scholarly journals Salicylate Functions as an Efflux Pump Inducer and Promotes the Emergence of Fluoroquinolone-Resistant Campylobacter jejuni Mutants

2011 ◽  
Vol 77 (20) ◽  
pp. 7128-7133 ◽  
Author(s):  
Zhangqi Shen ◽  
Xiao-Ying Pu ◽  
Qijing Zhang
Keyword(s):  
Efflux Pump ◽  
Fold Increase ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Mobility Shift ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Resistant Mutants ◽  
Promoter Dna ◽  
Electrophoretic Mobility Shift Assays

ABSTRACTSalicylate, a nonsteroidal anti-inflammatory compound, has been shown to increase the resistance ofCampylobacterto antimicrobials. However, the molecular mechanism underlying salicylate-induced resistance has not yet been established. In this study, we determined how salicylate increases antibiotic resistance and evaluated its impact on the development of fluoroquinolone-resistantCampylobactermutants. Transcriptional fusion assays, real-time quantitative reverse transcription-PCR (RT-PCR), and immunoblotting assays consistently demonstrated the induction of the CmeABC multidrug efflux pump by salicylate. Electrophoretic mobility shift assays further showed that salicylate inhibits the binding of CmeR (a transcriptional repressor of the TetR family) to the promoter DNA ofcmeABC, suggesting that salicylate inhibits the function of CmeR. The presence of salicylate in the culture medium not only decreased the susceptibility ofCampylobacterto ciprofloxacin but also resulted in an approximately 70-fold increase in the observed frequency of emergence of fluoroquinolone-resistant mutants under selection with ciprofloxacin. Together, these results indicate that inCampylobacter, salicylate inhibits the binding of CmeR to the promoter DNA and induces expression ofcmeABC, resulting in decreased susceptibility to antibiotics and in increased emergence of fluoroquinolone-resistant mutants under selection pressure.

scholarly journals TtgV Represses Two Different Promoters by Recognizing Different Sequences

2008 ◽  
Vol 191 (6) ◽  
pp. 1901-1909 ◽  
Author(s):  
Sandy Fillet ◽  
Marisela Vélez ◽  
Duo Lu ◽  
Xiaodong Zhang ◽  
María-Trinidad Gallegos ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Efflux Pump ◽  
Three Dimensional ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Mobility Shift ◽  
Alanine Substitution ◽  
Three Dimensional Models ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT Expression of the multidrug efflux pump ttgDEF and ttgGHI operons is modulated in vivo mainly by the TtgV repressor. TtgV is a multidrug recognition repressor that exhibits a DNA binding domain with a long interaction helix comprising residues 47 to 64. The pattern of expression of the two pumps is different in Pseudomonas putida: in the absence of effectors, the promoter for the ttgD gene is silent, whereas the ttgG gene is expressed at a high basal level. This correlates with the fact that TtgV exhibits a higher affinity for the ttgD operator (K D = 10 ± 1 nM) than for the ttgG (K D = 19 ± 1 nM) operator. Sequence analysis revealed that both operators are 40% identical, and mutational analysis of the ttgD and ttgG operators combined with electrophoretic mobility shift assays and in vivo expression analysis suggests that TtgV recognizes an inverted repeat with a high degree of palindromicity around the central axis. We generated a collection of alanine substitution mutants with substitutions between residues 47 and 64 of TtgV. The results of extensive combinations of promoter variants with these TtgV alanine substitution mutants revealed that TtgV modulates expression from ttgD and ttgG promoters through the recognition of both common and different sequences in the two promoters. In this regard, we found that TtgV mutants at residues 48, 50, 53, 54, 60, and 61 failed to bind ttgG but recognized the ttgD operator. TtgV residues R47, R52, L57, and T49 are critical for binding to both operators. Based on three-dimensional models, we propose that these residues contact nucleotides within the major groove of DNA.

scholarly journals FK506 Resistance ofSaccharomyces cerevisiaePdr5 andCandida albicansCdr1 Involves Mutations in the Transmembrane Domains and Extracellular Loops

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Koichi Tanabe ◽  
Michele Bonus ◽  
Susumu Tomiyama ◽  
Kunji Miyoshi ◽  
Minoru Nagi ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Drug Efflux ◽  
Pleiotropic Drug Resistance ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Content Type ◽  
Binding Domains ◽  
Contact Points ◽  
Extracellular Loops ◽  
Resistant Mutants

