Resistance-nodulation-division Pumps Research Articles

Repurposing Mitomycin C in Combination with Pentamidine or Gentamicin to Treat Infections with Multi-Drug-Resistant (MDR) Pseudomonas aeruginosa.

The aims of this study were (i) to determine if the combination of mitomycin C with pentamidine or existing antibiotics resulted in enhanced efficacy versus infections with MDR P. aeruginosa in vivo; and (ii) to determine if the doses of mitomycin C and pentamidine in combination can be reduced to levels that are non-toxic in humans but still retain antibacterial activity. Resistant clinical isolates of P. aeruginosa, a mutant strain over-expressing the MexAB-OprM resistance nodulation division (RND) efflux pump and a strain with three RND pumps deleted, were used. MIC assays indicated that all strains were sensitive to mitomycin C, but deletion of three RND pumps resulted in hypersensitivity and over-expression of MexAB-OprM caused some resistance. These results imply that mitomycin C is a substrate of the RND efflux pumps. Mitomycin C monotherapy successfully treated infected Galleria mellonella larvae, albeit at doses too high for human administration. Checkerboard and time-kill assays showed that the combination of mitomycin C with pentamidine, or the antibiotic gentamicin, resulted in synergistic inhibition of most P. aeruginosa strains in vitro. In vivo, administration of a combination therapy of mitomycin C with pentamidine, or gentamicin, to G. mellonella larvae infected with P. aeruginosa resulted in enhanced efficacy compared with monotherapies for the majority of MDR clinical isolates. Notably, the therapeutic benefit conferred by the combination therapy occurred with doses of mitomycin C close to those used in human medicine. Thus, repurposing mitomycin C in combination therapies to target MDR P. aeruginosa infections merits further investigation.

HME, NFE, and HAE-1 efflux pumps in Gram-negative bacteria: a comprehensive phylogenetic and ecological approach.

The three primary resistance-nodulation-cell division (RND) efflux pump families (heavy metal efflux [HME], nodulation factor exporter [NFE], and hydrophobe/amphiphile efflux-1 [HAE-1]) are almost exclusively found in Gram-negative bacteria and play a major role in resistance against metals and bacterial biocides, including antibiotics. Despite their significant societal interest, their evolutionary history and environmental functions are poorly understood. Here, we conducted a comprehensive phylogenetic and ecological study of the RND permease, the subunit responsible for the substrate specificity of these efflux pumps. From 920 representative genomes of Gram-negative bacteria, we identified 6205 genes encoding RND permeases with an average of 6.7 genes per genome. The HME family, which is involved in metal resistance, corresponds to a single clade (21.8% of all RND pumps), but the HAE-1 and NFE families had overlapping distributions among clades. We propose to restrict the HAE-1 family to two phylogenetic sister clades, representing 41.8% of all RND pumps and grouping most of the RND pumps involved in multidrug resistance. Metadata associated with genomes, analyses of previously published metagenomes, and quantitative Polymerase Chain Reaction (qPCR) analyses confirmed a significant increase in genes encoding HME permeases in metal-contaminated environments. Interestingly, and possibly related to their role in root colonization, genes encoding HAE-1 permeases were particularly abundant in the rhizosphere. In addition, we found that the genes encoding these HAE-1 permeases are significantly less abundant in marine environments, whereas permeases of a new proposed HAE-4 family are predominant in the genomes of marine strains. These findings emphasize the critical role of the RND pumps in bacterial resistance and adaptation to diverse ecological niches.

Acinetobacter baumannii Survival under Infection-Associated Stresses Depends on the Expression of Resistance-Nodulation-Division and Major Facilitator Superfamily Efflux Pumps.

