Discovering targeted inhibitors for Escherichia coli efflux pump fusion proteins using computational and structure-guided approaches.

Multidrug resistance pathogens causing infections and illness remain largely untreated clinically. Efflux pumps are one of the primary processes through which bacteria develop resistance by transferring antibiotics from the interior of their cells to the outside environment. Inhibiting these pumps by developing efficient derivatives appears to be a promising strategy for restoring antibiotic potency. This investigation explores literature-reported inhibitors of E. coli efflux pump fusion proteins AcrB-AcrA and identify potential chemical derivatives of these inhibitors to overcome the limitations. Using computational and structure-guided approaches, a study was conducted with the selected inhibitors (AcrA:25-AcrB:59) obtained by data mining and their derivatives (AcrA:857-AcrB:3891) to identify their inhibitory effect on efflux pump using virtual screening, molecular docking and density functional theory (DFT) calculations. The finding indicates that Compound 2 (ZINC000072136376) has shown better binding and a significant inhibitory effect on AcrA, while Compound 3 (ZINC000072266819) has shown stronger binding and substantial inhibition effect on both non-mutant and mutated AcrB subunits. The identified derivatives could exhibit a better inhibitor and provide a potential approach for restoring the actions of resistant antibiotics.

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.

Role of bacterial multidrug efflux pumps during infection.

Multidrug efflux pumps are protein complexes located in the cell envelope that enable bacteria to expel, not only antibiotics, but also a wide array of molecules relevant for infection. Hence, they are important players in microbial pathogenesis. On the one hand, efflux pumps can extrude exogenous compounds, including host-produced antimicrobial molecules. Through this extrusion, pathogens can resist antimicrobial agents and evade host defenses. On the other hand, efflux pumps also have a role in the extrusion of endogenous compounds, such as bacterial intercommunication signaling molecules, virulence factors or metabolites. Therefore, efflux pumps are involved in the modulation of bacterial behavior and virulence, as well as in the maintenance of the bacterial homeostasis under different stresses found within the host. This review delves into the multifaceted roles that efflux pumps have, shedding light on their impact on bacterial virulence and their contribution to bacterial infection. These observations suggest that strategies targeting bacterial efflux pumps could both reinvigorate the efficacy of existing antibiotics and modulate the bacterial pathogenicity to the host. Thus, a comprehensive understanding of bacterial efflux pumps can be pivotal for the development of new effective strategies for the management of infectious diseases.

Mass Spectrometry Investigation of Some ATP-Binding Cassette (ABC) Proteins.

Drug resistance remains one of the main causes of poor outcome in cancer therapy. It is also becoming evident that drug resistance to both chemotherapy and to antibiotics is driven by more than one mechanism. So far, there are at least eight recognized mechanisms behind such resistance. In this review, we choose to discuss one of these mechanisms, which is known to be partially driven by a class of transmembrane proteins known as ATP-binding cassette (ABC) transporters. In normal tissues, ABC transporters protect the cells from the toxic effects of xenobiotics, whereas in tumor cells, they reduce the intracellular concentrations of anticancer drugs, which ultimately leads to the emergence of multidrug resistance (MDR). A deeper understanding of the structures and the biology of these proteins is central to current efforts to circumvent resistance to both chemotherapy, targeted therapy, and antibiotics. Understanding the biology and the function of these proteins requires detailed structural and conformational information for this class of membrane proteins. For many years, such structural information has been mainly provided by X-ray crystallography and cryo-electron microscopy. More recently, mass spectrometry-based methods assumed an important role in the area of structural and conformational characterization of this class of proteins. The contribution of this technique to structural biology has been enhanced by its combination with liquid chromatography and ion mobility, as well as more refined labelling protocols and the use of more efficient fragmentation methods, which allow the detection and localization of labile post-translational modifications. In this review, we discuss the contribution of mass spectrometry to efforts to characterize some members of the ATP-binding cassette (ABC) proteins and why such a contribution is relevant to efforts to clarify the link between the overexpression of these proteins and the most widespread mechanism of chemoresistance.

