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 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 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.

Patupilone (epothilone B) inhibits growth and survival of multiple myeloma cells in vitro and in vivo

Blood ◽  
2005 ◽  
Vol 105 (1) ◽  
pp. 350-357 ◽  
Author(s):  
Boris Lin ◽  
Laurence Catley ◽  
Richard LeBlanc ◽  
Constantine Mitsiades ◽  
Renate Burger ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Growth Factor ◽  
Efflux Pump ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Growth And Survival ◽  
Epothilone B ◽  
Endothelial Growth Factor

Abstract In this study, we investigated the in vitro and in vivo efficacy of patupilone (epothilone B, EPO906), a novel nontaxane microtubule stabilizing agent, in treatment of multiple myeloma (MM). Patupilone directly inhibited growth and survival of MM cells, including those resistant to conventional chemotherapies, such as the taxane paclitaxel. Patupilone induced G2M arrest of MM cells, with subsequent apoptosis. Interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1), 2 known growth and survival factors for MM, did not protect MM.1S cells against patupilone-induced cell death. Proliferation of MM cells induced by adherence to bone marrow stromal cells (BMSCs) was also inhibited by patupilone and was paralleled by down-regulation of vascular endothelial growth factor (VEGF) secretion. Importantly, stimulation of cells from patients with MM, either with IL-6 or by adherence to BMSCs, enhanced the anti-proliferative and proapoptotic effects of patupilone. Moreover, patupilone was effective against MM cell lines that overexpress the MDR1/P-glycoprotein multidrug efflux pump. In addition, patupilone was effective in slowing tumor growth and prolonging median survival of mice that received orthotopical transplants with MM tumor cells. Taken together, these preclinical findings suggest that patupilone may be a safe and effective drug in the treatment of MM, providing the framework for clinical studies to improve patient outcome in MM. (Blood. 2005;105:350-357)

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 Structural, Biochemical, and In Vivo Characterization of MtrR-Mediated Resistance to Innate Antimicrobials by the Human Pathogen Neisseria gonorrhoeae

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Grace A. Beggs ◽  
Yaramah M. Zalucki ◽  
Nicholas Gene Brown ◽  
Sheila Rastegari ◽  
Rebecca K. Phillips ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Bile Salts ◽  
Efflux Pump ◽  
Binding Modes ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Content Type ◽  
Multiple Drugs

ABSTRACT Neisseria gonorrhoeae responds to host-derived antimicrobials by inducing the expression of the mtrCDE-encoded multidrug efflux pump, which expels microbicides, such as bile salts, fatty acids, and multiple extrinsically administered drugs, from the cell. In the absence of these cytotoxins, the TetR family member MtrR represses the mtrCDE genes. Although antimicrobial-dependent derepression of mtrCDE is clear, the physiological inducers of MtrR are unknown. Here, we report the crystal structure of an induced form of MtrR. In the binding pocket of MtrR, we observed electron density that we hypothesized was N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), a component of the crystallization reagent. Using the MtrR-CAPS structure as an inducer-bound template, we hypothesized that bile salts, which bear significant chemical resemblance to CAPS, are physiologically relevant inducers. Indeed, characterization of MtrR-chenodeoxycholate and MtrR-taurodeoxycholate interactions, both in vitro and in vivo, revealed that these bile salts, but not glyocholate or taurocholate, bind MtrR tightly and can act as bona fide inducers. Furthermore, two residues, W136 and R176, were shown to be important in binding chenodeoxycholate but not taurodeoxycholate, suggesting different binding modes of the bile salts. These data provide insight into a crucial mechanism utilized by the pathogen to overcome innate human defenses. IMPORTANCE Neisseria gonorrhoeae causes a significant disease burden worldwide, and a meteoric rise in its multidrug resistance has reduced the efficacy of antibiotics previously or currently approved for therapy of gonorrheal infections. The multidrug efflux pump MtrCDE transports multiple drugs and host-derived antimicrobials from the bacterial cell and confers survival advantage on the pathogen within the host. Transcription of the pump is repressed by MtrR but relieved by the cytosolic influx of antimicrobials. Here, we describe the structure of induced MtrR and use this structure to identify bile salts as physiological inducers of MtrR. These findings provide a mechanistic basis for antimicrobial sensing and gonococcal protection by MtrR through the derepression of mtrCDE expression after exposure to intrinsic and clinically applied antimicrobials.

