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.

Phylogenetic and functional characterisation of the Haemophilus influenzae multidrug efflux pump AcrB.

Multidrug resistance in Gram-negative bacteria can arise by the over-expression of multidrug efflux pumps, which can extrude a wide range of antibiotics. Here we describe the ancestral Haemophilus influenzae efflux pump AcrB (AcrB-Hi). We performed a phylogenetic analysis of hundreds of RND-type transporters. We found that AcrB-Hi is a relatively ancient efflux pump, which nonetheless can export the same range of antibiotics as its evolved colleague from Escherichia coli. AcrB-Hi was not inhibited by the efflux pump inhibitor ABI-PP, and could export bile salts weakly. This points to an environmental adaptation of RND transporters. We also explain the sensitivity of H. influenzae cells to ß-lactams and novobiocin by the outer membrane porin OmpP2. This porin counterbalances the AcrB-Hi efflux by leaking the drugs back into the cells. We hypothesise that multidrug recognition by RND-type pumps is not an evolutionarily acquired ability, and has been present since ancient promiscuous transporters.

Crystal structures of multidrug efflux pump MexB bound with high-molecular-mass compounds.

RND-type multidrug efflux pumps have two voluminous multisite drug-binding pockets named the proximal and distal binding pocket. High- and low-molecular-mass drugs bind to these proximal and distal pocket, respectively. Here, we report the crystal structures of MexB of Pseudomonas aeruginosa bound with high-molecular-mass compounds. Contrary to the expectations, lauryl maltose neopentyl glycol (LMNG, MW 1,005), which is a surfactant larger than the proximal pocket-binding drugs, was found to bind to the distal pocket: one of the two hydrophobic alkyl chains was inserted into the hydrophobic pit, which is the binding site of the efflux pump inhibitor ABI-PP. LMNG is a substrate of the MexAB-OprM system and competitively inhibits the export of other substrates by this system. However, LMNG does not inhibit the export of other substrates by the inhibitor-binding-pit mutant F178W, which retains the export activity of LMNG. The crystal structure of this mutant suggested that the alkyl chain of LMNG could no longer be inserted into the pit because of steric hindrance. We also determined the crystal structure of MexB containing the high-molecular-mass compound neopentyl glycol derivative C7NG (MW 1,028), the binding site of which overlapped with LMNG in the distal pocket, indicating that whether a substrate binds to the distal or proximal pockets is controlled not only by its molecular weight but also by its individual molecular characteristic.

Molecular mechanisms of AcrB-mediated multidrug export.

The over-expression of multidrug efflux pumps belonging to the Resistance-Nodulation-Division (RND) superfamily is one of the main causes of multidrug-resistance (MDR) in Gram-negative pathogenic bacteria. AcrB is the most thoroughly studied RND transporter and has functioned as a model for our understanding of efflux-mediated MDR. This multidrug-exporter can recognize and transport a wide range of structurally unrelated compounds (including antibiotics, dyes, bile salts and detergents), while it shows a strict inhibitor specificity. Here we discuss our current knowledge of AcrB, and include recent advances, regarding its structure, mechanism of drug transport, substrate recognition, different intramolecular entry pathways and the drug export driven by remote conformational coupling.

MFSD2B is a sphingosine 1-phosphate transporter in erythroid cells.

Sphingosine 1-phosphate (S1P) is an intercellular signaling molecule present in blood. Erythrocytes have a central role in maintaining the S1P concentration in the blood stream. We previously demonstrated that S1P is exported from erythrocytes by a glyburide-sensitive S1P transporter. However, the gene encoding the S1P transporter in erythrocytes is unknown. In this study, we found that the mouse erythroid cell line, MEDEP-E14, has S1P export activity and exhibits properties that are consistent with those of erythrocytes. Using microarray analysis of MEDEP-E14 cells and its parental cell line, E14TG2a, we identified several candidate genes for S1P export activity. Of those genes, only one gene, Mfsd2b, showed S1P transport activity. The properties of S1P release by MFSD2B were similar to those in erythrocytes. Moreover, knockout of MFSD2B in MEDEP-E14 cells decreased S1P export from the cells. These results strongly suggest that MFSD2B is a novel S1P transporter in erythroid cells.

Multiple entry pathways within the efflux transporter AcrB contribute to multidrug recognition.

