than previously considered.

Effect of NlpE overproduction on multidrug resistance in Escherichia coli.

NlpE, an outer membrane lipoprotein, functions during envelope stress responses in Gram-negative bacteria. In this study, we report that overproduction of NlpE increases multidrug and copper resistance through activation of the genes encoding the AcrD and MdtABC multidrug efflux pumps in Escherichia coli.

Macrophage ABCA5 deficiency influences cellular cholesterol efflux and increases susceptibility to atherosclerosis in female LDLr knockout mice.

OBJECTIVES: To determine the role of macrophage ATP-binding cassette transporter A5 (ABCA5) in cellular cholesterol homeostasis and atherosclerotic lesion development. METHODS AND RESULTS: Chimeras with dysfunctional macrophage ABCA5 (ABCA5(-M/-M)) were generated by transplantation of bone marrow from ABCA5 knockout (ABCA5(-/-)) mice into irradiated LDLr(-/-) mice. In vitro, bone marrow-derived macrophages from ABCA5(-M/-M) chimeras exhibited a 29% (P<0.001) decrease in cholesterol efflux to HDL, whereas a 21% (P=0.07) increase in cholesterol efflux to apoA-I was observed. Interestingly, expression of ABCA1, but not ABCG1, was up-regulated in absence of functional ABCA5 in macrophages. To induce atherosclerosis, the transplanted LDLr(-/-) mice were fed a high-cholesterol Western-type diet (WTD) for 6, 10, or 18weeks, allowing analysis of effects on initial as well as advanced lesion development. Atherosclerosis development was not affected in male ABCA5(-M/-M) chimeras after 6, 10, and 18weeks WTD feeding. However, female ABCA5(-M/-M) chimeras did develop significantly (P<0.05) larger aortic root lesions as compared with female controls after 6 and 10weeks WTD feeding. CONCLUSIONS: ABCA5 influences macrophage cholesterol efflux, and selective disruption of ABCA5 in macrophages leads to increased atherosclerotic lesion development in female LDLr(-/-) mice.

TolC dependency of multidrug efflux systems in Salmonella enterica serovar Typhimurium.

OBJECTIVES: TolC is a major outer membrane channel and it plays an important role in the excretion of a wide range of molecules, including antibiotics. A recent study has shown that Salmonella enterica serovar Typhimurium has nine functional drug efflux pumps; however, the TolC dependency of these efflux pumps remains to be studied in detail. The aim of this study was to investigate the TolC dependency of multidrug efflux pumps in this organism. METHODS: All genes encoding the drug efflux systems were cloned into the pUC118 vector. Constructed plasmids were transformed into DeltaacrB and DeltatolC mutants of S. enterica serovar Typhimurium ATCC 14028s, and then the drug susceptibilities of these transformants were determined. RESULTS: Plasmids carrying the acrAB, acrD, acrEF, mdsAB, mdtABC, emrAB or macAB genes did not confer resistance to the tolC mutant, whereas they conferred drug resistance to the acrB mutant. Only three plasmids carrying mdsABC, mdfA or mdtK conferred resistance to the tolC mutant. CONCLUSIONS: TolC is required for the function of seven drug efflux systems (AcrAB, AcrD, AcrEF, MdsAB, MdtABC, EmrAB and MacAB) in S. enterica serovar Typhimurium.

Impact of the RNA chaperone Hfq on multidrug resistance in Escherichia coli.

OBJECTIVES: Hfq is a bacterial RNA chaperone involved in the post-transcriptional regulation of many stress-inducible genes via small non-coding RNAs. Although Hfq is related to important phenotypes including virulence in many bacterial pathogens, its role in drug resistance is unknown. The aim of this study was to investigate the role of Hfq in bacterial multidrug resistance. METHODS: The hfq gene was inactivated in Escherichia coli by use of pKO3, which is a gene replacement vector. The drug susceptibility and drug accumulation of the hfq mutant were determined. The level of production of the AcrB multidrug efflux pump in this mutant was also measured. RESULTS: The hfq mutant was susceptible to acriflavine, benzalkonium, cefamandole, chloramphenicol, Crystal Violet, nalidixic acid, novobiocin, oxacillin and rhodamine 6G. E. coli cells were strongly stained with rhodamine 6G compared with the wild-type on deletion of hfq, indicating that Hfq affects the accumulation of the drug in bacterial cells. The deletion of the drug efflux gene acrB impairs the effect of hfq deletion on E. coli susceptibility. Furthermore, the level of AcrB protein production was reduced in the hfq mutant, whereas hfq deletion did not affect the promoter activity of the acrAB operon. CONCLUSIONS: These results indicate that Hfq regulates the drug efflux system at the post-transcriptional level and reveals the previously uncharacterized role of Hfq in bacterial multidrug resistance.

Indole enhances acid resistance in Escherichia coli.

As a stationary-phase signal, indole is secreted in large quantities by Escherichia coli on enriched media and has been shown to control several genes; however, its impact on acid resistance remains to be studied in detail. Real-time quantitative reverse transcription-polymerase chain reaction analysis revealed that indole increases the expression of the glutamine decarboxylase system that includes genes such as gadA, gadB, and gadC genes with no effect on the expression of other acid resistance systems such as arginine decarboxylase (adiA) and lysine decarboxylase (cadA, cadB, cadC, and ldcC). Indole also induces yhiE (gadE) that encodes the regulator required for expression of gadA, gadB, and gadC. These results suggest that indole enhances the survival of E. coli under acidic conditions by increasing the expression of acid resistance genes of the glutamine decarboxylase system, thus increasing its acid resistance.