ABSTRACTThe 23-membered-ring macrolide tacrolimus, a commonly used immunosuppressant, also known as FK506, is a broad-spectrum inhibitor and an efflux pump substrate of pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters. Little, however, is known about the molecular mechanism by which FK506 inhibits PDR transporter drug efflux. Thus, to obtain further insights we searched for FK506-resistant mutants ofSaccharomyces cerevisiaecells overexpressing either the endogenous multidrug efflux pump Pdr5 or itsCandida albicansorthologue, Cdr1. A simple but powerful screen gave 69 FK506-resistant mutants with, between them, 72 mutations in either Pdr5 or Cdr1. Twenty mutations were in just three Pdr5/Cdr1 equivalent amino acid positions, T550/T540 and T552/S542 of extracellular loop 1 (EL1) and A723/A713 of EL3. Sixty of the 72 mutations were either in the ELs or the extracellular halves of individual transmembrane spans (TMSs), while 11 mutations were found near the center of individual TMSs, mostly in predicted TMS-TMS contact points, and only two mutations were in the cytosolic nucleotide-binding domains of Pdr5. We propose that FK506 inhibits Pdr5 and Cdr1 drug efflux by slowing transporter opening and/or substrate release, and that FK506 resistance of Pdr5/Cdr1 drug efflux is achieved by modifying critical intramolecular contact points that, when mutated, enable the cotransport of FK506 with other pump substrates. This may also explain why the 35 Cdr1 mutations that caused FK506 insensitivity of fluconazole efflux differed from the 13 Cdr1 mutations that caused FK506 insensitivity of cycloheximide efflux.

scholarly journals Cryo-EM Structures of a Gonococcal Multidrug Efflux Pump Illuminate a Mechanism of Drug Recognition and Resistance

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Meinan Lyu ◽  
Mitchell A. Moseng ◽  
Jennifer L. Reimche ◽  
Concerta L. Holley ◽  
Vijaya Dhulipala ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Drug Resistance ◽  
Neisseria Gonorrhoeae ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Transmitted Infection ◽  
Content Type ◽  
Cryo Electron Microscopy

ABSTRACT Neisseria gonorrhoeae is an obligate human pathogen and causative agent of the sexually transmitted infection (STI) gonorrhea. The most predominant and clinically important multidrug efflux system in N. gonorrhoeae is the multiple transferrable resistance (Mtr) pump, which mediates resistance to a number of different classes of structurally diverse antimicrobial agents, including clinically used antibiotics (e.g., β-lactams and macrolides), dyes, detergents and host-derived antimicrobials (e.g., cationic antimicrobial peptides and bile salts). Recently, it has been found that gonococci bearing mosaic-like sequences within the mtrD gene can result in amino acid changes that increase the MtrD multidrug efflux pump activity, probably by influencing antimicrobial recognition and/or extrusion to elevate the level of antibiotic resistance. Here, we report drug-bound solution structures of the MtrD multidrug efflux pump carrying a mosaic-like sequence using single-particle cryo-electron microscopy, with the antibiotics bound deeply inside the periplasmic domain of the pump. Through this structural approach coupled with genetic studies, we identify critical amino acids that are important for drug resistance and propose a mechanism for proton translocation. IMPORTANCE Neisseria gonorrhoeae has become a highly antimicrobial-resistant Gram-negative pathogen. Multidrug efflux is a major mechanism that N. gonorrhoeae uses to counteract the action of multiple classes of antibiotics. It appears that gonococci bearing mosaic-like sequences within the gene mtrD, encoding the most predominant and clinically important transporter of any gonococcal multidrug efflux pump, significantly elevate drug resistance and enhance transport function. Here, we report cryo-electron microscopy (EM) structures of N. gonorrhoeae MtrD carrying a mosaic-like sequence that allow us to understand the mechanism of drug recognition. Our work will ultimately inform structure-guided drug design for inhibiting these critical multidrug efflux pumps.

scholarly journals Multidrug Adaptive Resistance of Pseudomonas aeruginosa Swarming Cells