Multidrug efflux transporters are major contributors to the antibiotic resistance of Acinetobacter baumannii in clinical settings. Previous studies showed that these transporters are tightly integrated into the physiology of A. baumannii and have diverse functions. However, for many of the efflux pumps, such functions remain poorly defined. In this study, we characterized two putative drug efflux pumps, AmfAB and AmfCD (Acinetobacter Major Facilitator), that are homologous to EmrAB-like transporters from Escherichia coli and other Gram-negative bacteria. These pumps comprise the Major Facilitator Superfamily (MFS) transporters AmfB and AmfD and the periplasmic membrane fusion proteins AmfA and AmfC, respectively. We inactivated and overproduced these pumps in the wild-type ATCC 17978 strain and its derivative strains lacking the major efflux pumps from the Resistance-Nodulation-Division (RND) superfamily and characterized antibiotic susceptibilities and growth of the strains under stresses typical during human infections. We found that neither AmfAB nor AmfCD contribute to the antibiotic non-susceptibility phenotypes of A. baumannii. The two pumps, however, are critical for the adaptation and growth of the bacterium under acidic stress, whereas AmfCD also contributes to growth under conditions of low iron, high temperature, and in the presence of bile salts. These functions are dependent on the presence of the RND pumps, the inactivation of which further diminishes A. baumannii survival and growth. Our results suggest that MFS transporters contribute to stress survival by affecting the permeability properties of the A. baumannii cell envelope.

RND pumps across the genus Acinetobacter: AdeIJK is the universal efflux pump.

Acinetobacter are generally soil-dwelling organisms that can also cause serious human infections. A. baumannii is one of the most common causative agents of Acinetobacter infections and is often multidrug resistant. However, an additional 25 species within the genus have also been associated with infection. A. baumannii encodes six resistance nodulation division (RND) efflux pumps, the most clinically relevant class of efflux pumps for antibiotic export, but the distribution and types of RND efflux pumps across the genus is currently unknown. Sixty-four species making up the genus Acinetobacter were searched for RND systems within their genomes. We also developed a novel method using conserved RND residues to predict the total number of RND proteins including currently undescribed RND pump proteins. The total number of RND proteins differed both within a species and across the genus. Species associated with infection tended to encode more pumps. AdeIJK/AdeXYZ was found in all searched species of Acinetobacter, and through genomic, structural and phenotypic work we show that these genes are actually homologues of the same system. This interpretation is further supported by structural analysis of the potential drug-binding determinants of the associated RND-transporters, which reveal their close similarity to each other, and distinctiveness from other RND-pumps in Acinetobacter, such as AdeB. Therefore, we conclude that AdeIJK is the fundamental RND system for species in the genus Acinetobacter. AdeIJK can export a broad range of antibiotics and provides crucial functions within the cell, for example lipid modulation of the cell membrane, and therefore it is likely that all Acinetobacter require AdeIJK for survival and homeostasis. In contrast, additional RND systems, such as AdeABC and AdeFGH, were only found in a subset of Acinetobacter that are associated with infection. By understanding the roles and mechanisms of RND efflux systems in Acinetobacter, treatments for infections can avoid efflux-mediated resistance and improve patient outcomes.

Identification of Promoter Region Markers Associated With Altered Expression of Resistance-Nodulation-Division Antibiotic Efflux Pumps in Acinetobacter baumannii.

Genetic alterations leading to the constitutive upregulation of specific efflux pumps contribute to antibacterial resistance in multidrug resistant bacteria. The identification of such resistance markers remains one of the most challenging tasks of genome-level resistance predictors. In this study, 487 non-redundant genetic events were identified in upstream zones of three operons coding for resistance-nodulation-division (RND) efflux pumps of 4,130 Acinetobacter baumannii isolates. These events included insertion sequences, small indels, and single nucleotide polymorphisms. In some cases, alterations explicitly modified the expression motifs described for these operons, such as the promoter boxes, operators, and Shine-Dalgarno sequences. In addition, changes in DNA curvature and mRNA secondary structures, which are structural elements that regulate expression, were also calculated. According to their influence on RND upregulation, the catalog of upstream modifications were associated with “experimentally verified,” “presumed,” and “probably irrelevant” degrees of certainty. For experimental verification, DNA of upstream sequences independently carrying selected markers, three for each RND operon, were fused to a luciferase reporter plasmid system. Five out of the nine selected markers tested showed significant increases in expression with respect to the wild-type sequence control. In particular, a 25-fold expression increase was observed with the ISAba1 insertion sequence upstream the adeABC pump. Next, overexpression of each of the three multi-specific RND pumps was linked to their respective antibacterial substrates by a deep A. baumannii literature screen. Consequently, a data flow framework was then developed to link genomic upregulatory RND determinants to potential antibiotic resistance. Assignment of potential increases in minimal inhibitory concentrations at the “experimentally verified” level was permitted for 42 isolates to 7–8 unrelated antibacterial agents including tigecycline, which is overlooked by conventional resistome predictors. Thus, our protocol may represent a time-saving filter step prior to laborious confirmation experiments for efflux-driven resistance. Altogether, a computational-experimental pipeline containing all components required for identifying the upstream regulatory resistome is proposed. This schema may provide the foundational stone for the elaboration of tools approaching antibiotic efflux that complement routine resistome predictors for preventing antimicrobial therapy failure against difficult-to-threat bacteria.

