scholarly journals Functional and Genetic Characterization of the Tap Efflux Pump in Mycobacterium bovis BCG

2012 ◽  
Vol 56 (4) ◽  
pp. 2074-2083 ◽  
Author(s):  
Santiago Ramón-García ◽  
Virginie Mick ◽  
Elisa Dainese ◽  
Carlos Martín ◽  
Charles J. Thompson ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Stationary Phase ◽  
Mycobacterium Bovis ◽  
Efflux Pump ◽  
Expression Patterns ◽  
Efflux Pumps ◽  
Gene Encoding ◽  
Content Type ◽  
Active Efflux ◽  
Bacterial Physiology

ABSTRACTEfflux pumps extrude a wide variety of chemically unrelated compounds conferring multidrug resistance and participating in numerous physiological processes.Mycobacterium tuberculosispossesses many efflux pumps, and their roles in drug resistance and physiology are actively investigated. In this work we found thattapmutant cells showed changes in morphology and a progressive loss of viability upon subcultivation in liquid medium. Transcriptome analysis inMycobacterium bovisBCG revealed that disruption of theRv1258cgene, encoding the Tap efflux pump, led to an extensive change in gene expression patterns during stationary phase, with no changes during exponential growth. In stationary phase, Tap inactivation triggered a general stress response and led to a general repression of genes involved in cell wall biosynthesis, in particular the formation of the peptidoglycan; this suggested the accumulation of an unknown Tap substrate that reaches toxic concentrations during stationary phase. We also found that both disruption and overexpression oftapaltered susceptibility to many clinically approved antibiotics inM. bovisBCG. Acriflavine and tetracycline accumulation assays and carbonyl cyanidem-chlorophenylhydrazone (CCCP) potentiation experiments demonstrated that this phenotype was due to an active efflux mechanism. These findings emphasize the important role of the Tap efflux pump in bacterial physiology and intrinsic drug resistance.

scholarly journals The Evolutionary Conservation of Escherichia coli Drug Efflux Pumps Supports Physiological Functions

2020 ◽  
Vol 202 (22) ◽  
Author(s):  
Tanisha Teelucksingh ◽  
Laura K. Thompson ◽  
Georgina Cox
Keyword(s):  
Escherichia Coli ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Evolutionary Conservation ◽  
Drug Efflux ◽  
Physiological Functions ◽  
Content Type ◽  
Bacterial Physiology ◽  
E Coli ◽  
Drug Efflux Pumps

ABSTRACT Bacteria harness an impressive repertoire of resistance mechanisms to evade the inhibitory action of antibiotics. One such mechanism involves efflux pump-mediated extrusion of drugs from the bacterial cell, which significantly contributes to multidrug resistance. Intriguingly, most drug efflux pumps are chromosomally encoded components of the intrinsic antibiotic resistome. In addition, in terms of xenobiotic detoxification, bacterial efflux systems often exhibit significant levels of functional redundancy. Efflux pumps are also considered to be highly conserved; however, the extent of conservation in many bacterial species has not been reported and the majority of genes that encode efflux pumps appear to be dispensable for growth. These observations, in combination with an increasing body of experimental evidence, imply alternative roles in bacterial physiology. Indeed, the ability of efflux pumps to facilitate antibiotic resistance could be a fortuitous by-product of ancient physiological functions. Using Escherichia coli as a model organism, we here evaluated the evolutionary conservation of drug efflux pumps and we provide phylogenetic analysis of the major efflux families. We show the E. coli drug efflux system has remained relatively stable and the majority (∼80%) of pumps are encoded in the core genome. This analysis further supports the importance of drug efflux pumps in E. coli physiology. In this review, we also provide an update on the roles of drug efflux pumps in the detoxification of endogenously synthesized substrates and pH homeostasis. Overall, gaining insight into drug efflux pump conservation, common evolutionary ancestors, and physiological functions could enable strategies to combat these intrinsic and ancient elements.

scholarly journals Role of smeU1VWU2X Operon in Alleviation of Oxidative Stresses and Occurrence of Sulfamethoxazole-Trimethoprim-Resistant Mutants in Stenotrophomonas maltophilia