2019 ◽  
Vol 64 (3) ◽  
Author(s):  
Shannon R. Coleman ◽  
Travis Blimkie ◽  
Reza Falsafi ◽  
Robert E. W. Hancock
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Efflux Pump ◽  
Iron Acquisition ◽  
Polymyxin B ◽  
Rna Seq ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Content Type ◽  
Adaptive Resistance

ABSTRACT Swarming surface motility is a complex adaptation leading to multidrug antibiotic resistance and virulence factor production in Pseudomonas aeruginosa. Here, we expanded previous studies to demonstrate that under swarming conditions, P. aeruginosa PA14 is more resistant to multiple antibiotics, including aminoglycosides, β-lactams, chloramphenicol, ciprofloxacin, tetracycline, trimethoprim, and macrolides, than swimming cells, but is not more resistant to polymyxin B. We investigated the mechanism(s) of swarming-mediated antibiotic resistance by examining the transcriptomes of swarming cells and swarming cells treated with tobramycin by transcriptomics (RNA-Seq) and reverse transcriptase quantitative PCR (qRT-PCR). RNA-Seq of swarming cells (versus swimming) revealed 1,581 dysregulated genes, including 104 transcriptional regulators, two-component systems, and sigma factors, numerous upregulated virulence and iron acquisition factors, and downregulated ribosomal genes. Strain PA14 mutants in resistome genes that were dysregulated under swarming conditions were tested for their ability to swarm in the presence of tobramycin. In total, 41 mutants in genes dysregulated under swarming conditions were shown to be more resistant to tobramycin under swarming conditions, indicating that swarming-mediated tobramycin resistance was multideterminant. Focusing on two genes downregulated under swarming conditions, both prtN and wbpW mutants were more resistant to tobramycin, while the prtN mutant was additionally resistant to trimethoprim under swarming conditions; complementation of these mutants restored susceptibility. RNA-Seq of swarming cells treated with subinhibitory concentrations of tobramycin revealed the upregulation of the multidrug efflux pump MexXY and downregulation of virulence factors.

scholarly journals CmeR Functions as a Pleiotropic Regulator and Is Required for Optimal Colonization of Campylobacter jejuni In Vivo

2008 ◽  
Vol 190 (6) ◽  
pp. 1879-1890 ◽  
Author(s):  
Baoqing Guo ◽  
Ying Wang ◽  
Feng Shi ◽  
Yi-Wen Barton ◽  
Paul Plummer ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Capsular Polysaccharide ◽  
Efflux Pump ◽  
Loss Of Function ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Reverse Transcription Pcr ◽  
Dicarboxylate Transport

ABSTRACT CmeR functions as a transcriptional repressor modulating the expression of the multidrug efflux pump CmeABC in Campylobacter jejuni. To determine if CmeR also regulates other genes in C. jejuni, we compared the transcriptome of the cmeR mutant with that of the wild-type strain using a DNA microarray. This comparison identified 28 genes that showed a ≥2-fold change in expression in the cmeR mutant. Independent real-time quantitative reverse transcription-PCR experiments confirmed 27 of the 28 differentially expressed genes. The CmeR-regulated genes encode membrane transporters, proteins involved in C4-dicarboxylate transport and utilization, enzymes for biosynthesis of capsular polysaccharide, and hypothetical proteins with unknown functions. Among the genes whose expression was upregulated in the cmeR mutant, Cj0561c (encoding a putative periplasmic protein) showed the greatest increase in expression. Subsequent experiments demonstrated that this gene is strongly repressed by CmeR. The presence of the known CmeR-binding site, an inverted repeat of TGTAAT, in the promoter region of Cj0561c suggests that CmeR directly inhibits the transcription of Cj0561c. Similar to expression of cmeABC, transcription of Cj0561c is strongly induced by bile compounds, which are normally present in the intestinal tracts of animals. Inactivation of Cj0561c did not affect the susceptibility of C. jejuni to antimicrobial compounds in vitro but reduced the fitness of C. jejuni in chickens. Loss-of-function mutation of cmeR severely reduced the ability of C. jejuni to colonize chickens. Together, these findings indicate that CmeR governs the expression of multiple genes with diverse functions and is required for Campylobacter adaptation in the chicken host.

scholarly journals SAGA/ADA Complex Subunit Ada2 Is Required for Cap1- but Not Mrr1-Mediated Upregulation of the Candida albicans Multidrug Efflux PumpMDR1