A role for the periplasmic adaptor protein AcrA in vetting substrate access to the RND efflux transporter AcrB

Tripartite resistance-nodulation-division (RND) efflux pumps, such as AcrAB-TolC of Salmonella Typhimurium, contribute to antibiotic resistance and comprise an inner membrane RND-transporter, an outer membrane factor, and a periplasmic adaptor protein (PAP). The role of the PAP in the assembly and active transport process remains poorly understood. Here, we identify the functionally critical residues involved in PAP-RND-transporter binding between AcrA and AcrB and show that the corresponding RND-binding residues in the closely related PAP AcrE, are also important for its interaction with AcrB. We also report a residue in the membrane-proximal domain of AcrA, that when mutated, differentially affects the transport of substrates utilising different AcrB efflux channels, namely channels 1 and 2. This supports a potential role for the PAP in sensing the substrate-occupied state of the proximal binding pocket of the transporter and substrate vetting. Understanding the PAP’s role in the assembly and function of tripartite RND pumps can guide novel ways to inhibit their function to combat antibiotic resistance.

Membrane Efflux Pumps of Pathogenic Vibrio Species: Role in Antimicrobial Resistance and Virulence

Infectious diseases caused by bacterial species of the Vibrio genus have had considerable significance upon human health for centuries. V. cholerae is the causative microbial agent of cholera, a severe ailment characterized by profuse watery diarrhea, a condition associated with epidemics, and seven great historical pandemics. V. parahaemolyticus causes wound infection and watery diarrhea, while V. vulnificus can cause wound infections and septicemia. Species of the Vibrio genus with resistance to multiple antimicrobials have been a significant health concern for several decades. Mechanisms of antimicrobial resistance machinery in Vibrio spp. include biofilm formation, drug inactivation, target protection, antimicrobial permeability reduction, and active antimicrobial efflux. Integral membrane-bound active antimicrobial efflux pump systems include primary and secondary transporters, members of which belong to closely related protein superfamilies. The RND (resistance-nodulation-division) pumps, the MFS (major facilitator superfamily) transporters, and the ABC superfamily of efflux pumps constitute significant drug transporters for investigation. In this review, we explore these antimicrobial transport systems in the context of Vibrio spp. pathogenesis and virulence.

Artesunate interrupts the self-transcriptional activation of MarA to inhibit RND family pumps of Escherichia coli

Resistance-Nodulation-Division (RND) family pumps are responsible for producing multidrug resistance in Escherichia coli; however, there has been little study of targeted inhibitors of RNDs. In the present study, we investigated the inhibition of RND pumps by artesunate (AS) in E. coli, and further investigated the mechanism with respect to MarA, a regulator of RNDs. Although AS had no direct antibacterial effect, it showed a synergistic effect in combination with β-lactams against E. coli ATCC35218 in vitro and in vivo, suggesting it possesses antibacterial enhancement activity. Notably, AS, alone or in combination with β-lactams, downregulated the mRNA expression levels of marA, soxS, and rob, known as the marA-soxS-rob regulon, which then decreased the expression levels of RNDs, thereby increased ampicillin accumulation within ATCC35218. Using gene-deletion strains, we found that the antibacterial sensitization effect of AS persisted in wildtype bacteria, but was completely lost in the strain lacking marA, and decreased in the strain lacking soxS or rob, suggesting marA plays a crucial role in the sensitization of AS. Critically, we showed that AS inhibited the binding of MarA to the promoter of marA itself, not acrB, resulting in decreased mRNA expression of both acrB and marA. Mechanistically, we found AS directly bound to the central cavity of MarA through the R59 and K62 residues, and thus altered the charge distribution of MarA to interrupt the recognition between MarA and its promoter. We concluded that AS interrupts the self-transcriptional activation of MarA, thereby inhibits MarA-dependent mRNA expression of marA, acrAB, and tolC, and also certain other RNDs and regulatory genes related to MarA. Therefore, AS is a novel inhibitor of RND pumps that acts on the regulator MarA.

