scholarly journals Drug Efflux by a Small Multidrug Resistance Protein Is Inhibited by a Transmembrane Peptide

2012 ◽  
Vol 56 (7) ◽  
pp. 3911-3916 ◽  
Author(s):  
Bradley E. Poulsen ◽  
Charles M. Deber
Keyword(s):  
Multidrug Resistance ◽  
Rate Constant ◽  
Halobacterium Salinarum ◽  
Resistant Bacteria ◽  
Bacterial Cells ◽  
Multidrug Resistance Proteins ◽  
Content Type ◽  
Hydrophobic Peptides ◽  
Small Multidrug Resistance ◽  
Efflux Rate Constant

ABSTRACTDrug-resistant bacteria use several families of membrane-embedded transporters to remove antibiotics from the cell. One such family is the small multidrug resistance proteins (SMRs) that, because of their relatively small size (ca. 110 residues with four transmembrane [TM] helices), must form (at least) dimers to efflux drugs. Here, we use a Lys-tagged synthetic peptide with exactly the same sequence as TM4 of the full-length SMR Hsmr fromHalobacterium salinarum[TM4 sequence: AcA(Sar)3-VAGVVGLALIVAGVVVLNVAS-KKK (Sar =N-methylglycine)] to compete with and disrupt the native TM4-TM4 interactions believed to constitute the locus of Hsmr dimerization. Using a cellular efflux assay of the fluorescent SMR substrate ethidium bromide, we determined that bacterial cells containing Hsmr are able to remove cellular ethidium via first-order exponential decay with a rate constant (k) of 10.1 × 10−3± 0.7 × 10−3s−1. Upon treatment of the cells with the TM4 peptide, we observed a saturable ∼60% decrease in the efflux rate constant to 3.7 × 10−3± 0.2 × 10−3s−1. In corresponding experiments with control peptides, including scrambled sequences and a sequence withd-chirality, a decrease in ethidium efflux either was not observed or was marginal, likely from nonspecific effects. The designed peptides did not evoke bacterial lysis, indicating that they act via the α-helicity and membrane insertion propensities of the native TM4 helix. Our overall results suggest that this approach could conceivably be used to design hydrophobic peptides for disruption of key TM-TM interactions of membrane proteins and represent a valuable route to the discovery of new therapeutics.

scholarly journals Peptide-Based Efflux Pump Inhibitors of the Small Multidrug Resistance Protein from Pseudomonas aeruginosa

2019 ◽  
Vol 63 (9) ◽  
Author(s):  
Chloe J Mitchell ◽  
Tracy A. Stone ◽  
Charles M. Deber
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Multidrug Resistance ◽  
Efflux Pump ◽  
Membrane Integrity ◽  
Resistant Bacteria ◽  
Binding Motif ◽  
Sequence Motif ◽  
Content Type ◽  
Binding Interface ◽  
Small Multidrug Resistance

ABSTRACT Bacteria have acquired multiple mechanisms to evade the lethal effects of current therapeutics, hindering treatment of bacterial infections, such as those caused by the pathogen Pseudomonas aeruginosa, which is responsible for nosocomial and cystic fibrosis lung infections. One resistance mechanism involves membrane-embedded multidrug efflux pumps that can effectively extrude an array of substrates, including common antibiotics, dyes, and biocides. Among these is a small multidrug resistance (SMR) efflux protein, consisting of four transmembrane (TM) helices, that functions as an antiparallel dimer. TM helices 1 to 3 (TM1 to TM3) comprise the substrate binding pocket, while TM4 contains a GG7 heptad sequence motif that mediates the SMR TM4-TM4 dimerization. In the present work, we synthesized a series of peptides containing the residues centered on the TM4-TM4 binding interface found in the P. aeruginosa SMR (PAsmr), typified by Ac-Ala-(Sar)3-LLGIGLIIAGVLV-KKK-NH2 (helix-helix interaction residues are underlined). Here, the acetylated N-terminal sarcosine (N-methyl-Gly) tag [Ac-Ala-(Sar)3] promotes membrane penetration, while the C-terminal Lys tag promotes selectivity for the negatively charged bacterial membranes. This peptide was observed to competitively disrupt PAsmr-mediated efflux, as measured by efflux inhibition of the fluorescent dye ethidium bromide, while having no effect on cell membrane integrity. Alternatively, a corresponding peptide in which the TM4 binding motif is scrambled was inactive in this assay. In addition, when Escherichia coli cells expressing PAsmr were combined with sublethal concentrations of several biocides, growth was significantly inhibited when peptide was added, notably, by up to 95% with the disinfectant benzylalkonium chloride. These results demonstrate promise for an efflux pump inhibitor to address the increasing threat of antibiotic-resistant bacteria.