2014 ◽  
Vol 58 (9) ◽  
pp. 5102-5110 ◽  
Author(s):  
Bernardo Ramírez-Zavala ◽  
Selene Mogavero ◽  
Eva Schöller ◽  
Christoph Sasse ◽  
P. David Rogers ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Candida Albicans ◽  
Efflux Pump ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Gain Of Function ◽  
Content Type ◽  
Zinc Cluster ◽  
Fluconazole Resistant

ABSTRACTOverexpression of the multidrug efflux pumpMDR1is one mechanism by which the pathogenic yeastCandida albicansdevelops resistance to the antifungal drug fluconazole. The constitutive upregulation ofMDR1in fluconazole-resistant, clinicalC. albicansisolates is caused by gain-of-function mutations in the zinc cluster transcription factor Mrr1. It has been suggested that Mrr1 activatesMDR1transcription by recruiting Ada2, a subunit of the SAGA/ADA coactivator complex. However,MDR1expression is also regulated by the bZIP transcription factor Cap1, which mediates the oxidative stress response inC. albicans. Here, we show that a hyperactive Mrr1 containing a gain-of-function mutation promotesMDR1overexpression independently of Ada2. In contrast, a C-terminally truncated, hyperactive Cap1 causedMDR1overexpression in a wild-type strain but only weakly in mutants lackingADA2. In the presence of benomyl or H2O2, compounds that induceMDR1expression in an Mrr1- and Cap1-dependent fashion,MDR1was upregulated with the same efficiency in wild-type andada2Δ cells. These results indicate that Cap1, but not Mrr1, recruits Ada2 to theMDR1promoter to induce the expression of this multidrug efflux pump and that Ada2 is not required forMDR1overexpression in fluconazole-resistantC. albicansstrains containing gain-of-function mutations in Mrr1.

scholarly journals A New Natural Product Analog of Blasticidin S Reveals Cellular Uptake Facilitated by the NorA Multidrug Transporter

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Jack R. Davison ◽  
Katheryn M. Lohith ◽  
Xiaoning Wang ◽  
Kostyantyn Bobyk ◽  
Sivakoteswara R. Mandadapu ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Bacterial Strains ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Gene Encoding ◽  
Content Type ◽  
Bacterial Membranes ◽  
Development Of Resistance ◽  
Antibiotic Compounds ◽  
Blasticidin S

ABSTRACT The permeation of antibiotics through bacterial membranes to their target site is a crucial determinant of drug activity but in many cases remains poorly understood. During screening efforts to discover new broad-spectrum antibiotic compounds from marine sponge samples, we identified a new analog of the peptidyl nucleoside antibiotic blasticidin S that exhibited up to 16-fold-improved potency against a range of laboratory and clinical bacterial strains which we named P10. Whole-genome sequencing of laboratory-evolved strains of Staphylococcus aureus resistant to blasticidin S and P10, combined with genome-wide assessment of the fitness of barcoded Escherichia coli knockout strains in the presence of the antibiotics, revealed that restriction of cellular access was a key feature in the development of resistance to this class of drug. In particular, the gene encoding the well-characterized multidrug efflux pump NorA was found to be mutated in 69% of all S. aureus isolates resistant to blasticidin S or P10. Unexpectedly, resistance was associated with inactivation of norA, suggesting that the NorA transporter facilitates cellular entry of peptidyl nucleosides in addition to its known role in the efflux of diverse compounds, including fluoroquinolone antibiotics.

scholarly journals Molecular Mechanism of MBX2319 Inhibition of Escherichia coli AcrB Multidrug Efflux Pump and Comparison with Other Inhibitors

2014 ◽  
Vol 58 (10) ◽  
pp. 6224-6234 ◽  
Author(s):  
Attilio V. Vargiu ◽  
Paolo Ruggerone ◽  
Timothy J. Opperman ◽  
Son T. Nguyen ◽  
Hiroshi Nikaido
Keyword(s):  
Efflux Pump ◽  
Efflux Pumps ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Efflux Pump Inhibitor ◽  
Content Type ◽  
X Ray Crystallography ◽  
Resistance Nodulation Division ◽  
Hydrophobic Portion ◽  
Dynamics Simulations

ABSTRACTEfflux 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 silicostructure-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 ofEnterobacteriaceaespecies, 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.

