Clove Buds Volatile Compounds: Inhibitory Activity on Mycobacterium Growth and Molecular Docking on Mmr Efflux Pump Drug Resistance.

Syzygium aromaticum is used in traditional and modern medicine for its various and outstanding pharmacological properties. Here, we studied the chemical composition of hexane extract and non-polar fractions (NPF) obtained from the maceration and fractionation of clove buds, in order to evaluate their in vitro antimycobacterial activity, as well as their contribution against efflux pump (EP) resistance through molecular docking experiments. The gas chromatography-mass spectrometry (GC-MS) analysis of the volatile profiles revealed the presence of eugenol, followed by eugenyl acetate, and ß-caryophyllene as common major compounds. According to Resazurin microtiter assay (REMA), Mycobacterium tuberculosis H37 Rv strain was sensitive to all volatile samples at concentration range between 10 and 100 µg/mL. The NPF of ethanol extract was the best inhibitor with a MIC=10 µg/mL. The in silico study revealed a strong binding affinity between eugenol and Mmr EP protein (-8.1 Kcal/mol), involving two binding modes of hydrogen bond and π-alkyl interactions. The non-polarity character of clove volatile constituents, and their potential additive or synergistic effects could be responsible for the antimycobacterial activity. In addition, these findings suggest the benefic effect of eugenol in the management of mycobacterium drug resistance, whether as potential inhibitor of Mmr drug EP, or modulator during combination therapy.

The C2H2 Transcription Factor SltA Contributes to Azole Resistance by Coregulating the Expression of the Drug Target Erg11A and the Drug Efflux Pump Mdr1 in Aspergillus fumigatus.

The emergence of azole-resistant fungal pathogens has posed a great threat to public health worldwide. Although the molecular mechanism of azole resistance has been extensively investigated, the potential regulators of azole resistance remain largely unexplored. In this study, we identified a new function of the fungal specific C2H2 zinc finger transcription factor SltA (involved in the salt tolerance pathway) in the regulation of azole resistance of the human fungal pathogen Aspergillus fumigatus A lack of SltA results in an itraconazole hypersusceptibility phenotype. Transcriptional profiling combined with LacZ reporter analysis and electrophoretic mobility shift assays (EMSA) demonstrated that SltA is involved in its own transcriptional regulation and also regulates the expression of genes related to ergosterol biosynthesis (erg11A, erg13A, and erg24A) and drug efflux pumps (mdr1, mfsC, and abcE) by directly binding to the conserved 5'-AGGCA-3' motif in their promoter regions, and this binding is dependent on the conserved cysteine and histidine within the C2H2 DNA binding domain of SltA. Moreover, overexpression of erg11A or mdr1 rescues sltA deletion defects under itraconazole conditions, suggesting that erg11A and mdr1 are related to sltA-mediated itraconazole resistance. Most importantly, deletion of SltA in laboratory-derived and clinical azole-resistant isolates significantly attenuates drug resistance. Collectively, we have identified a new function of the transcription factor SltA in regulating azole resistance by coordinately mediating the key azole target Erg11A and the drug efflux pump Mdr1, and targeting SltA may provide a potential strategy for intervention of clinical azole-resistant isolates to improve the efficiency of currently approved antifungal drugs.

Gene interaction network studies to decipher the multi-drug resistance mechanism in Salmonella enterica serovar Typhi CT18 reveal potential drug targets.

Salmonella enterica subsp. enterica serovar Typhi, a human enteric pathogen causing typhoid fever, developed resistance to multiple antibiotics over the years. The current study was dedicated to understand the multi-drug resistance (MDR) mechanism of S. enterica serovar Typhi CT18 and to identify potential drug targets that could be exploited for new drug discovery. We have employed gene interaction network analysis for 44 genes which had 275 interactions. Clustering analysis resulted in three highly interconnecting clusters (C1-C3). Functional enrichment analysis revealed the presence of drug target alteration and three different multi-drug efflux pumps in the bacteria that were associated with antibiotic resistance. We found seven genes (arnA,B,C,D,E,F,T) conferring resistance to Cationic Anti-Microbial Polypeptide (CAMP) molecules by membrane Lipopolysaccharide (LPS) modification, while macB was observed to be an essential controlling hub of the network and played a crucial role in MacAB-TolC efflux pump. Further, we identified five genes (mdtH, mdtM, mdtG, emrD and mdfA) which were involved in Major Facilitator Superfamily (MFS) efflux system and acrAB contributed towards AcrAB-TolC efflux pump. All three efflux pumps were seen to be highly dependent on tolC gene. The five genes, namely tolC, macB, acrA, acrB and mdfA which were involved in multiple resistance pathways, can act as potential drug targets for successful treatment strategies. Therefore, this study has provided profound insights into the MDR mechanism in S. Typhi CT18. Our results will be useful for experimental biologists to explore new leads for S. enterica.