Microbial Primer: Multidrug efflux pumps.

Multidrug efflux pumps are molecular machines that sit in the bacterial cell membrane and pump molecules out from either the periplasm or cytoplasm to outside the cell. While involved in a variety of biological roles, they are primarily known for their contribution to antibiotic resistance by limiting the intracellular accumulation of antimicrobial compounds within bacteria. These transporters are often overexpressed in clinical isolates, leading to multidrug-resistant phenotypes. Efflux pumps are classified into several families based on their structure and understanding the characteristics of each family is important for the development of novel therapies to restore antibiotic potency.

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.

Antibiotic Resistance in Pseudomonas.

Pseudomonas is a bacterial genus, with a bona fide environmental habitat that comprises different species, some of them causing diseases in humans and plants, as well as some strains with biotechnological potential. Amongst them, Pseudomonas aeruginosa is currently one of the most important nosocomial pathogens. In addition, this microorganism is a prevalent cause of chronic infections in cystic fibrosis patients and in people suffering from chronic obstructive pulmonary disease. The success of P. aeruginosa in colonising different habitats largely relies on its metabolic versatility and robustness. Besides, this bacterial pathogen harbours in its core genome a large set of virulence determinants that allows it to colonise/infect a variety of hosts, from unicellular organisms to humans. Nevertheless, these are not just the only conditions needed for infecting patients at hospitals. Taking into consideration that infected patients are regularly under antibiotic treatment, the ability to avoid antibiotics' action is also needed. In this sense, P. aeruginosa displays a characteristic low susceptibility to several antibiotics currently used in therapy. This is due to the reduced permeability of its cellular envelopes and the presence in its genome of an arrangement of genes encoding multidrug efflux pumps and antibiotic-inactivating enzymes that contribute to its resilience to antibiotics. Besides intrinsic resistance, P. aeruginosa is able to evolve towards antibiotic resistance through mutations (particularly relevant in the case of chronic infections) and via acquisition of antibiotic resistance genes. It is worth mentioning that acquired resistance is not the only venue that P. aeruginosa has for avoiding the action of antibiotics. Transient resistance can also confer this phenotype. Indeed, the induction of the expression of intrinsic resistance genes by conditions or compounds that P. aeruginosa could face during infection can compromise the effectiveness of antibiotics for treating such infections. In addition, tolerant cells able to survive during the exposure to bactericidal antibiotics without an increase in their antibiotic resistance phenotype are found as well in these patients, and they are the prelude of the evolution towards antibiotic resistance. Finally, P. aeruginosa biofilms, frequently encountered in the lungs of cystic fibrosis patients, in prostheses, or in catheters, present low antibiotic susceptibility and are associated with recalcitrance and disease worsening.

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.

Characterization of a Unique Outer Membrane Protein Required for Oxidative Stress Resistance and Virulence of Francisella tularensis.

Francisella tularensis, the causative agent of tularemia, lacks typical bacterial virulence factors and toxins but still exhibits extreme virulence. The bacterial multidrug efflux systems consist of an inner membrane, a transmembrane membrane fusion protein, and an outer membrane (OM) component that form a contiguous channel for the secretion of a multitude of bacterial products. Francisella contains three orthologs of the OM proteins; two of these, termed TolC and FtlC, are important for tularemia pathogenesis. The third OM protein, SilC, is homologous to the silver cation efflux protein of other bacterial pathogens. The silC gene (FTL_0686) is located on an operon encoding an Emr-type multidrug efflux pump of F. tularensis The role of SilC in tularemia pathogenesis is not known. In this study, we investigated the role of SilC in secretion and virulence of F. tularensis by generating a silC gene deletion (ΔsilC) mutant and its transcomplemented strain. Our results demonstrate that the ΔsilC mutant exhibits increased sensitivity to antibiotics, oxidants, silver, diminished intramacrophage growth, and attenuated virulence in mice compared to wild-type F. tularensis However, the secretion of antioxidant enzymes of F. tularensis is not impaired in the ΔsilC mutant. The virulence of the ΔsilC mutant is restored in NADPH oxidase-deficient mice, indicating that SilC resists oxidative stress in vivo Collectively, this study demonstrates that the OM component SilC serves a specialized role in virulence of F. tularensis by conferring resistance against oxidative stress and silver.IMPORTANCEFrancisella tularensis, the causative agent of a fatal human disease known as tularemia, is a category A select agent and a potential bioterror agent. The virulence mechanisms of Francisella are not completely understood. This study investigated the role of a unique outer membrane protein, SilC, of a multidrug efflux pump in the virulence of F. tularensis This is the first report demonstrating that the OM component SilC plays an important role in efflux of silver and contributes to the virulence of F. tularensis primarily by providing resistance against oxidative stress. Characterization of these unique virulence mechanisms will provide an understanding of the pathogenesis of tularemia and identification of potential targets for the development of effective therapeutics and prophylactics for protection from this lethal disease.