scholarly journals MepR, a Repressor of the Staphylococcus aureus MATE Family Multidrug Efflux Pump MepA, Is a Substrate-Responsive Regulatory Protein

2006 ◽  
Vol 50 (4) ◽  
pp. 1276-1281 ◽  
Author(s):  
Glenn W. Kaatz ◽  
Carmen E. DeMarco ◽  
Susan M. Seo
Keyword(s):  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Regulatory Protein ◽  
Inducible Promoter ◽  
Multidrug Efflux ◽  
Recognition Sequence ◽  
Multidrug Efflux Pump ◽  
Mobility Shift ◽  
Repressive Effect ◽  
Operator Interaction

ABSTRACT The mepRAB gene cluster of Staphylococcus aureus encodes a MarR family repressor (MepR; known to repress mepA expression), a MATE family multidrug efflux pump (MepA), and a protein of unknown function (MepB). In this report, we show that MepR also is autoregulatory, repressing the expression of its own gene. Exposure of strains containing a mepR::lacZ fusion with mepR provided in trans under the control of an inducible promoter, or a mepA::lacZ fusion alone, to subinhibitory concentrations of MepA substrates resulted in variably increased expression mainly of mepA. Mobility shift assays revealed that MepR binds upstream of mepR and mepA, with an apparently higher affinity for the mepA binding site. MepA substrates abrogated MepR binding to each site in a differential manner, with the greatest effect observed on the MepR-mepA operator interaction. DNase I footprinting identified precise binding sites which included promoter motifs, inverted repeats, and transcription start sites for mepR and mepA, as well as a conserved GTTAG motif, which may be a signature recognition sequence for MepR. Analogous to other multidrug efflux pump regulatory proteins such as QacR, the substrate-MepR interaction likely results in its dissociation from its mepA, and in a more limited fashion its mepR, operator sites and relief of its repressive effect. The enhanced effect of substrates on mepA compared to mepR expression, and on the MepR-mepA operator interaction, results in significant relief of mepA and relative maintenance of mepR repression, leading to increased MepA protein unimpeded by MepR when the need for detoxification exists.

scholarly journals Toxic Electrophiles Induce Expression of the Multidrug Efflux Pump MexEF-OprN in Pseudomonas aeruginosa through a Novel Transcriptional Regulator, CmrA

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Paulo Juarez ◽  
Katy Jeannot ◽  
Patrick Plésiat ◽  
Catherine Llanes
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Redox Homeostasis ◽  
Transcriptional Regulator ◽  
Significant Loss ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Content Type ◽  
Constitutive Overexpression

ABSTRACT The multidrug efflux system MexEF-OprN is produced at low levels in wild-type strains of Pseudomonas aeruginosa. However, in so-called nfxC mutants, mutational alteration of the gene mexS results in constitutive overexpression of the pump, along with increased resistance of the bacterium to chloramphenicol, fluoroquinolones, and trimethoprim. In this study, analysis of in vitro-selected chloramphenicol-resistant clones of strain PA14 led to the identification of a new class of MexEF-OprN-overproducing mutants (called nfxC2) exhibiting alterations in an as-yet-uncharacterized gene, PA14_38040 (homolog of PA2047 in strain PAO1). This gene is predicted to encode an AraC-like transcriptional regulator and was called cmrA (for chloramphenicol resistance activator). In nfxC2 mutants, the mutated CmrA increases its proper gene expression and upregulates the operon mexEF-oprN through MexS and MexT, resulting in a multidrug resistance phenotype without significant loss in bacterial virulence. Transcriptomic experiments demonstrated that CmrA positively regulates a small set of 11 genes, including PA14_38020 (homolog of PA2048), which is required for the MexS/T-dependent activation of mexEF-oprN. PA2048 codes for a protein sharing conserved domains with the quinol monooxygenase YgiN from Escherichia coli. Interestingly, exposure of strain PA14 to toxic electrophilic molecules (glyoxal, methylglyoxal, and cinnamaldehyde) strongly activates the CmrA pathway and upregulates MexEF-OprN and, thus, increases the resistance of P. aeruginosa to the pump substrates. A picture emerges in which MexEF-OprN is central in the response of the pathogen to stresses affecting intracellular redox homeostasis.

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