scholarly journals Induction of Pseudomonas syringae pv. tomato DC3000 MexAB-OprM Multidrug Efflux Pump by Flavonoids Is Mediated by the Repressor PmeR

2011 ◽  
Vol 24 (10) ◽  
pp. 1207-1219 ◽  
Author(s):  
Paola Vargas ◽  
Antonia Felipe ◽  
Carmen Michán ◽  
María-Trinidad Gallegos
Keyword(s):  
Pseudomonas Syringae ◽  
Efflux Pump ◽  
Regulatory Gene ◽  
Multidrug Efflux ◽  
Tomato Dc3000 ◽  
Multidrug Efflux Pump ◽  
Tomato Plants ◽  
In Vivo Experiments

In this study, we have analyzed the expression of the Pseudomonas syringae pv. tomato DC3000 mexAB-oprM efflux pump operon and of the regulatory gene pmeR, and we have investigated the role of the PmeR protein on transcription from both promoters. We demonstrate that mexAB-oprM and pmeR are expressed in vivo at a relatively high and moderate basal level, respectively, which, in both cases, increases in the presence of different flavonoids and other compounds, such as butyl and methylparaben. We show that PmeR is the local repressor of the mexAB-oprM promoter and is able to regulate its own expression. The mechanism for this regulation includes binding to a pseudopalindromic operator site which overlaps both mexAB-oprM and pmeR promoters. We have also proven that flavonoids are able to interact with PmeR and induce a conformational change that interferes with the DNA binding ability of PmeR, thereby modulating mexAB-oprM and pmeR expression. Finally, we demonstrate by in vivo experiments that the PmeR/MexAB-OprM system contributes to the colonization of tomato plants. These results provide new insight into a transcriptional regulator and a transport system that play essential roles in the ability of P. syringae pv. tomato DC3000 to resist the action of flavonoids produced by the host.

Lipid-Layer Crystallization and Preliminary Three-Dimensional Structural Analysis of AcrA, the Periplasmic Component of a Bacterial Multidrug Efflux Pump

2001 ◽  
Vol 136 (1) ◽  
pp. 81-88 ◽  
Author(s):  
Agustin J Avila-Sakar ◽  
Shahram Misaghi ◽  
Elizabeth M Wilson-Kubalek ◽  
Kenneth H Downing ◽  
Helen Zgurskaya ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Efflux Pump ◽  
Three Dimensional ◽  
Multidrug Efflux ◽  
Lipid Layer ◽  
Multidrug Efflux Pump

scholarly journals Lincomycin Resistance Mutations in Two Regions Immediately Downstream of the −10 Region of lmr Promoter Cause Overexpression of a Putative Multidrug Efflux Pump in Bacillus subtilis Mutants

2003 ◽  
Vol 47 (1) ◽  
pp. 432-435 ◽  
Author(s):  
Miyuki Kumano ◽  
Masaya Fujita ◽  
Kouji Nakamura ◽  
Makiko Murata ◽  
Reiko Ohki ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Efflux Pump ◽  
Resistance Mutations ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Lincomycin Resistance

ABSTRACT We isolated 19 lincomycin-resistant Bacillus subtilis mutants by expressing lmrB encoding a putative multidrug efflux protein. Eighteen of the mutants altered at two regions (−3 to −1 and +15) immediately downstream of the −10 region of the lmr promoter increased lmr transcription in vivo and in vitro.

scholarly journals Low-Level Resistance of Staphylococcus aureus to Thrombin-Induced Platelet Microbicidal Protein 1 In Vitro Associated with qacA Gene Carriage Is Independent of Multidrug Efflux Pump Activity

2006 ◽  
Vol 50 (7) ◽  
pp. 2448-2454 ◽  
Author(s):  
A. S. Bayer ◽  
L. I. Kupferwasser ◽  
M. H. Brown ◽  
R. A. Skurray ◽  
S. Grkovic ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cytoplasmic Membrane ◽  
Efflux Pump ◽  
Multidrug Transporter ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Pump Activity ◽  
The Impact