The S-layer biogenesis system of Synechocystis 6803: Role of Sll1180 and Sll1181 (E. coli HlyB and HlyD analogs) as type-I secretion components for Sll1951 export.

Multiple secretion pathways are known for export of protein(s) forming the S-layer in bacteria. The unicellular model cyanobacterium Synechocystis sp. strain PCC 6803 (hereafter S. 6803) also possesses a well-defined S-layer composed of Sll1951 protein. However, the mechanism of its secretion is not completely understood. In the present study, the putative T1SS (Type I secretion system) components, Sll1180 and Sll1181 [inner membrane ABC transporter and membrane fusion protein (MFP), respectively] were characterized for their role in Sll1951 secretion. The corresponding ORFs i.e. sll1180 and sll1181 were inactivated by insertion of a spectinomycin resistance gene. The viability of the homozygous mutants of both the genes indicated dispensability of the corresponding proteins under the experimental conditions. Interestingly, the culture supernatants of the mutants i.e. Δsll1180 and Δsll1181, lacked Sll1951 as observed on SDS-PAGE and confirmed by mass spectrometry. Immunofluorescence delineated a distinct outer ring of Sll1951 in S. 6803 cells only that was further iterated by transmission and scanning electron microscopy. The loss of S-layer imparted an aggregative phenotype to both the mutants. Surprisingly, Δsll1181 cells showed increased sensitivity to different antibiotics indicating a role in multidrug efflux. This is the first report establishing Sl1180 and Sll1181 proteins as partners of the previously characterized Slr1270, for Sll1951 secretion and thus S-layer biogenesis in S. 6803. Sll1181 (in conjunction with Slr1270) also acts as MFP in multidrug efflux along with a yet uncharacterized inner membrane protein.

The highly conserved domain of RND multidrug efflux pumps in pathogenic Gram-negative bacteria.

RND (Resistance-Nodulation-Division) family transporters have a vital role in both intrinsic and acquired multi-drug resistance in Gram-negative bacteria. It is important to find a conserved domain in the RND family between different pathogenic bacteria for diagnostic and therapeutic purpose. Total sequences of three-component system RND efflux pumps were retrieved from NCBI nucleotide and protein database and were subjected to conservation and variation analysis using the multiple sequence alignment feature of the CLC workbench. The phylogenetic tree for main transporters was drawn and the three-dimensional structure was also evaluated. From the sequence conservation analysis, highly conserved residues with 282 base pair (94 amino acid) long were identified. The location of the highly conserved domain is positioned in the domain 1 crystallographic structure of AcrB Escherichia coli and MexB Pseudomonas aeruginosa. The main transporter component phylogenetic tree shows the clusters of different genotypes and their evolutionary association.  Each of three components of RND proteins is crucial for drug efflux, and the absence of even one component makes the entire complex totally nonfunctional. Therefore, this highly conserved region can be used to disable the RND multidrug efflux pumps. In addition, this highly conserved can also be used for diagnostic aspects.

ATP-Binding Cassette Transporter VcaM from Vibrio cholerae is Dependent on the Outer Membrane Factor Family for Its Function.