2017 ◽  
Vol 62 (2) ◽  
Author(s):  
Chao-Jung Wu ◽  
Tsu-Ting Chiu ◽  
Yi-Tsung Lin ◽  
Yi-Wei Huang ◽  
Li-Hua Li ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stenotrophomonas Maltophilia ◽  
Efflux Pump ◽  
Acquired Resistance ◽  
Resistant Mutant ◽  
Efflux Pumps ◽  
Content Type ◽  
Fusion Construct ◽  
Bacterial Physiology ◽  
Stress Alleviation

ABSTRACT Overexpression of resistance-nodulation-division (RND)-type efflux pumps is an important mechanism for bacteria to combat antimicrobials. RND efflux pumps are also critical for bacterial physiology, such as oxidative stress tolerance. Stenotrophomonas maltophilia, a multidrug-resistant opportunistic pathogen, harbors eight RND-type efflux pump operons. Of these, the smeU1VWU2X operon is unique for its possession of two additional genes, smeU1 and smeU2, which encode proteins of the short-chain dehydrogenase/reductase (SDR) family. Overexpression of the SmeVWX pump is known to contribute to the acquired resistance to chloramphenicol, quinolone, and tetracycline; however, SmeU1 and SmeU2 are little involved in this phenotype. In the study described in this article, we further linked the smeU1VWU2X operon to oxidative stress alleviation and sulfamethoxazole-trimethoprim (SXT)-resistant mutant occurrence. The smeU1VWU2X operon was inducibly expressed upon challenge with menadione (MD), plumbagin (PL), and hydrogen peroxide (H2O2), as verified by the use of the chromosomal smeU1VWU2X-xylE transcriptional fusion construct and quantitative real-time PCR (qRT-PCR). The MD-mediated smeU1VWU2X upexpression was totally dependent on SoxR and partially relied on SmeRv but was less relevant to OxyR. SmeRv, but not SoxR and OxyR, played a regulatory role in the H2O2-mediated smeU1VWU2X upexpression. The significance of smeU1VWU2X upexpression was investigated with respect to oxidative stress alleviation and SXT-resistant mutant occurrence. Overexpression of the smeU1VWU2X operon contributed to the alleviation of MD-mediated oxidative stress. Of the encoded proteins, the SmeVWX pump and SmeU2, rather than SmeU1, participated in MD tolerance. Furthermore, we also demonstrated that the MD-mediated expression of the smeU1VWU2X operon decreased the SXT resistance frequency when S. maltophilia was grown in a reactive oxygen species (ROS)-rich environment.

scholarly journals Small RNA Regulation of TolC, the Outer Membrane Component of Bacterial Multidrug Transporters

2016 ◽  
Vol 198 (7) ◽  
pp. 1101-1113 ◽  
Author(s):  
Ashley Parker ◽  
Susan Gottesman
Keyword(s):  
Stationary Phase ◽  
Outer Membrane ◽  
Small Rna ◽  
Efflux Pump ◽  
Outer Membrane Proteins ◽  
Efflux Pumps ◽  
Toxic Compounds ◽  
Content Type ◽  
Phenotypic Microarray ◽  
Mrna Targets