scholarly journals Functional phenotyping of small multidrug resistance proteins from Staphylococcus aureus and Francisella tularensis reveals functional homology to EmrE

2021 ◽  
Author(s):  
Peyton J Spreacker ◽  
Will F Beeninga ◽  
Brooke L Young ◽  
Colin J Porter ◽  
Katherine A Henzler-Wildman
Keyword(s):  
Staphylococcus Aureus ◽  
Multidrug Resistance ◽  
Substrate Specificity ◽  
Francisella Tularensis ◽  
Bacterial Species ◽  
Toxic Metabolite ◽  
Bacterial Cells ◽  
Multidrug Resistance Proteins ◽  
Antibiotic Development ◽  
Small Multidrug Resistance

Small multidrug resistance (SMR) transporters efflux toxic substrates from bacterial cells. These transporters were recently divided into two subfamilies: the GdX-like and EmrE-like SMRs. The EmrE-like subfamily of SMRs is predicted to contain transporters that are highly promiscuous in both substrate specificity and mechanism based on extensive characterization of the founding member of this subfamily, EmrE. However, there is only limited functional analysis of other members of this family from pathogenic strains such as Staphylococcus aureus and Francisella tularensis. Here, we use a small compound screen to explore the substrate specificity and diversity of EmrE-subfamily SMRs from these two bacterial species and confirm that they are functionally more like EmrE than the GdX-like subfamily of toxic-metabolite transporters. The results of these experiments lay the foundation for understanding the complex substrate specificity profiles of SMR family transporters and assess the potential for targeting these transporters for future antibiotic development, either broadly or in a species-specific manner.

scholarly journals Guanidine Riboswitch-Regulated Efflux Transporters Protect Bacteria against Ionic Liquid Toxicity

2019 ◽  
Vol 201 (13) ◽  
Author(s):  
Douglas A. Higgins ◽  
John M. Gladden ◽  
Jeff A. Kimbrel ◽  
Blake A. Simmons ◽  
Steven W. Singer ◽  
...  
Keyword(s):  
Ionic Liquid ◽  
Multidrug Resistance ◽  
Critical Role ◽  
Efflux Transporters ◽  
Imidazolium Ionic Liquid ◽  
Content Type ◽  
Small Multidrug Resistance ◽  
Cellulosic Sugars ◽  
Industrial Strains ◽  
Biochemical Production

ABSTRACT Plant cell walls contain a renewable, nearly limitless supply of sugar that could be used to support microbial production of commodity chemicals and biofuels. Imidazolium ionic liquid (IIL) solvents are among the best reagents for gaining access to the sugars in this otherwise recalcitrant biomass. However, the sugars from IIL-treated biomass are inevitably contaminated with residual IILs that inhibit growth in bacteria and yeast, blocking biochemical production by these organisms. IIL toxicity is, therefore, a critical roadblock in many industrial biosynthetic pathways. Although several IIL-tolerant (IILT) bacterial and yeast isolates have been identified in nature, few genetic mechanisms have been identified. In this study, we identified two IILT Bacillus isolates as well as a spontaneous IILT Escherichia coli lab strain that are tolerant to high levels of two widely used IILs. We demonstrate that all three IILT strains contain one or more pumps of the small multidrug resistance (SMR) family, and two of these strains contain mutations that affect an adjacent regulatory guanidine riboswitch. Furthermore, we show that the regulation of E. coli sugE by the guanidine II riboswitch can be exploited to promote IIL tolerance by the simple addition of guanidine to the medium. Our results demonstrate the critical role that transporter genes play in IIL tolerance in their native bacterial hosts. The study presented here is another step in engineering IIL tolerance into industrial strains toward overcoming this key gap in biofuels and industrial biochemical production processes. IMPORTANCE This study identifies bacteria that are tolerant to ionic liquid solvents used in the production of biofuels and industrial biochemicals. For industrial microbiology, it is essential to find less-harmful reagents and microbes that are resistant to their cytotoxic effects. We identified a family of small multidrug resistance efflux transporters, which are responsible for the tolerance of these strains. We also found that this resistance can be caused by mutations in the sequences of guanidine-specific riboswitches that regulate these efflux pumps. Extending this knowledge, we demonstrated that guanidine itself can promote ionic liquid tolerance. Our findings will inform genetic engineering strategies that improve conversion of cellulosic sugars into biofuels and biochemicals in processes where low concentrations of ionic liquids surpass bacterial tolerance.