Vibrio cholerae ATP-binding cassette transporter VcaM (V. cholerae ABC multidrug resistance pump) has previously been shown to confer resistance to a variety of medically important drugs. In this study, we set to analyse its properties both in vitro in detergent-solubilised state and in vivo to differentiate its dependency on auxiliary proteins for its function. We report the first detailed kinetic parameters of purified VcaM and the rate of phosphate (Pi) production. To determine the possible functional dependencies of VcaM on the tripartite efflux pumps we then utilized different E. coli strains lacking the principal secondary transporter AcrB (Acriflavine resistance protein), as well as cells lacking the outer membrane factor (OMF) TolC (Tolerance to colicins). Consistent with the ATPase function of VcaM we found it to be susceptible to sodium orthovanadate (NaOV), however, we also found a clear dependency of VcaM function on TolC. Inhibitors targeting secondary active transporters had no effects on either VcaM-conferred resistance or Hoechst 33342 accumulation, suggesting that VcaM might be capable of engaging with the TolC-channel without periplasmic mediation by additional transporters. Our findings are indicative of VcaM being capable of a one-step substrate translocation from cytosol to extracellular space utilising the TolC-channel, making it the only multidrug ABC-transporter outside of the MacB-family with demonstrable TolC-dependency.

CmeABC Multidrug Efflux Pump Contributes to Antibiotic Resistance and Promotes Campylobacter jejuni Survival and Multiplication in Acanthamoeba polyphaga.

Campylobacter jejuni is a foodborne pathogen that is recognized as the leading cause of human bacterial gastroenteritis. The widespread use of antibiotics in medicine and in animal husbandry has led to an increased incidence of antibiotic resistance in Campylobacter In addition to a role in multidrug resistance (MDR), the Campylobacter CmeABC resistance-nodulation-division (RND)-type efflux pump may be involved in virulence. As a vehicle for pathogenic microorganisms, the protozoan Acanthamoeba is a good model for investigations of bacterial survival in the environment and the molecular mechanisms of pathogenicity. The interaction between C. jejuni 81-176 and Acanthamoeba polyphaga was investigated in this study by using a modified gentamicin protection assay. In addition, a possible role for the CmeABC MDR pump in this interaction was explored. Here we report that this MDR pump is beneficial for the intracellular survival and multiplication of C. jejuni in A. polyphaga but is dispensable for biofilm formation and motility.IMPORTANCE The endosymbiotic relationship between amoebae and microbial pathogens may contribute to persistence and spreading of the latter in the environment, which has significant implications for human health. In this study, we found that Campylobacter jejuni was able to survive and to multiply inside Acanthamoeba polyphaga; since these microorganisms can coexist in the same environment (e.g., on poultry farms), the latter may increase the risk of infection with Campylobacter Our data suggest that, in addition to its role in antibiotic resistance, the CmeABC MDR efflux pump plays a role in bacterial survival within amoebae. Furthermore, we demonstrated synergistic effects of the CmeABC MDR efflux pump and TetO on bacterial resistance to tetracycline. Due to its role in both the antibiotic resistance and the virulence of C. jejuni, the CmeABC MDR efflux pump could be considered a good target for the development of antibacterial drugs against this pathogen.

Multidrug efflux pumps as main players in intrinsic and acquired resistance to antimicrobials.

Multidrug efflux pumps constitute a group of transporters that are ubiquitously found in any organism. In addition to other functions with relevance for the cell physiology, efflux pumps contribute to the resistance to compounds used for treating different diseases, including resistance to anticancer drugs, antibiotics or antifungal compounds. In the case of antimicrobials, efflux pumps are major players in both intrinsic and acquired resistance to drugs currently in use for the treatment of infectious diseases. One important aspect not fully explored of efflux pumps consists on the identification of effectors able to induce their expression. Indeed, whereas the analysis of clinical isolates have shown that mutants overexpressing these resistance elements are frequently found, less is known on the conditions that may trigger expression of efflux pumps, hence leading to transient induction of resistance in vivo, a situation that is barely detectable using classical susceptibility tests. In the current article we review the structure and mechanisms of regulation of the expression of bacterial and fungal efflux pumps, with a particular focus in those for which a role in clinically relevant resistance has been reported.