ABSTRACTBacteria use multidrug efflux pumps to export drugs and toxic compounds out of the cell. One of the most important efflux pumps inEscherichia coliis the AcrAB-TolC system. Small regulatory RNAs (sRNAs) are known to be major posttranscriptional regulators that can enhance or repress translation by binding to the 5′ untranslated region (UTR) of mRNA targets with the help of a chaperone protein, Hfq. In this study, we investigated the expression ofacrA,acrB, andtolCtranslational fusions using 27 Hfq-dependent sRNAs overexpressed from plasmids. No significant sRNA regulation ofacrAoracrBwas detected. SdsR (also known as RyeB), an abundant and well-conserved stationary-phase sRNA, was found to repress the expression oftolC, the gene encoding the outer membrane protein of many multidrug resistance efflux pumps. This repression was shown to be by direct base pairing occurring upstream from the ribosomal binding site. SdsR overexpression and its regulation oftolCwere found to reduce resistance to novobiocin and crystal violet. Our results suggest that additional targets for SdsR exist that contribute to increased antibiotic sensitivity and reduced biofilm formation. In an effort to identify phenotypes associated with single-copy SdsR and its regulation oftolC, the effect of a deletion ofsdsRor mutations intolCthat should block SdsR pairing were investigated using a Biolog phenotypic microarray. However, no significant phenotypes were identified. Therefore, SdsR appears to modulate rather than act as a major regulator of its targets.IMPORTANCEAcrAB-TolC is a major efflux pump present inE. coliand Gram-negative bacteria used to export toxic compounds; the pump confers resistance to many antibiotics of unrelated classes. In this study, we found that SdsR, a small RNA expressed in stationary phase, repressed the expression oftolC, resulting in increased sensitivity to some antibiotics. This extends the findings of previous studies showing that sRNAs contribute to the regulation of many outer membrane proteins; manipulating or enhancing their action might help in sensitizing bacteria to antibiotics.

scholarly journals Coordinate Hyperproduction of SmeZ and SmeJK Efflux Pumps Extends Drug Resistance in Stenotrophomonas maltophilia

2012 ◽  
Vol 57 (1) ◽  
pp. 655-657 ◽  
Author(s):  
Virginia C. Gould ◽  
Aki Okazaki ◽  
Matthew B. Avison
Keyword(s):  
Drug Resistance ◽  
Clinical Isolate ◽  
Clinical Relevance ◽  
Stenotrophomonas Maltophilia ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Content Type ◽  
Resistance Nodulation Division

ABSTRACTAStenotrophomonas maltophiliamutant that coordinately hyper-expresses three resistance nodulation division-type efflux pump genes,smeZ,smeJ, andsmeK, has been identified. SmeZ is responsible for elevating aminoglycoside MICs; SmeJ and SmeK are jointly responsible for elevating tetracycline, minocycline, and ciprofloxacin MICs and conferring levofloxacin resistance. One clinical isolate with this same phenotype was identified from a sample of six, and the isolate also coordinately hyper-expressessmeZandsmeJK, confirming the clinical relevance of our findings.

scholarly journals Efflux Pumps of Mycobacterium tuberculosis Play a Significant Role in Antituberculosis Activity of Potential Drug Candidates

2012 ◽  
Vol 56 (5) ◽  
pp. 2643-2651 ◽  
Author(s):  
Meenakshi Balganesh ◽  
Neela Dinesh ◽  
Sreevalli Sharma ◽  
Sanjana Kuruppath ◽  
Anju V. Nair ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Vital Role ◽  
Major Facilitator Superfamily ◽  
Content Type ◽  
Active Efflux ◽  
Drug Candidates ◽  
Small Multidrug Resistance ◽  
Major Facilitator

ABSTRACTActive efflux of drugs mediated by efflux pumps that confer drug resistance is one of the mechanisms developed by bacteria to counter the adverse effects of antibiotics and chemicals. To understand these efflux mechanisms inMycobacterium tuberculosis, we generated knockout (KO) mutants of four efflux pumps of the pathogen belonging to different classes. We measured the MICs and kill values of two different compound classes on the wild type (WT) and the efflux pump (EP) KO mutants in the presence and absence of the efflux inhibitors verapamil andl-phenylalanyl-l-arginyl-β-naphthylamide (PAβN). Among the pumps studied, the efflux pumps belonging to the ABC (ATP-binding cassette) class, encoded byRv1218c, and the SMR (small multidrug resistance) class, encoded byRv3065, appear to play important roles in mediating the efflux of different chemical classes and antibiotics. Efflux pumps encoded byRv0849andRv1258calso mediate the efflux of these compounds, but to a lesser extent. Increased killing is observed in WTM. tuberculosiscells by these compounds in the presence of either verapamil or PAβN. The efflux pump KO mutants were more susceptible to these compounds in the presence of efflux inhibitors. We have shown that these four efflux pumps ofM. tuberculosisplay a vital role in mediating efflux of different chemical scaffolds. Inhibitors of one or several of these efflux pumps could have a significant impact in the treatment of tuberculosis. The identification and characterization ofRv0849, a new efflux pump belonging to the MFS (major facilitator superfamily) class, are reported.