An efflux inhibitor of the MacAB pump in Salmonella enterica serovar Typhimurium.

Multidrug efflux pumps play an important role in bacterial multidrug resistance by actively excreting antibiotics. The ATP-binding cassette-type drug efflux pump MacAB was originally reported as a macrolide-specific pump. MacAB is also known to be required for the virulence of Salmonella enterica serovar Typhimurium following oral infection in mice. Here, we performed a screening of inhibitors of Salmonella MacAB and found a compound that increased the susceptibility of a MacAB-expressing strain to macrolides. It was previously reported that MacAB is required to resist peroxide-mediated killing in vitro and that a supernatant of wild-type Salmonella rescues the growth defect of a macAB mutant in H2 O2 . In this study, we also found that the MacAB inhibitor reduced the ability of the supernatant to rescue Salmonella cells in H2 O2 . This compound could lead to a better understanding of the function of MacAB.

Targeting the multidrug transporter Patched potentiates chemotherapy efficiency on adrenocortical carcinoma in vitro and in vivo.

One of the crucial challenges in the clinical management of cancer is the resistance to chemotherapeutics. We recently demonstrated that the Hedgehog receptor Patched, which is overexpressed in many recurrent and metastatic cancers, is a multidrug transporter for chemotherapeutic agents such as doxorubicin. The present work provides evidences that Patched is expressed in adrenocortical carcinoma (ACC) patients, and is a major player of the doxorubicin efflux and the doxorubicin resistance in the human ACC cell line H295R. We discovered that methiothepin inhibits the doxorubicin efflux activity of Patched. This drug-like molecule enhances the cytotoxic, pro-apoptotic, antiproliferative and anticlonogenic effects of doxorubicin on ACC cells which endogenously overexpress Patched, and thereby mitigates the resistance of these cancer cells to doxorubicin. Moreover, we report that in mice the combination of methiothepin with doxorubicin prevents the development of xenografted ACC tumors more efficiently than doxorubicin alone by enhancing the accumulation of doxorubicin specifically in tumors without obvious undesirable side effects. Our results suggest that the use of an inhibitor of Patched drug efflux such as methiothepin in combination with doxorubicin could be a promising therapeutic option for adrenocortical carcinoma, and most likely also for other Patched-expressing cancers.

Antimicrobial activity of nanoemulsion on drug-resistant bacterial pathogens.

The appearance of drug-resistant (DR) bacteria in the community is a crucial development, and is associated with increased morbidity, mortality, healthcare costs, and antibiotic use. Natural oil nanoemulsions (NEs) have potential for antimicrobial applications. In the present study, we determined the antimicrobial activity of an NE against DR bacterial pathogens in vitro. The NE comprised Cleome viscosa essential oil, Tween 80 nonionic surfactant, and water. We found that an NE with a droplet size of 7 nm and an oil:surfactant (v/v) ratio of 1:3 was effective against methicillin-resistant Staphylococcus aureus (MRSA), DR Streptococcus pyogenes, and DR extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Fourier-transform infrared (FTIR) spectroscopy revealed that NE treatment modified the functional groups of lipids, proteins, and nucleic acids in DR bacterial cells. Scanning electron microscopy (SEM) showed damage to the cell membranes and walls of NE-treated DR bacteria. These alterations were caused by bioactive compounds with wide-spectrum enzyme-inhibiting activity in the NE, such as ß-sitosterol, demecolcine, campesterol, and heneicosyl formate. The results suggest that the nanoemulsion is effective against DR bacteria, and acts by inhibiting the drug efflux mechanism of DR strains.

Evaluation of a series of 2-napthamide derivatives as inhibitors of the drug efflux pump AcrB for the reversal of antimicrobial resistance.