scholarly journals RadD Contributes to R-Loop Avoidance in Sub-MIC Tobramycin

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Veronica Negro ◽  
Evelyne Krin ◽  
Sebastian Aguilar Pierlé ◽  
Thibault Chaze ◽  
Quentin Giai Gianetto ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Coupling Factor ◽  
Resistant Bacteria ◽  
Bacterial Cells ◽  
Dna Breaks ◽  
Bacterial Populations ◽  
Content Type ◽  
Double Strand Dna Breaks ◽  
Translation Coupling ◽  
Recombination Pathway

ABSTRACTWe have previously identifiedVibrio choleraemutants in which the stress response to subinhibitory concentrations of aminoglycoside is altered. One gene identified, VC1636, encodes a putative DNA/RNA helicase, recently named RadD inEscherichia coli. Here we combined extensive genetic characterization and high-throughput approaches in order to identify partners and molecular mechanisms involving RadD. We show that double-strand DNA breaks (DSBs) are formed upon subinhibitory tobramycin treatment in the absence ofradDandrecBCDand that formation of these DSBs can be overcome by RNase H1 overexpression. Loss of RNase H1, or of the transcription-translation coupling factor EF-P, is lethal in theradDdeletion mutant. We propose that R-loops are formed upon sublethal aminoglycoside treatment, leading to the formation of DSBs that can be repaired by the RecBCD homologous recombination pathway, and that RadD counteracts such R-loop accumulation. We discuss how R-loops that can occur upon translation-transcription uncoupling could be the link between tobramycin treatment and DNA break formation.IMPORTANCEBacteria frequently encounter low concentrations of antibiotics. Active antibiotics are commonly detected in soil and water at concentrations much below lethal concentration. Although sub-MICs of antibiotics do not kill bacteria, they can have a major impact on bacterial populations by contributing to the development of antibiotic resistance through mutations in originally sensitive bacteria or acquisition of DNA from resistant bacteria. It was shown that concentrations as low as 100-fold below the MIC can actually lead to the selection of antibiotic-resistant cells. We seek to understand how bacterial cells react to such antibiotic concentrations usingE. coli, the Gram-negative bacterial paradigm, andV. cholerae, the causative agent of cholera. Our findings shed light on the processes triggered at the DNA level by antibiotics targeting translation, how damage occurs, and what the bacterial strategies are to respond to such DNA damage.

scholarly journals Roles of P-Glycoprotein, Bcrp, and Mrp2 in Biliary Excretion of Spiramycin in Mice

2007 ◽  
Vol 51 (9) ◽  
pp. 3230-3234 ◽  
Author(s):  
Xianbin Tian ◽  
Jun Li ◽  
Maciej J. Zamek-Gliszczynski ◽  
Arlene S. Bridges ◽  
Peijin Zhang ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Rate Constant ◽  
Knockout Mice ◽  
Biliary Excretion ◽  
Cancer Resistance ◽  
Multidrug Resistance Proteins ◽  
Wild Type ◽  
P Glycoprotein ◽  
Hepatobiliary Disposition

ABSTRACT The multidrug resistance proteins P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and multidrug resistance-associated protein 2 (Mrp2) are the three major canalicular transport proteins responsible for the biliary excretion of most drugs and metabolites. Previous in vitro studies demonstrated that P-gp transported macrolide antibiotics, including spiramycin, which is eliminated primarily by biliary excretion. Bcrp was proposed to be the primary pathway for spiramycin secretion into breast milk. In the present study, the contributions of P-gp, Bcrp, and Mrp2 to the biliary excretion of spiramycin were examined in single-pass perfused livers of male C57BL/6 wild-type, Bcrp-knockout, and Mrp2-knockout mice in the presence or absence of GF120918 (GW918), a P-gp and Bcrp inhibitor. Spiramycin was infused to achieve steady-state conditions, followed by a washout period, and parameters governing spiramycin hepatobiliary disposition were recovered by using pharmacokinetic modeling. In the absence of GW918, the rate constant governing spiramycin biliary excretion was decreased in Mrp2− knockout mice (0.0013 ± 0.0009 min−1) relative to wild-type mice (0.0124 ± 0.0096 min−1). These data are consistent with the ∼8-fold decrease in the recovery of spiramycin in the bile of Mrp2-knockout mice and suggest that Mrp2 is the major canalicular transport protein responsible for spiramycin biliary excretion. Interestingly, biliary recovery of spiramycin in Bcrp-knockout mice was increased in both the absence and presence of GW918 compared to wild-type mice. GW918 significantly decreased the rate constant for spiramycin biliary excretion and the rate constant for basolateral efflux of spiramycin. In conclusion, the biliary excretion of spiramycin in mice is mediated primarily by Mrp2 with a modest P-gp component.