AcrB is the major multidrug exporter in Escherichia coli. Although several substrate-entrances have been identified, the specificity of these various transport paths remains unclear. Here we present evidence for a substrate channel (channel 3)  from the central cavity of the AcrB trimer, which is connected directly to the deep pocket without first passing the switch-loop and the proximal pocket . Planar aromatic cations, such as ethidium, prefer channel 3 to channels 1 and 2. The efflux through channel 3 increases by targeted mutations and is not in competition with the export of drugs such as minocycline and erythromycin through channels 1 and 2. A switch-loop mutant, in which the pathway from the proximal to the deep pocket is hindered, can export only channel 3-utilizing drugs. The usage of multiple entrances thus contributes to the recognition and transport of a wide range of drugs with different physicochemical properties.

Crystallographic Analysis of Drug and Inhibitor-Binding Structure of RND-Type Multidrug Exporter AcrB in Physiologically Relevant Asymmetric Crystals.

Xenobiotic extruding pumps have recently been known to be widely distributed in living organisms from mammalian to bacteria as a host-defense mechanism in cellular level. These pumps not only confer multidrug resistance of cancer cells and pathogenic bacteria but also cause hereditary diseases through the mutation. Our purposes are to elucidate the molecular structures and mechanisms of these xenobiotic exporters.We had succeeded to determine the crystal structure of bacterial major multidrug exporter AcrB at 3.5 Å resolution (Murakami et al., Nature 419:587-593, 2002) and elucidated the structural bases of substrate recognition that the pump recognize the places and thus act as a "membrane vacuum cleaner." After that we also determined the crystal structure of the drug-binding form of AcrB in space group C2 in which asymmetric unit contains structurally asymmetric homo-trimer of AcrB (Murakami et al., Nature 443:173-179, 2006; Nakashima et al., Nature 480:565-569, 2011; Nakashima et al., Nature 500:120-126, 2013). Analyses revealed the existence of a specific mechanism to recognize numerous substrates that the multisite binding is the base of multidrug recognition rather than induced-fit, and functional-rotation mechanism in which three monomers undergo a strictly coordinated sequential conformational change cycle of access, binding, and extrusion. Determination of physiological asymmetric AcrB structure was crucially important to understand these transport mechanisms.

Hoisting-Loop in Bacterial Multidrug Exporter AcrB Is a Highly Flexible Hinge That Enables the Large Motion of the Subdomains.

The overexpression of RND-type exporters is one of the main causes of multidrug resistance (MDR) in Gram-negative pathogens. In RND transporters, such as Escherichia coli's main efflux pump AcrB, drug efflux occurs in the porter domain, while protons flow through the transmembrane domain: remote conformational coupling. At the border of a transmembrane helix (TM8) and subdomain PC2, there is a loop which makes a hoisting movement by a random-coil-to-α-helix change, and opens and closes a drug channel entrance. This loop is supposed to play a key role in the allosteric conformational coupling between the transmembrane and porter domain. Here we show the results of a series of flexibility loop-mutants of AcrB. We determined the crystal structure of a three amino acid truncated loop mutant, which is still a functional transporter, and show that the short α-helix between Cß15 and the loop unwinds to a random coil in the access and binding monomers and in the extrusion monomer it makes a partially stretched coil-to-helix change. The loop has undergone compensatory conformational changes and still facilitates the opening and closing of the channel. In addition, more flexible mutated loops (proline mutated and significantly elongated) can still function during export. The flexibility in this region is however limited, as an even more truncated mutant (six amino acid deletion) becomes mostly inactive. We found that the hoisting-loop is a highly flexible hinge that enables the conformational energy transmission passively.

Structural Analysis and New Drug Development against Multidrug Efflux Pumps.

Multidrug efflux pumps 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. ABI-PP (a pryridopyrimidine derivative) is a MexB-specific inhibitor that does not inhibit MexY; MexB and MexY are principal pumps in Pseudomonas aeruginosa. We previously found that drugs were exported through tandem proximal and distal multisite drug-binding pockets. Here we describe the first inhibitor-bound structures of pumps. ABI-PP binds tightly to a narrow pit located in the distal pocket and sterically hinders the functional rotation. Phenylalanine is located at the edge of this pit in MexB and contributes to the tight binding of the inhibitor molecule. On the other hand, the voluminous side chain of tryptophan located at the corresponding position in MexY prevents inhibitor binding. For the development of universal inhibitors of MexB and MexY, it is important to avoid the steric hindrance of tryptophan in MexY. Now we are developing clinically useful inhibitors on the basis of the structural information obtained. Started from the ABI-PP structure, we designed many compounds that can bind to the inhibitor-binding pits of MexB and MexY. Some of designed compounds were actually synthesized and their inhibitory activity determined. Finally, we obtained some lead compounds that showed complete prevention of the growth of strains expressing MexB and MexY with low concentrations of antibiotics.