Drug efflux pumps confer multidrug resistance to dangerous pathogens which makes these pumps important drug targets. We have synthesised a novel series of compounds based on a 2-naphthamide pharmacore aimed at inhibiting the efflux pumps from Gram-negative bacteria. The archeatypical transporter AcrB from Escherichia coli was used as model efflux pump as AcrB is widely conserved throughout Gram-negative organisms. The compounds were tested for their antibacterial action, ability to potentiate the action of antibiotics and for their ability to inhibit Nile Red efflux by AcrB. None of the compounds were antimicrobial against E. coli wild type cells. Most of the compounds were able to inhibit Nile Red efflux indicating that they are substrates of the AcrB efflux pump. Three compounds were able to synergise with antibiotics and reverse resistance in the resistant phenotype. Compound A3, 4-(isopentyloxy)-2-naphthamide, reduced the MICs of erythromycin and chloramphenicol to the MIC levels of the drug sensitive strain that lacks an efflux pump. A3 had no effect on the MIC of the non-substrate rifampicin indicating that this compound acts specifically through the AcrB efflux pump. A3 also does not act through non-specific mechanisms such as outer membrane or inner membrane permeabilisation and is not cytotoxic against mammalian cell lines. Therefore, we have designed and synthesised a novel chemical compound with great potential to further optimisation as inhibitor of drug efflux pumps.

Studies on Acinetobacter baumannii involving multiple mechanisms of carbapenem resistance.

AIMS: Characterize the genetic type and resistance mechanisms of 16 carbapenem-resistant Acinetobacter baumannii (CRAB) isolates recovered between January 2010 and March 2011 from US tertiary-care hospital. METHODS AND RESULTS: A modified Hodge test demonstrated the presence of carbapenemases, but meropenem and ethylenediaminetetraacetic acid (EDTA) double-disc synergy tests and PCR for metallo-ß-lactamase (MBL) genes were negative. The genes of ampC ß-lactamase and efflux pump of adeABC and adeIJK were detected. The presence of oxacillinase (OXA)-like genes, blaOXA-51-like , blaOXA-23-like and blaOXA-40-like genes, and insertion sequence ISAba1 in promoter region of blaOXA-51-like and blaOXA-23-like genes were detected; and confirmed by RT-PCR analyses. The sequencing of blaOXA-51-like genes revealed two major alleles, blaOXA-66-like (blaOXA-82 ) and blaOXA-113 from 31·2 to 68·8% of isolates respectively. The blaOXA-23 and blaOXA-72 genes showed high expression and found co-harbouring blaOXA-51-like gene preceded by ISAba-1. All CRAB isolates revealed significant reduction in carO transcription, indicated downregulation of CarO porin system, a potentially independent mechanism of carbapenam resistance. Sequencing of carO gene from representative isolates showed no ISAba1 insertional inactivation. Pulsed-field gel electrophoresis revealed a clonal relationship. CONCLUSIONS: CRAB exhibited diversity of mechanisms of carbapenem resistance, and clonal relationship. SIGNIFICANCE AND IMPACT OF THE STUDY: Studies on distinct outbreaks of CRAB are alarming situation for clinicians.

The Q loops of the human multidrug resistance transporter ABCB1 are necessary to couple drug binding to the ATP catalytic cycle.

For a primary active pump, such as the human ATP-binding-cassette (ABC) transporter ABCB1, coupling of drug-binding by the two transmembrane domains (TMDs) to the ATP catalytic cycle of the two nucleotide-binding domains (NBDs) is fundamental to the transport mechanism, but is poorly understood at the biochemical level. Structure data suggest that signals are transduced through intracellular loops of the TMDs that slot into grooves on the NBDs. At the base of these grooves is the Q loop. We therefore mutated the eponymous glutamine in one or both NBD Q loops and measured the effect on conformation and function by using a conformation-sensitive antibody (UIC2) and a fluorescent drug (Bodipy-verapamil), respectively. We showed that the double mutant is trapped in the inward-open state, which binds the drug, but cannot couple to the ATPase cycle. Our data also describe marked redundancy within the transport mechanism, because single-Q-loop mutants are functional for Bodipy-verapamil transport. This result allowed us to elucidate transduction pathways from twin drug-binding cavities to the Q loops using point mutations to favor one cavity over the other. Together, the data show that the Q loop is the central flexion point where the aspect of the drug-binding cavities is coupled to the ATP catalytic cycle.