Threading the Needle: Small-Molecule Targeting of a Xenobiotic Receptor to Ablate Escherichia coli Polysaccharide Capsule Expression Without Altering Antibiotic Resistance.

BACKGROUND: Uropathogenic Escherichia coli (UPEC), a leading cause of urinary tract and invasive infections worldwide, is rapidly acquiring multidrug resistance, hastening the need for selective new anti-infective agents. Here we demonstrate the molecular target of DU011, our previously discovered potent, nontoxic, small-molecule inhibitor of UPEC polysaccharide capsule biogenesis and virulence. METHODS: Real-time polymerase chain reaction analysis and a target-overexpression drug-suppressor screen were used to localize the putative inhibitor target. A thermal shift assay quantified interactions between the target protein and the inhibitor, and a novel DNase protection assay measured chemical inhibition of protein-DNA interactions. Virulence of a regulatory target mutant was assessed in a murine sepsis model. RESULTS: MprA, a MarR family transcriptional repressor, was identified as the putative target of the DU011 inhibitor. Thermal shift measurements indicated the formation of a stable DU011-MprA complex, and DU011 abrogated MprA binding to its DNA promoter site. Knockout of mprA had effects similar to that of DU011 treatment of wild-type bacteria: a loss of encapsulation and complete attenuation in a murine sepsis model, without any negative change in antibiotic resistance. CONCLUSIONS: MprA regulates UPEC polysaccharide encapsulation, is essential for UPEC virulence, and can be targeted without inducing antibiotic resistance.

Identification and functional characterization of Penicillium marneffei pleiotropic drug resistance transporters ABC1 and ABC2.

Penicilliosis caused by the dimorphic fungus Penicillium marneffei is an endemic, AIDS-defining illness and, after tuberculosis and cryptococcosis, the third most common opportunistic infection of AIDS patients in tropical Southeast Asia. Untreated, patients have poor prognosis; however, primary amphotericin B treatment followed by prolonged itraconazole prophylaxis is effective. To identify ATP-binding cassette (ABC) transporters that may play a role in potential multidrug resistance of P. marneffei, we identified and classified all 46 P. marneffei ABC transporters from the genome sequence. PmABC1 and PmABC2 were most similar to the archetype Candida albicans multidrug efflux pump gene CDR1. P. marneffei Abc1p (PmAbc1p) was functionally expressed in Saccharomyces cerevisiae, although at rather low levels, and correctly localized to the plasma membrane, causing cells to be fourfold to eightfold more resistant to azoles and many other xenobiotics than untransformed cells. P. marneffei Abc2p (PmAbc2p) was expressed at similarly low levels, but it had no efflux activity and did not properly localize to the plasma membrane. Interestingly, PmAbc1p mislocalized and lost its transport activity when cells were shifted to 37 °C. We conclude that expression of PmAbc1p in S. cerevisiae confers resistance to several xenobiotics indicating that PmAbc1p may be a multidrug efflux pump.

Bacterial multidrug efflux pumps: mechanisms, physiology and pharmacological exploitations.

Multidrug resistance (MDR) refers to the capability of bacterial pathogens to withstand lethal doses of structurally diverse drugs which are capable of eradicating non-resistant strains. MDR has been identified as a major threat to the public health of human being by the World Health Organization (WHO). Among the four general mechanisms that cause antibiotic resistance including target alteration, drug inactivation, decreased permeability and increased efflux, drug extrusion by the multidrug efflux pumps serves as an important mechanism of MDR. Efflux pumps not only can expel a broad range of antibiotics owing to their poly-substrate specificity, but also drive the acquisition of additional resistance mechanisms by lowering intracellular antibiotic concentration and promoting mutation accumulation. Over-expression of multidrug efflux pumps have been increasingly found to be associated with clinically relevant drug resistance. On the other hand, accumulating evidence has suggested that efflux pumps also have physiological functions in bacteria and their expression is subject tight regulation in response to various of environmental and physiological signals. A comprehensive understanding of the mechanisms of drug extrusion, and regulation and physiological functions of efflux pumps is essential for the development of anti-resistance interventions. In this review, we summarize the development of these research areas in the recent decades and present the pharmacological exploitation of efflux pump inhibitors as a promising anti-drug resistance intervention.