scholarly journals Antileishmanial Aminopyrazoles: Studies into Mechanisms and Stability of Experimental Drug Resistance

2020 ◽  
Vol 64 (9) ◽  
Author(s):  
M. Van den Kerkhof ◽  
D. Mabille ◽  
S. Hendrickx ◽  
P. Leprohon ◽  
C. E. Mowbray ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Copy Number ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Copy Number Variations ◽  
Content Type ◽  
Development Of Resistance ◽  
Efflux Pump Inhibitors

ABSTRACT Current antileishmanial treatment is hampered by limitations, such as drug toxicity and the risk of treatment failure, which may be related to parasitic drug resistance. Given the urgent need for novel drugs, the Drugs for Neglected Diseases initiative (DNDi) has undertaken a drug discovery program, which has resulted in the identification of aminopyrazoles, a highly promising antileishmanial chemical series. Multiple experiments have been performed to anticipate the propensity for resistance development. Resistance selection was performed by successive exposure of Leishmania infantum promastigotes (in vitro) and intracellular amastigotes (both in vitro and in golden Syrian hamsters). The stability of the resistant phenotypes was assessed after passage in mice and Lutzomyia longipalpis sandflies. Whole-genome sequencing (WGS) was performed to identify mutated genes, copy number variations (CNVs), and somy changes. The potential role of efflux pumps (the MDR and MRP efflux pumps) in the development of resistance was assessed by coincubation of aminopyrazoles with specific efflux pump inhibitors (verapamil, cyclosporine, and probenecid). Repeated drug exposure of amastigotes did not result in the emergence of drug resistance either in vitro or in vivo. Selection at the promastigote stage, however, was able to select for parasites with reduced susceptibility (resistance index, 5.8 to 24.5). This phenotype proved to be unstable after in vivo passage in mice and sandflies, suggesting that nonfixed alterations are responsible for the elevated resistance. In line with this, single nucleotide polymorphisms and indels identified by whole-genome sequencing could not be directly linked to the decreased drug susceptibility. Copy number variations were absent, whereas somy changes were detected, which may have accounted for the transient acquisition of resistance. Finally, aminopyrazole activity was not influenced by the MDR and MRP efflux pump inhibitors tested. The selection performed does not suggest the rapid development of resistance against aminopyrazoles in the field. Karyotype changes may confer elevated levels of resistance, but these do not seem to be stable in the vertebrate and invertebrate hosts. MDR/MRP efflux pumps are not likely to significantly impact the activity of the aminopyrazole leads.

scholarly journals Predictive Rules of Efflux Inhibition and Avoidance in Pseudomonas aeruginosa

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jitender Mehla ◽  
Giuliano Malloci ◽  
Rachael Mansbach ◽  
Cesar A. López ◽  
Ruslan Tsivkovski ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Predictive Models ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Resistant Bacteria ◽  
Natural Environments ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Active Efflux