Modulators of Candida albicans Membrane Drug Transporters: A Lucrative Portfolio for the Development of Effective Antifungals.

The escalating prevalence of membrane drug transporters and drug efflux pumps in pathogenic yeast like Candida albicans necessitates a comprehensive understanding of their roles in MDR. The overexpression of drug transporter families, ABC and MFS, implicated in MDR through drug efflux and poses a significant challenge in the diagnosis and treatment of fungal infection. Various mechanisms have been proposed for MDR; however, the upregulation of ABC and MFS superfamily transporters is most noticeable in MDR. The direct inhibition of these transporters seems an efficient strategy to overcome this problem. The goal of the article is to present an overview of the prospect of utilizing these modulators of C. albicans drug transports as effective antifungal molecules against MDR addressing a critical gap in the field. The review tries to address to prevent drug extrusion by modulating the expression of drug transporters of C. albicans. The review discussed the progress in identifying potent, selective, and non-toxic modulators of these transporters to develop some effective antifungals and overcome MDR. We reviewed major studies in this area and found that recent work has shifted toward the exploration of natural compounds as potential modulators to restore drug sensitivity in MDR fungal cells. The focus of this review is to survey and interpret current research information on modulators of C. albicans drug transporters from natural sources emphasizing those compounds that are potent antifungal agents.

A novel member of drug/metabolite transporter (DMT) family efflux pump, SA00565, contributes to tetracycline antibiotics resistance in Staphylococcus aureus USA300.

Drug efflux systems have recently been recognized as a significant mechanism responsible for multidrug resistance in bacteria. In this study, we described the identification and characterization of a new chromosomally encoded efflux pump (SA00565) in Staphylococcus aureus. SA00565, which belongs to the drug/metabolite transporter (DMT) superfamily, was predicted to be a 10-transmembrane segment transporter. To evaluate the role of sa00565 in resistance, we generated sa00565 gene deletion mutant (Δsa00565) and assessed its susceptibility to 35 different antibiotic treatments. Our results demonstrated that the Δsa00565 mutant exhibited reduced resistance to tetracycline and doxycycline, with 64-fold and 12-fold decreased MICs, respectively. The mechanism of SA00565-mediated tetracycline resistance was demonstrated that SA00565 possesses the capability to efficiently extrud intracellular tetracycline into the environment. The efflux activity of SA00565 was further validated using EtBr accumulation and efflux assays. In summary, our study uncovered a previously unknown function of a DMT family transporter, which serves as a tetracycline efflux pump, thereby contributing to tetracycline resistance in S. aureus.IMPORTANCEIn this study, we addressed the significance of drug efflux systems in multidrug resistance of Staphylococcus aureus, focusing on the unexplored efflux pump SA00565 in the drug/metabolite transporter (DMT) superfamily. Through phylogenetic analysis, gene knockout, and overexpression experiments, we identified the role of SA00565 in antibiotic resistance. The Δsa00565 mutant showed increased susceptibility to tetracycline and doxycycline in disk diffusion assays, with significantly lower MICs compared to the WT. Remarkably, intracellular tetracycline concentration in the mutant was two- to threefold higher, indicating SA00565 actively eliminates intracellular tetracycline. Our findings emphasize the pivotal contribution of SA00565 to tetracycline antibiotic resistance in S. aureus, shedding light on its functional attributes within the DMT superfamily and providing valuable insights for combating multidrug resistance.

RND Efflux Pump Induction: A Crucial Network Unveiling Adaptive Antibiotic Resistance Mechanisms of Gram-Negative Bacteria.

The rise of multi-drug-resistant (MDR) pathogenic bacteria presents a grave challenge to global public health, with antimicrobial resistance ranking as the third leading cause of mortality worldwide. Understanding the mechanisms underlying antibiotic resistance is crucial for developing effective treatments. Efflux pumps, particularly those of the resistance-nodulation-cell division (RND) superfamily, play a significant role in expelling molecules from bacterial cells, contributing to the emergence of multi-drug resistance. These are transmembrane transporters naturally produced by Gram-negative bacteria. This review provides comprehensive insights into the modulation of RND efflux pump expression in bacterial pathogens by numerous and common molecules (bile, biocides, pharmaceuticals, additives, plant extracts, etc.). The interplay between these molecules and efflux pump regulators underscores the complexity of antibiotic resistance mechanisms. The clinical implications of efflux pump induction by non-antibiotic compounds highlight the challenges posed to public health and the urgent need for further investigation. By addressing antibiotic resistance from multiple angles, we can mitigate its impact and preserve the efficacy of antimicrobial therapies.