ABSTRACT Thrombin-induced platelet microbial protein 1 (tPMP-1), a cationic antimicrobial polypeptide released from thrombin-stimulated rabbit platelets, targets the Staphylococcus aureus cytoplasmic membrane to initiate its microbicidal effects. In vitro resistance to tPMP-1 correlates with survival advantages in vivo. In S. aureus, the plasmid-carried qacA gene encodes a multidrug transporter, conferring resistance to organic cations (e.g., ethidium [Et]) via proton motive force (PMF)-energized export. We previously showed that qacA also confers a tPMP-1-resistant (tPMP-1r) phenotype in vitro. The current study evaluated whether (i) transporters encoded by the qacB and qacC multidrug resistance genes also confer tPMP-1r and (ii) tPMP-1r mediated by qacA is dependent on efflux pump activity. In contrast to tPMP-1r qacA-bearing strains, the parental strain and its isogenic qacB- and qacC-containing strains were tPMP-1 susceptible (tPMP-1s). Efflux pump inhibition by cyanide m-chlorophenylhydrazone abrogated Etr, but not tPMP-1r, in the qacA-bearing strain. In synergy assays, exposure of the qacA-bearing strain to tPMP-1 did not affect the susceptibility of Et (ruling out Et-tPMP-1 cotransport). The following cytoplasmic membrane parameters did not differ significantly between the qacA-bearing and parental strains: contents of the major phospholipids; asymmetric distributions of the positively charged species, lysyl-phosphotidylglycerol; fatty acid composition; and relative surface charge. Of note, the qacA-bearing strain exhibited greater membrane fluidity than that of the parental, qacB-, or qacC-bearing strain. In conclusion, among these families of efflux pumps, only the multidrug transporter encoded by qacA conferred a tPMP-1r phenotype. These data suggest that qacA-encoded tPMP-1r results from the impact of a specific transporter upon membrane structure or function unrelated to PMF-dependent peptide efflux.

scholarly journals Critical Role of Multidrug Efflux Pump CmeABC in Bile Resistance and In Vivo Colonization of Campylobacter jejuni

2003 ◽  
Vol 71 (8) ◽  
pp. 4250-4259 ◽  
Author(s):  
Jun Lin ◽  
Orhan Sahin ◽  
Linda Overbye Michel ◽  
Qijing Zhang
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Efflux Pump ◽  
In Vitro Growth ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Bile Resistance

ABSTRACT CmeABC functions as a multidrug efflux pump contributing to the resistance of Campylobacter to a broad range of antimicrobials. In this study, we examined the role of CmeABC in bile resistance and its contribution to the adaptation of Campylobacter jejuni in the intestinal tract of the chicken, a natural host and a major reservoir for Campylobacter. Inactivation of cmeABC drastically decreased the resistance of Campylobacter to various bile salts. Addition of choleate (2 mM) in culture medium impaired the in vitro growth of the cmeABC mutants but had no effect on the growth of the wild-type strain. Bile concentration varied in the duodenum, jejunum, and cecum of chicken intestine, and the inhibitory effect of the intestinal extracts on the in vitro growth of Campylobacter was well correlated with the total bile concentration in the individual sections of chicken intestine. When inoculated into chickens, the wild-type strain colonized the birds as early as day 2 postinoculation with a density as high as 107 CFU/g of feces. In contrast, the cmeABC mutants failed to colonize any of the inoculated chickens throughout the study. The minimum infective dose for the cmeABC mutant was at least 2.6 × 104-fold higher than that of the wild-type strain. Complementation of the cmeABC mutants with a wild-type cmeABC allele in trans fully restored the in vitro growth in bile-containing media and the in vivo colonization to the levels of the wild-type strain. Immunoblotting analysis indicated that CmeABC is expressed and immunogenic in chickens experimentally infected with C. jejuni. Together, these findings provide compelling evidence that CmeABC, by mediating resistance to bile salts in the intestinal tract, is required for successful colonization of C. jejuni in chickens. Inhibition of CmeABC function may not only control antibiotic resistance but also prevent the in vivo colonization of pathogenic Campylobacter.

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