Inactivation of AdeABC and AdeIJK efflux pumps elicits specific nonoverlapping transcriptional and phenotypic responses in Acinetobacter baumannii.

Multidrug resistant (MDR) strains of Acinetobacter baumannii present a serious clinical challenge. The development of antibiotic resistance in this species is enabled by efflux pumps of the Resistance-Nodulation-Division (RND) superfamily of proteins creating an efficient permeability barrier for antibiotics. At least three RND pumps, AdeABC, AdeIJK, and AdeFGH are encoded in the A. baumannii genome and are reported to contribute to antibiotic resistance in clinical isolates. In this study, we analyzed the contributions of AdeABC and AdeIJK in antibiotic resistance and growth physiology of the two MDR strains, AYE and AB5075. We found that not only the two pumps have nonoverlapping substrate specificities, their inactivation leads to specific nonoverlapping changes in gene expression as determined by RNA sequencing and confirmed by gene knockouts and growth phenotypes. Our results suggest that inactivation of AdeIJK elicits broader changes in the abundances of mRNAs and this response is modified in the absence of AdeB. In contrast, inactivation of AdeB leads to a focused cellular response, which is not sensitive to the activity of AdeIJK. We identified additional efflux pumps and transcriptional regulators that contribute to MDR phenotype of clinical A. baumannii isolates.

Defining the RND-binding residues of AcrA

The resistance-nodulation-division (RND) family of efflux pumps confer clinically relevant antibiotic resistance in Gram-negative bacteria, such as Salmonella enterica. RND pumps, including AcrB, are organized as tri-partite systems, consisting of an inner membrane RND pump, a periplasmic adaptor protein (PAP) and an outer membrane channel. Previously, inactivation of the PAPs AcrA and AcrE in S. enterica has been shown to significantly increase susceptibility to antimicrobials and reduce virulence. Therefore, PAPs are seen as attractive targets for the development of efflux pump inhibitors. However, the role of PAPs in the assembly of tri-partite pumps and the residues involved in PAP-RND pump binding is poorly understood. In this study, point mutations in the predicted RND binding residues of AcrA were generated by site-directed mutagenesis. The point mutants were characterised phenotypically through ethidium bromide efflux assays and antimicrobial susceptibility testing. Furthermore, Western blotting was used to verify that the phenotypic effect of the point mutations was not due to destabilisation of the AcrA protein. Point mutations in certain residues, such as G58, F292, R294 and G363 were found to significantly impair efflux activity and increase susceptibility to various antibiotics and dyes, suggesting an important role for these AcrA residues in RND pump binding. Western blotting confirmed that these point mutants were stable and exhibited similar expression levels to the wild-type. These residues could be important targets for the design and development of PAP inhibitors to restore the activity of existing antibiotics and reduce virulence of Salmonella.

An RND transporter in the monoterpene metabolism of Castellaniella defragrans

The betaproteobacterium Castellaniella defragrans 65Phen grows on monoterpenes at concentrations toxic to many bacteria. Tolerance mechanisms include modifications of the membrane fatty acid composition and the mineralization of monoterpenes. In this study, we characterized an efflux transporter associated to the monoterpene metabolism. The inner-membrane transporter AmeD (apolar monoterpene efflux) affiliated to the HAE3 (hydrophobe/amphiphile efflux) family within the Resistance-Nodulation-Division (RND) superfamily. RND pumps of the HAE3 family are known for transporting substrates into the periplasm. AmeD is co-expressed with the outer membrane protein AmeA and the periplasmic proteins AmeB and AmeC, suggesting an export channel into the environment similar to HAE1-type RND exporters. Proteins AmeABCD are encoded within a genetic island involved in the metabolism of acyclic and cyclic monoterpenes. The deletion of ameABCD translated into a decrease in tolerance to monoterpenes in liquid cultures. The addition of acetate as cosubstrate in limonene-containing cultures partially alleviated monoterpene toxicity in the deletion mutant. Accumulation of Nile Red in cells of C. defragrans required dissipation of the proton motive force with carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells lacking AmeABCD accumulated more Nile Red, suggesting an export function of the proteins. Our observations suggest that the tetrapartite RND transporter AmeABCD acts as an exporter during monoterpene detoxification in C. defragrans.