ABSTRACT Antibiotic-resistant bacteria rapidly spread in clinical and natural environments and challenge our modern lifestyle. A major component of defense against antibiotics in Gram-negative bacteria is a drug permeation barrier created by active efflux across the outer membrane. We identified molecular determinants defining the propensity of small peptidomimetic molecules to avoid and inhibit efflux pumps in Pseudomonas aeruginosa, a human pathogen notorious for its antibiotic resistance. Combining experimental and computational protocols, we mapped the fate of the compounds from structure-activity relationships through their dynamic behavior in solution, permeation across both the inner and outer membranes, and interaction with MexB, the major efflux transporter of P. aeruginosa. We identified predictors of efflux avoidance and inhibition and demonstrated their power by using a library of traditional antibiotics and compound series and by generating new inhibitors of MexB. The identified predictors will enable the discovery and optimization of antibacterial agents suitable for treatment of P. aeruginosa infections. IMPORTANCE Efflux pump avoidance and inhibition are desired properties for the optimization of antibacterial activities against Gram-negative bacteria. However, molecular and physicochemical interactions defining the interface between compounds and efflux pumps remain poorly understood. We identified properties that correlate with efflux avoidance and inhibition, are predictive of similar features in structurally diverse compounds, and allow researchers to distinguish between efflux substrates, inhibitors, and avoiders in P. aeruginosa. The developed predictive models are based on the descriptors representative of different clusters comprising a physically intuitive combination of properties. Molecular shape (represented by acylindricity), amphiphilicity (anisotropic polarizability), aromaticity (number of aromatic rings), and the partition coefficient (LogD) are physicochemical predictors of efflux inhibitors, whereas interactions with Pro668 and Leu674 residues of MexB distinguish between inhibitors/substrates and efflux avoiders. The predictive models and efflux rules are applicable to compounds with unrelated chemical scaffolds and pave the way for development of compounds with the desired efflux interface properties.

scholarly journals Substrate Specificities and Efflux Efficiencies of RND Efflux Pumps ofAcinetobacter baumannii

2018 ◽  
Vol 200 (13) ◽  
pp. e00049-18 ◽  
Author(s):  
Inga V. Leus ◽  
Jon W. Weeks ◽  
Vincent Bonifay ◽  
Lauren Smith ◽  
Sophie Richardson ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Antibacterial Activities ◽  
Permeability Barrier ◽  
Bacterial Cells ◽  
Content Type ◽  
Active Efflux ◽  
Rnd Efflux Pumps ◽  
The Impact

ABSTRACTAntibiotic-resistantAcinetobacter baumanniicauses infections that are extremely difficult to treat. A significant role in these resistance profiles is attributed to multidrug efflux pumps, especially those belonging to the resistance-nodulation-cell division (RND) superfamily of transporters. In this study, we analyzed functions and properties of RND efflux pumps inA. baumanniiATCC 17978. This strain is susceptible to antibiotics and does not contain mutations that are commonly selected upon exposure to high concentrations of antibiotics. We constructed derivatives of ATCC 17978 lacking chromosomally encoded RND pumps and complemented these strains by the plasmid-borne genes. We analyzed the substrate selectivities and efficiencies of the individual pumps in the context of native outer membranes and their hyperporinated variants. Our results show that inactivation of AdeIJK provides the strongest potentiation of antibiotic activities, whereas inactivation of AdeFGH triggers the overexpression of AdeAB. The plasmid-borne overproduction complements the hypersusceptible phenotypes of the efflux deletion mutants to the levels of the parental ATCC 17978. Only a few antibiotics strongly benefitted from the overproduction of efflux pumps and antibacterial activities of some of those depended on the synergistic interaction with the low permeability barrier of the outer membrane. Either overproduction or inactivation of efflux pumps change dramatically the lipidome of ATCC 17978. We conclude that efflux pumps ofA. baumanniiare tightly integrated into physiology of this bacterium and that clinical levels of antibiotic resistance inA. baumanniiisolates are unlikely to be reached solely due to the overproduction of RND efflux pumps.IMPORTANCERND-type efflux pumps are important contributors in development of clinical antibiotic resistance inA. baumannii. However, their specific roles and the extent of contribution to antibiotic resistance remain unclear. We analyzed antibacterial activities of antibiotics in strains with different permeability barriers and found that the role of active efflux in antibiotic resistance ofA. baumanniiis limited to a few select antibiotics. Our results further show that the impact of efflux pump overproduction on antibiotic susceptibility is significantly lower than the previously reported for clinical isolates. Additional mechanisms of resistance, in particular those that improve the permeability barriers of bacterial cells and act synergistically with active efflux pumps are likely involved in antibiotic resistance of clinicalA. baumanniiisolates.

scholarly journals A Combination Fluorescence Assay Demonstrates Increased Efflux Pump Activity as a Resistance Mechanism in Azole-Resistant Vaginal Candida albicans Isolates