Engineered Biosensors for Diagnosing Multidrug Resistance in Microbial and Malignant Cells.

To curtail pathogens or tumors, antimicrobial or antineoplastic drugs have been developed. These drugs target microbial/cancer growth and survival, thereby improving the host's health. In attempts to evade the detrimental effects of such drugs, these cells have evolved several mechanisms over time. Some variants of the cells have developed resistances against multiple drugs or antimicrobial agents. Such microorganisms or cancer cells are said to exhibit multidrug resistance (MDR). The drug resistance status of a cell can be determined by analyzing several genotypic and phenotypic changes, which are brought about by significant physiological and biochemical alterations. Owing to their resilient nature, treatment and management of MDR cases in clinics is arduous and requires a meticulous approach. Currently, techniques such as plating and culturing, biopsy, gene sequencing, and magnetic resonance imaging are prevalent in clinical practices for determining drug resistance status. However, the major drawbacks of using these methods lie in their time-consuming nature and the problem of translating them into point-of-care or mass-detection tools. To overcome the shortcomings of conventional techniques, biosensors with a low detection limit have been engineered to provide quick and reliable results conveniently. These devices are highly versatile in terms of analyte range and quantities that can be detected to report drug resistance in a given sample. A brief introduction to MDR, along with a detailed insight into recent biosensor design trends and use for identifying multidrug-resistant microorganisms and tumors, is presented in this review.

Porphyromonas gingivalis resistance and virulence: An integrated functional network analysis.

BACKGROUND: The gram-negative bacteria Porphyromonas gingivalis (PG) is the most prevalent cause of periodontal diseases and multidrug-resistant (MDR) infections. Periodontitis and MDR infections are severe due to PG's ability to efflux antimicrobial and virulence factors. This gives rise to colonisation, biofilm development, evasion, and modulation of the host defence system. Despite extensive studies on the MDR efflux pump in other pathogens, little is known about the efflux pump and its association with the virulence factor in PG. Prolonged infection of PG leads to complete loss of teeth and other systemic diseases. This necessitates the development of new therapeutic interventions to prevent and control MDR. OBJECTIVE: The study aims to identify the most indispensable proteins that regulate both resistance and virulence in PG, which could therefore be used as a target to fight against the MDR threat to antibiotics. METHODS: We have adopted a hierarchical network-based approach to construct a protein interaction network. Firstly, individual networks of four major efflux pump proteins and two virulence regulatory proteins were constructed, followed by integrating them into one. The relationship between proteins was investigated using a combination of centrality scores, k-core network decomposition, and functional annotation, to computationally identify the indispensable proteins. RESULTS: Our study identified four topologically significant genes, PG_0538, PG_0539, PG_0285, and PG_1797, as potential pharmacological targets. PG_0539 and PG_1797 were identified to have significant associations between the efflux pump and virulence genes. This type of underpinning research may help in narrowing the drug spectrum used for treating periodontal diseases, and may also be exploited to look into antibiotic resistance and pathogenicity in bacteria other than PG.

Inhibition of the drug efflux activity of Ptch1 as a promising strategy to overcome chemotherapy resistance in cancer cells.

The development of inhibitors of key biological mechanisms involved in multidrug resistance (MDR) burden meets an important medical need but still represents a challenging task. Major MDR targets in both bacterial and cancer cells are multidrug efflux systems. Several aspects should be considered in the attempt to design efficient inhibitors of these systems such as toxicity, stability, permeability as a few examples. In order to successfully design promising new compounds, a full understanding of the efflux mechanism is required, from both biological and structural points of view. It is nowadays well established that the success rate in classical drug design and biological evaluation improves when combined with in silico methodologies. In this review, we focus on the biological evaluation and molecular mechanistic insights of inhibitors of the drug efflux activity of the Hedgehog receptor Patched1 (Ptch1). Ptch1 is known to be over-expressed in many types of cancers, but its activity and role in the resistance to chemotherapy of cancer cells have been highlighted only recently. Remarkably, due to its peculiar efflux mechanism, inhibition of Ptch1 was shown to be particularly relevant for improving the efficacy of chemotherapy without concomitant toxicity for healthy cells or potential side effects. To date, three compounds have been identified as efficient Ptch1 inhibitors, namely astemizole, methiothepin and panicein A hydroquinone. Due to the chemical and structural differences of these molecules, the hit-to-lead drug design is not straightforward. This review describes how the merging of in vitro, in vivo and in silico studies provides molecular details that could contribute to the rational design of new Ptch1 inhibitors.