AdeIJK Pump-Specific Inhibitors Effective against Multidrug Resistant Acinetobacter baumannii.

Multidrug-resistant Acinetobacter baumannii is a serious threat pathogen rapidly spreading in clinics and causing a range of complicated human infections. The major contributor to A. baumannii antibiotic resistance is the overproduction of AdeIJK and AdeABC multidrug efflux pumps of the resistance-nodulation-division (RND) superfamily of proteins. The dominant role of efflux in antibiotic resistance and the relatively high permeability of the A. baumannii outer membrane to amphiphilic compounds make this pathogen a promising target for the discovery of clinically relevant efflux pump inhibitors. In this study, we identified 4,6-diaminoquoniline analogs with inhibitory activities against A. baumannii AdeIJK efflux pump and followed up on these compounds with a focused synthetic program to improve the target specificity and to reduce cytotoxicity. We identified several candidates that potentiate antibacterial activities of antibiotics erythromycin, tetracycline, and novobiocin not only in the laboratory antibiotic susceptible strain A. baumannii ATCC17978 but also in multidrug-resistant clinical isolates AB5075 and AYE. The best analogs potentiated the activities of antibiotics in low micromolar concentrations, did not have antibacterial activities on their own, inhibited AdeIJK-mediated efflux of its fluorescent substrate ethidium ion, and had low cytotoxicity in A549 human lung epithelial cells.

Major facilitator superfamily efflux pumps in human pathogens: Role in multidrug resistance and beyond.

The major facilitator superfamily (MFS) of proteins constitutes a large group of related solute transporters found across all known living taxa of organisms. The transporters of the MFS contain an extremely diverse array of substrates, including ions, molecules of intermediary metabolism, and structurally different antimicrobial agents. First discovered over 30 years ago, the MFS represents an important collection of integral membrane transporters. Bacterial microorganisms expressing multidrug efflux pumps belonging to the MFS are considered serious pathogens, accounting for alarming morbidity and mortality numbers annually. This review article considers recent advances in the structure-function relationships, the transport mechanism, and modulation of MFS multidrug efflux pumps within the context of drug resistance mechanisms of bacterial pathogens of public health concerns.

Unraveling the ligand specificity and promiscuity of the Staphylococcus aureus NorA efflux pump: a computational study.

Staphylococcus aureus, a gram-positive bacterial pathogen, develops antibiotic resistance partly through enhanced activity of transmembrane multi-drug efflux pump proteins like NorA. Being a prominent member of the Major Facilitator Superfamily (MFS), NorA transports various small molecules including hydrophilic fluoroquinolone antibiotics across the cell membrane. Intriguingly, NorA is inhibited by a structurally diverse set of small molecule inhibitors as well, indicating a highly promiscuous ligand/inhibitor recognition. Our study aims to elucidate the structural facets of this promiscuity. Known NorA inhibitors were grouped into five clusters based on chemical class and docked into ligand binding pockets on NorA conformations generated via molecular dynamics simulations. We discovered that several key residues, such as I23, E222, and F303, are involved in inhibitor binding. Additionally, residues I244, T223, F303, and F140 were identified as prominent in interactions with specific ligand clusters. Our findings suggest that NorA's substrate binding site, encompassing residues aiding ligand recognition based on chemical nature, facilitates the recognition of chemically diverse ligands. This insight into NorA's structural promiscuity in ligand recognition not only enhances understanding of antibiotic resistance mechanisms in S. aureus but also sets the stage for the development of more effective efflux pump inhibitors, vital for combating multidrug resistance.Communicated by Ramaswamy H. Sarma.