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.

A Microfluidic Channel Method for Rapid Drug-Susceptibility Testing of Pseudomonas aeruginosa.

The recent global increase in the prevalence of antibiotic-resistant bacteria and lack of development of new therapeutic agents emphasize the importance of selecting appropriate antimicrobials for the treatment of infections. However, to date, the development of completely accelerated drug susceptibility testing methods has not been achieved despite the availability of a rapid identification method. We proposed an innovative rapid method for drug susceptibility testing for Pseudomonas aeruginosa that provides results within 3 h. The drug susceptibility testing microfluidic (DSTM) device was prepared using soft lithography. It consisted of five sets of four microfluidic channels sharing one inlet slot, and the four channels are gathered in a small area, permitting simultaneous microscopic observation. Antimicrobials were pre-introduced into each channel and dried before use. Bacterial suspensions in cation-adjusted Mueller-Hinton broth were introduced from the inlet slot and incubated for 3 h. Susceptibilities were microscopically evaluated on the basis of differences in cell numbers and shapes between drug-treated and control cells, using dedicated software. The results of 101 clinically isolated strains of P. aeruginosa obtained using the DSTM method strongly correlated with results obtained using the ordinary microbroth dilution method. Ciprofloxacin, meropenem, ceftazidime, and piperacillin caused elongation in susceptible cells, while meropenem also induced spheroplast and bulge formation. Morphological observation could alternatively be used to determine the susceptibility of P. aeruginosa to these drugs, although amikacin had little effect on cell shape. The rapid determination of bacterial drug susceptibility using the DSTM method could also be applicable to other pathogenic species, and it could easily be introduced into clinical laboratories without the need for expensive instrumentation.

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.

Structural basis of RND-type multidrug exporters.

Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient ejection of drugs from the cell. Substrates are transported through dual multidrug-binding pockets via the peristaltic motion of the substrate translocation channel. Although there are no clinically available inhibitors of bacterial multidrug exporters, efforts to develop inhibitors based on structural information are underway.

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.

Streptococcus pneumoniae invades erythrocytes and utilizes them to evade human innate immunity.

Streptococcus pneumoniae, a Gram-positive bacterium, is a major cause of invasive infection-related diseases such as pneumonia and sepsis. In blood, erythrocytes are considered to be an important factor for bacterial growth, as they contain abundant nutrients. However, the relationship between S. pneumoniae and erythrocytes remains unclear. We analyzed interactions between S. pneumoniae and erythrocytes, and found that iron ion present in human erythrocytes supported the growth of Staphylococcus aureus, another major Gram-positive sepsis pathogen, while it partially inhibited pneumococcal growth by generating free radicals. S. pneumoniae cells incubated with human erythrocytes or blood were subjected to scanning electron and confocal fluorescence microscopic analyses, which showed that the bacterial cells adhered to and invaded human erythrocytes. In addition, S. pneumoniae cells were found associated with human erythrocytes in cultures of blood from patients with an invasive pneumococcal infection. Erythrocyte invasion assays indicated that LPXTG motif-containing pneumococcal proteins, erythrocyte lipid rafts, and erythrocyte actin remodeling are all involved in the invasion mechanism. In a neutrophil killing assay, the viability of S. pneumoniae co-incubated with erythrocytes was higher than that without erythrocytes. Also, H2O2 killing of S. pneumoniae was nearly completely ineffective in the presence of erythrocytes. These results indicate that even when S. pneumoniae organisms are partially killed by iron ion-induced free radicals, they can still invade erythrocytes. Furthermore, in the presence of erythrocytes, S. pneumoniae can more effectively evade antibiotics, neutrophil phagocytosis, and H2O2 killing.

Design of a large-scale femtoliter droplet array for single-cell analysis of drug-tolerant and drug-resistant bacteria.

Single-cell analysis is a powerful method to assess the heterogeneity among individual cells, enabling the identification of very rare cells with properties that differ from those of the majority. In this Methods Article, we describe the use of a large-scale femtoliter droplet array to enclose, isolate, and analyze individual bacterial cells. As a first example, we describe the single-cell detection of drug-tolerant persisters of Pseudomonas aeruginosa treated with the antibiotic carbenicillin. As a second example, this method was applied to the single-cell evaluation of drug efflux activity, which causes acquired antibiotic resistance of bacteria. The activity of the MexAB-OprM multidrug efflux pump system from Pseudomonas aeruginosa was expressed in Escherichia coli and the effect of an inhibitor D13-9001 were assessed at the single cell level.

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.

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.