Efflux Pump AcrAB Confers Decreased Susceptibility to Piperacillin-Tazobactam and Ceftolozane-Tazobactam in Tigecycline-Non-Susceptible Klebsiella pneumoniae.

INTRODUCTION: Drug efflux pumps are critical for resistance in Gram-negative organisms, but there are limited data on the role they play in decreased susceptibility to ß-lactam/ß-lactamase inhibitor combinations. In this study, we aimed to investigate the impact of efflux pump AcrAB on piperacillin-tazobactam (TZP) and ceftolozane-tazobactam (C/T) susceptibility in tigecycline-non-susceptible Klebsiella pneumoniae (TNSKP) strains. METHODS: A tigecycline gradient was used to obtain various TNSKP strains, and in conjunction with the gradient derived strains, a TNSKP clinical strain (TNSKP24) was also included. Minimum inhibitory concentrations (MICs) of antibiotics were determined by the broth microdilution method, and whole-genome sequencing (WGS) was carried out to analyze genomic changes. PCR and sequencing were performed to confirm mutations in ramR, acrR, and the intergenic region of ramR-romA, and qRT-PCR was applied to evaluate levels of gene expression. In-frame acrB knockout and complementation were performed in 3 TNSKP strains. RESULTS: Two derivatives of K. pneumoniae K2606 (K2606-4 and K2606-16) and TNSKP24 overexpressed efflux pump AcrAB were obtained for further study. The MICs of TZP and C/T exhibited a 4- to 8-fold increase in K2606-4 and K2606-16, respectively, when compared with K2606 (TZP, 2/4 µg/mL; C/T, 0.25/4 µg/mL). Deletion of acrB decreased the MICs of TZP and C/T by 4- to 16-fold in TNSKP24, K2606-4, and K2606-16, respectively, and complementation of acrB increased the MICs of these agents. MICs of clavulanate, sulbactam, and avibactam in the presence of ß-lactam compounds did not change after acrB deletion and subsequent introduction of complementation mutants. CONCLUSION: This study highlights that decreased susceptibility to TZP and C/T could be caused by the multidrug efflux pump AcrAB in TNSKP strains.

A Putative Efflux Transporter of the ABC Family, YbhFSR, in Escherichia coli Functions in Tetracycline Efflux and Na+(Li+)/H+ Transport.

ATP-binding cassette transporters are ubiquitous in almost all organisms. The Escherichia coli genome is predicted to encode 69 ABC transporters. Eleven of the ABC transporters are presumed to be exporters, of which seven are possible drug export transporters. There has been minimal research on the function of YbhFSR, which is one of the putative drug resistance exporters. In this study, the ybhF gene of this transporter was characterized. Overexpression and knockout strains of ybhF were constructed. The ATPase activity of YbhF was studied using the malachite green assay, and the efflux abilities of knockout strains were demonstrated by using ethidium bromide (EB) as a substrate. The substrates of YbhFSR efflux, examined with the minimum inhibitory concentration (MIC), were determined to be tetracycline, oxytetracycline, chlortetracycline, doxycycline, EB, and Hoechst33342. Furthermore, tetracycline and EB efflux and accumulation experiments confirmed that the substrates of YbhFSR were tetracyclines and EB. The MIC assay and the fluorescence test results showed that tetracyclines are likely to be the major antibiotic substrate of YbhFSR. The existence of the signature NatA motif suggested that YbhFSR may also function as a Na+/H+ transporter. Overexpression of YbhF in E. coli KNabc lacking crucial Na+/H+ transporters conferred tolerance to NaCl, LiCl, and an alkaline pH. Together, the results showed that YbhFSR exhibited dual functions as a drug efflux pump and a Na+ (Li+)/H+ antiporter.

Development of novel nanoantibiotics using an outer membrane vesicle-based drug efflux mechanism.