The hydrophobic trap—the Achilles heel of RND efflux pumps

Resistance–nodulation–division (RND) superfamily efflux pumps play a major role in multidrug resistance (MDR) of Gram-negative pathogens by extruding diverse classes of antibiotics from the cell. There has been considerable interest in developing efflux pump inhibitors (EPIs) of RND pumps as adjunctive therapies. The primary challenge in EPI discovery has been the highly hydrophobic, poly-specific substrate binding site of the target. Recent findings have identified the hydrophobic trap, a narrow phenylalanine-lined groove in the substrate-binding site, as the “Achilles heel” of the RND efflux pumps. In this review, we will examine the hydrophobic trap as an EPI target and two chemically distinct series of EPIs that bind there.

Lack of AcrB Efflux Function Confers Loss of Virulence on Salmonella enterica Serovar Typhimurium.

ABSTRACTAcrAB-TolC is the paradigm resistance-nodulation-division (RND) multidrug resistance efflux system in Gram-negative bacteria, with AcrB being the pump protein in this complex. We constructed a nonfunctional AcrB mutant by replacing D408, a highly conserved residue essential for proton translocation. Western blotting confirmed that the AcrB D408A mutant had the same native level of expression of AcrB as the parental strain. The mutant had no growth deficiencies in rich or minimal medium. However, compared with wild-type SL1344, the mutant had increased accumulation of Hoechst 33342 dye and decreased efflux of ethidium bromide and was multidrug hypersusceptible. The D408A mutant was attenuated in vivo in mouse and Galleria mellonella models and showed significantly reduced invasion into intestinal epithelial cells and macrophages in vitro. A dose-dependent inhibition of invasion was also observed when two different efflux pump inhibitors were added to the wild-type strain during infection of epithelial cells. RNA sequencing (RNA-seq) revealed downregulation of bacterial factors necessary for infection, including those in the Salmonella pathogenicity islands 1, 2, and 4; quorum sensing genes; and phoPQ. Several general stress response genes were upregulated, probably due to retention of noxious molecules inside the bacterium. Unlike loss of AcrB protein, loss of efflux function did not induce overexpression of other RND efflux pumps. Our data suggest that gene deletion mutants are unsuitable for studying membrane transporters and, importantly, that inhibitors of AcrB efflux function will not induce expression of other RND pumps.

Evidence that a novel quaternary compound and its organic N-chloramine derivative do not select for resistant mutants of Pseudomonas aeruginosa

Pseudomonas aeruginosa is well known for causing hospital-acquired infections that are often difficult to treat because of its high intrinsic and acquired resistance to antibiotics. Resistance-nodulation-division (RND) efflux pumps are the major contributors to the intrinsic multidrug resistance (MDR) in this organism. Various biocides used in hospital settings have been shown to select for RND-pump-overexpressing mutants of P. aeruginosa that show cross-resistance to clinically relevant antibiotics. Therefore, finding biocides that do not select for multidrug-resistant mutants is important in controlling the spread of bacteria such as P. aeruginosa. To evaluate the potential of a novel quaternary ammonium compound and its N-chloramine derivative in selecting for MDR mutants of P. aeruginosa. P. aeruginosa PA01 was cultured in the presence of increasing concentrations of the quaternary ammonium compound and its N-chloramine derivative respectively, and one mutant each selected. Susceptibility of the mutants to both compounds as well as antibiotics was tested. Susceptibility of P. aeruginosa strains with deletions in RND pumps was also tested for both compounds to determine whether they are a substrate of these pumps. Expression of mexB, mexD, and mexY genes in the mutants was analysed using quantitative reverse transcriptase-polymerase chain reaction to determine whether the compounds can select for pump-overexpressing mutants. We show that whereas both compounds can be pumped by the MexCD-OprJ pump, they neither select for mutants that overexpress RND pumps nor for mutants that display cross-resistance to antibiotics. These compounds are promising candidates to be used as disinfectants in hospital settings.

The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria.