2016 ◽  
Vol 60 (10) ◽  
pp. 5858-5866 ◽  
Author(s):  
Somanon Bhattacharya ◽  
Jack D. Sobel ◽  
Theodore C. White
Keyword(s):  
Candida Albicans ◽  
Efflux Pump ◽  
Pathogenic Fungus ◽  
Resistance Mechanism ◽  
Efflux Pumps ◽  
Resistant Isolate ◽  
Vaginal Infections ◽  
Content Type ◽  
Qrt Pcr ◽  
Pump Activity

ABSTRACTCandida albicansis a pathogenic fungus causing vulvovaginal candidiasis (VVC). Azole drugs, such as fluconazole, are the most common treatment for these infections. Recently, azole-resistant vaginalC. albicansisolates have been detected in patients with recurring and refractory vaginal infections. However, the mechanisms of resistance in vaginalC. albicansisolates have not been studied in detail. In oral and systemic resistant isolates, overexpression of the ABC transporters Cdr1p and Cdr2p and the major facilitator transporter Mdr1p is associated with resistance. Sixteen fluconazole-susceptible and 22 fluconazole-resistant vaginalC. albicansisolates were obtained, including six matched sets containing a susceptible and a resistant isolate, from individual patients. Using quantitative real-time reverse transcriptase PCR (qRT-PCR), 16 of 22 resistant isolates showed overexpression of at least one efflux pump gene, while only 1 of 16 susceptible isolates showed such overexpression. To evaluate the pump activity associated with overexpression, an assay that combined data from two separate fluorescent assays using rhodamine 6G and alanine β-naphthylamide was developed. The qRT-PCR results and activity assay results were in good agreement. This combination of two fluorescent assays can be used to study efflux pumps as resistance mechanisms in clinical isolates. These results demonstrate that efflux pumps are a significant resistance mechanism in vaginalC. albicansisolates.

scholarly journals Magnitude of Voriconazole Resistance in Clinical and Environmental Isolates of Aspergillus flavus and Investigation into the Role of Multidrug Efflux Pumps

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Raees A. Paul ◽  
Shivaprakash M. Rudramurthy ◽  
Manpreet Dhaliwal ◽  
Pankaj Singh ◽  
Anup K. Ghosh ◽  
...  
Keyword(s):  
Aspergillus Flavus ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Azole Resistance ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Environmental Isolates ◽  
Content Type ◽  
Multidrug Efflux Pumps

ABSTRACT The magnitude of azole resistance in Aspergillus flavus and its underlying mechanism is obscure. We evaluated the frequency of azole resistance in a collection of clinical (n = 121) and environmental isolates (n = 68) of A. flavus by the broth microdilution method. Six (5%) clinical isolates displayed voriconazole MIC greater than the epidemiological cutoff value. Two of these isolates with non-wild-type MIC were isolated from same patient and were genetically distinct, which was confirmed by amplified fragment length polymorphism analysis. Mutations associated with azole resistance were not present in the lanosterol 14-α demethylase coding genes (cyp51A, cyp51B, and cyp51C). Basal and voriconazole-induced expression of cyp51A homologs and various efflux pump genes was analyzed in three each of non-wild-type and wild-type isolates. All of the efflux pump genes screened showed low basal expression irrespective of the azole susceptibility of the isolate. However, the non-wild-type isolates demonstrated heterogeneous overexpression of many efflux pumps and the target enzyme coding genes in response to induction with voriconazole (1 μg/ml). The most distinctive observation was approximately 8- to 9-fold voriconazole-induced overexpression of an ortholog of the Candida albicans ATP binding cassette (ABC) multidrug efflux transporter, Cdr1, in two non-wild-type isolates compared to those in the reference strain A. flavus ATCC 204304 and other wild-type strains. Although the dominant marker of azole resistance in A. flavus is still elusive, the current study proposes the possible role of multidrug efflux pumps, especially that of Cdr1B overexpression, in contributing azole resistance in A. flavus.

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