Conventionally used antibiotics are present in low concentrations at the infection site and require multiple administrations to sustain a continuous bactericidal effect, which not only increases their systemic toxicity but also results in bacterial drug resistance. In this study, we first identified an interesting drug resistance mechanism mediated by bacterial outer membrane vesicles (OMVs) and then designed novel antibiotic-loaded OMVs using this mechanism. We show that these antibiotic-loaded OMVs can effectively enter and kill pathogenic bacteria in vitro. In a mouse model of intestinal bacterial infection, one low-dose oral administration of antibiotic-loaded OMVs showed that the drug was retained in the intestine for 36 h, and no systemic spread was detected 12 h after drug administration. The antibiotic-loaded OMVs significantly reduced the bacterial load in the small intestine and feces of infected mice. Safety experiments confirmed that the antibiotic-loaded OMVs had excellent biocompatibility. This study extends the application range of OMVs and provides new ideas for the development of antibacterial drugs.

Astemizole Sensitizes Adrenocortical Carcinoma Cells to Doxorubicin by Inhibiting Patched Drug Efflux Activity.

Adrenocortical carcinoma (ACC) presents a high risk of relapse and metastases with outcomes not improving despite extensive research and new targeted therapies. We recently showed that the Hedgehog receptor Patched is expressed in ACC, where it strongly contributes to doxorubicin efflux and treatment resistance. Here, we report the identification of a new inhibitor of Patched drug efflux, the anti-histaminergic drug astemizole. We show that astemizole enhances the cytotoxic, proapoptotic, antiproliferative and anticlonogenic effects of doxorubicin on ACC cells at concentrations of astemizole or doxorubicin that are not effective by themselves. Our results suggest that a low concentration of astemizole sensitizes ACC cells to doxorubicin, which is a component of the standard treatment for ACC composed of etoposide, doxorubicin, cisplatin and mitotane (EDPM). Patched uses the proton motive force to efflux drugs. This makes its function specific to cancer cells, thereby avoiding toxicity issues that are commonly observed with inhibitors of ABC multidrug transporters. Our data provide strong evidence that the use of astemizole or a derivative in combination with EDPM could be a promising therapeutic option for ACC by increasing the treatment effectiveness at lower doses of EDPM, which would reduce the severe side effects of this regimen.

Mutations in the TolC Periplasmic Domain Affect Substrate Specificity of the AcrAB-TolC Pump.

TolC and the other members of the outer membrane factor (OMF) family are outer membrane proteins forming trimeric channels that serve as a conduit for most actively effluxed substrates in Gram-negative bacteria by providing a key component in a multitude of tripartite efflux-pumps. Current models of tripartite pump assembly ascribe substrate selection to the inner-membrane transporter and periplasmic-adapter protein (PAP) assembly, suggesting that TolC is a passive, non-selective channel. While the membrane-embedded portion of the protein adopts a porin-like fold, the periplasmic domain of TolC presents a unique "alpha-barrel" architecture. This alpha-barrel consists of pseudo-continuous α-helices forming curved coiled-coils, whose tips form α-helical hairpins, relaxation of which results in a transition of TolC from a closed to an open-aperture state allowing effective efflux of substrates through its channel. Here, we analyzed the effects of site-directed mutations targeting the alpha-barrel of TolC, of the principal tripartite efflux-pump Escherichia coli AcrAB-TolC, on the activity and specificity of efflux. Live-cell functional assays with these TolC mutants revealed that positions both at the periplasmic tip of, and partway up the TolC coiled-coil alpha-barrel domain are involved in determining the functionality of the complex. We report that mutations affecting the electrostatic properties of the channel, particularly the D371V mutation, significantly impact growth even in the absence of antibiotics, causing hyper-susceptibility to all tested efflux-substrates. These results suggest that inhibition of TolC functionality is less well-tolerated than deletion of tolC, and such inhibition may have an antibacterial effect. Significantly and unexpectedly, we identified antibiotic-specific phenotypes associated with novel TolC mutations, suggesting that substrate specificity may not be determined solely by the transporter protein or the PAP, but may reside at least partially with the TolC-channel. Furthermore, some of the effects of mutations are difficult to reconcile with the currently prevalent tip-to-tip model of PAP-TolC interaction due to their location higher-up on the TolC alpha-barrel relative to the proposed PAP-docking sites. Taken together our results suggest a possible new role for TolC in vetting of efflux substrates, alongside its established role in tripartite complex assembly.