The global emergence of multidrug-resistant Gram-negative bacteria is a growing threat to antibiotic therapy. The chromosomally encoded drug efflux mechanisms that are ubiquitous in these bacteria greatly contribute to antibiotic resistance and present a major challenge for antibiotic development. Multidrug pumps, particularly those represented by the clinically relevant AcrAB-TolC and Mex pumps of the resistance-nodulation-division (RND) superfamily, not only mediate intrinsic and acquired multidrug resistance (MDR) but also are involved in other functions, including the bacterial stress response and pathogenicity. Additionally, efflux pumps interact synergistically with other resistance mechanisms (e.g., with the outer membrane permeability barrier) to increase resistance levels. Since the discovery of RND pumps in the early 1990s, remarkable scientific and technological advances have allowed for an in-depth understanding of the structural and biochemical basis, substrate profiles, molecular regulation, and inhibition of MDR pumps. However, the development of clinically useful efflux pump inhibitors and/or new antibiotics that can bypass pump effects continues to be a challenge. Plasmid-borne efflux pump genes (including those for RND pumps) have increasingly been identified. This article highlights the recent progress obtained for organisms of clinical significance, together with methodological considerations for the characterization of MDR pumps.

Functional analysis of Vibrio vulnificus RND efflux pumps homologous to Vibrio cholerae VexAB and VexCD, and to Escherichia coli AcrAB.

Resistance-nodulation-division (RND) efflux pumps are associated with multidrug resistance in many gram-negative pathogens. The genome of Vibrio vulnificus encodes 11 putative RND pumps homologous to those of Vibrio cholerae and Escherichia coli. In this study, we analyzed three putative RND efflux pumps, showing homology to V. cholerae VexAB and VexCD and to E. coli AcrAB, for their functional roles in multidrug resistance of V. vulnificus. Deletion of the vexAB homolog resulted in increased susceptibility of V. vulnificus to bile acid, acriflavine, ethidium bromide, and erythromycin, whereas deletion of acrAB homologs rendered V. vulnificus more susceptible to acriflavine only. Deletion of vexCD had no effect on susceptibility of V. vulnificus to these chemicals. Upon exposure to these antibacterial chemicals, expression of tolCV1 and tolCV2, which are putative outer membrane factors of RND efflux pumps, was induced, whereas expression levels of vexAB, vexCD, and acrAB homologs were not significantly changed. Our results show that the V. vulnificus homologs of VexAB largely contributed to in vitro antimicrobial resistance with a broad substrate specificity that was partially redundant with the AcrAB pump homologs.

A linkage between SmeIJK efflux pump, cell envelope integrity, and σE-mediated envelope stress response in Stenotrophomonas maltophilia.

Resistance nodulation division (RND) efflux pumps, such as the SmeIJK pump of Stenotrophomonas maltophilia, are known to contribute to the multidrug resistance in Gram-negative bacteria. However, some RND pumps are constitutively expressed even though no antimicrobial stresses occur, implying that there should be some physical implications for these RND pumps. In this study, the role of SmeIJK in antimicrobials resistance, envelope integrity, and σE-mediated envelope stress response (ESR) of S. maltophilia was assessed. SmeIJK was involved in the intrinsic resistance of S. maltophilia KJ to aminoglycosides and leucomycin. Compared with the wild-type KJ, the smeIJK deletion mutant exhibited growth retardation in the MH medium, an increased sensitivity to membrane-damaging agents (MDAs), as well as activation of an σE-mediated ESR. Moreover, the expression of smeIJK was further induced by sub-lethal concentrations of MDAs or surfactants in an σE-dependent manner. These data collectively suggested an alternative physiological role of smeIJK in cell envelope integrity maintenance and σE-mediated ESR beyond the efflux of antibiotics. Because of the necessity of the physiological role of SmeIJK in protecting S. maltophilia from the envelope stress, smeIJK is constitutively expressed, which, in turn, contributes the intrinsic resistance to aminoglycoside and leucomycin. This is the first demonstration of the linkage among RND-type efflux pump, cell envelope integrity, and σE-mediated ESR in S. maltophilia.

Molecular mechanism of MBX2319 inhibition of Escherichia coli AcrB multidrug efflux pump and comparison with other inhibitors.

Efflux pumps of the resistance nodulation division (RND) superfamily, such as AcrB, make a major contribution to multidrug resistance in Gram-negative bacteria. The development of inhibitors of the RND pumps would improve the efficacy of current and next-generation antibiotics. To date, however, only one inhibitor has been cocrystallized with AcrB. Thus, in silico structure-based analysis is essential for elucidating the interaction between other inhibitors and the efflux pumps. In this work, we used computer docking and molecular dynamics simulations to study the interaction between AcrB and the compound MBX2319, a novel pyranopyridine efflux pump inhibitor with potent activity against RND efflux pumps of Enterobacteriaceae species, as well as other known inhibitors (D13-9001, 1-[1-naphthylmethyl]-piperazine, and phenylalanylarginine-β-naphthylamide) and the binding of doxorubicin to the efflux-defective F610A variant of AcrB. We also analyzed the binding of a substrate, minocycline, for comparison. Our results show that MBX2319 binds very tightly to the lower part of the distal pocket in the B protomer of AcrB, strongly interacting with the phenylalanines lining the hydrophobic trap, where the hydrophobic portion of D13-9001 was found to bind by X-ray crystallography. Additionally, MBX2319 binds to AcrB in a manner that is similar to the way in which doxorubicin binds to the F610A variant of AcrB. In contrast, 1-(1-naphthylmethyl)-piperazine and phenylalanylarginine-β-naphthylamide appear to bind to somewhat different areas of the distal pocket in the B protomer of AcrB than does MBX2319. However, all inhibitors (except D13-9001) appear to distort the structure of the distal pocket, impairing the proper binding of substrates.

MexY-promoted aminoglycoside resistance in Pseudomonas aeruginosa: involvement of a putative proximal binding pocket in aminoglycoside recognition.

ABSTRACTThe resistance-nodulation-division (RND) family multidrug efflux system MexXY-OprM is a major determinant of aminoglycoside resistance in Pseudomonas aeruginosa, although the details of aminoglycoside recognition and export by MexY, the substrate-binding RND component of this efflux system, have not been elucidated. To identify regions/residues of MexY important for aminoglycoside resistance, plasmid-borne mexY was mutagenized and mutations that impaired MexY-promoted aminoglycoside (streptomycin) resistance were identified in a ΔmexY strain of P. aeruginosa. Sixty-one streptomycin-sensitive mexY mutants were recovered; among these, 7 unique mutations that yielded wild-type levels of MexY expression were identified. These mutations compromised resistance to additional aminoglycosides and to other antimicrobials and occurred in both the transmembrane and periplasmic regions of the protein. Mapping of the mutated residues onto a 3-dimensional structure of MexY modeled on Escherichia coli AcrB revealed that these tended to occur in regions implicated in general pump operation (transmembrane domain) and MexY trimer assembly (docking domain) and, thus, did not provide insights into aminoglycoside recognition. A region corresponding to a proximal binding pocket connected to a periplasm-linked cleft, part of a drug export pathway of AcrB, was identified in MexY and proposed to play a role in aminoglycoside recognition. To test this, selected residues (K79, D133, and Y613) within this pocket were mutagenized and the impact on aminoglycoside resistance was assessed. Mutations of D133 and Y613 compromised aminoglycoside resistance, while, surprisingly, the K79 mutation enhanced aminoglycoside resistance, confirming a role for this putative proximal binding pocket in aminoglycoside recognition and export.

Resistance-Nodulation-Division Multidrug Efflux Pumps in Gram-Negative Bacteria: Role in Virulence.

Resistance-Nodulation-Division (RND) efflux pumps are one of the most important determinants of multidrug resistance (MDR) in Gram-negative bacteria. With an ever increasing number of Gram-negative clinical isolates exhibiting MDR phenotypes as a result of the activity of RND pumps, it is clear that the design of novel effective clinical strategies against such pathogens must be grounded in a better understanding of these pumps, including their physiological roles. To this end, recent evidence suggests that RND pumps play an important role in the virulence of Gram-negative pathogens. In this review, we discuss the important role RND efflux pumps play in different facets of virulence including colonization, evasion of host defense mechanisms, and biofilm formation. These studies provide key insights that may ultimately be applied towards strategies used in the design of effective therapeutics against MDR Gram negative bacterial pathogens.