scholarly journals Photocatalytic Protein Damage by Silver Nanoparticles Circumvents Bacterial Stress Response and Multidrug Resistance

mSphere ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Tianyuan Shi ◽  
Qiuxia Wei ◽  
Zhen Wang ◽  
Gong Zhang ◽  
Xuesong Sun ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Drug Resistance ◽  
Silver Nanoparticles ◽  
Protein Aggregation ◽  
Antibacterial Properties ◽  
Resistance Mechanisms ◽  
Resistant Bacteria ◽  
Bacterial Cells ◽  
Content Type ◽  
Bactericidal Mechanism

ABSTRACT Silver nanoparticles (AgNPs) are known for their broad-spectrum antibacterial properties, especially against antibiotic-resistant bacteria. However, the bactericidal mechanism of AgNPs remains unclear. In this study, we found that the bactericidal ability of AgNPs is induced by light. In contrast to previous postulates, visible light is unable to trigger silver ion release from AgNPs or to promote AgNPs to induce reactive oxygen species (ROS) in Escherichia coli. In fact, we revealed that light excited AgNPs to induce protein aggregation in a concentration-dependent manner in E. coli, indicating that the bactericidal ability of AgNPs relies on the light-catalyzed oxidation of cellular proteins via direct binding to proteins, which was verified by fluorescence spectra. AgNPs likely absorb the light energy and transfer it to the proteins, leading to the oxidation of proteins and thus promoting the death of the bacteria. Isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics revealed that the bacteria failed to develop effective resistance to the light-excited AgNPs. This direct physical mechanism is unlikely to be counteracted by any known drug resistance mechanisms of bacteria and therefore may serve as a last resort against drug resistance. This mechanism also provides a practical hint regarding the antimicrobial application of AgNPs—light exposure improves the efficacy of AgNPs. IMPORTANCE Although silver nanoparticles (AgNPs) are well known for their antibacterial properties, the mechanism by which they kill bacterial cells remains a topic of debate. In this study, we uncovered the bactericidal mechanism of AgNPs, which is induced by light. We tested the efficacy of AgNPs against a panel of antimicrobial-resistant pathogens as well as Escherichia coli under conditions of light and darkness and revealed that light excited the AgNPs to promote protein aggregation within the bacterial cells. Our report makes a significant contribution to the literature because this mechanism bypasses microbial drug resistance mechanisms, thus presenting a viable option for the treatment of multidrug-resistant bacteria.

scholarly journals Riboswitch-Associated Guanidinium-Selective Efflux Pumps Frequently Transmitted on Proteobacterial Plasmids Increase Escherichia coli Biofilm Tolerance to Disinfectants

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Carmine J. Slipski ◽  
Taylor R. Jamieson ◽  
George G. Zhanel ◽  
Denice C. Bay
Keyword(s):  
Escherichia Coli ◽  
Multidrug Resistance ◽  
Quaternary Ammonium ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Cloning And Expression ◽  
Upstream Region ◽  
Ammonium Compound ◽  
Content Type ◽  
Small Multidrug Resistance

ABSTRACT Members of the small multidrug resistance (SMR) efflux pump family known as SugE (recently renamed Gdx) are known for their narrow substrate selectivity to small guanidinium (Gdm+) compounds and disinfectant quaternary ammonium compounds (QACs). Gdx members have been identified on multidrug resistance plasmids in Gram-negative bacilli, but their functional role remains unclear, as few have been characterized. Here, we conducted a survey of sequenced proteobacterial plasmids that encoded one or more SugE/Gdx sequences in an effort to (i) identify the most frequently represented Gdx member(s) on these plasmids and their sequence diversity, (ii) verify if Gdx sequences possess a Gdm+ riboswitch that regulates their translation similarly to chromosomally encoded Gdx members, and (iii) determine the antimicrobial susceptibility profile of the most predominate Gdx member to various QACs and antibiotics in Escherichia coli strains BW25113 and KAM32. The results of this study determined 14 unique SugE sequences, but only one Gdx sequence, annotated as “SugE(p),” predominated among the >140 plasmids we surveyed. Enterobacterales plasmids carrying sugE(p) possessed a guanidine II riboswitch similar to the upstream region of E. coli gdx. Cloning and expression of sugE(p), gdx, and emrE sequences into a low-copy-number expression vector (pMS119EH) revealed significant increases in QAC resistance to a limited range of detergent-like QACs only when gdx and sugE(p) transformants were grown as biofilms. These findings suggest that sugE(p) presence on proteobacterial plasmids may be driven by species that frequently encounter Gdm+ and QAC exposure. IMPORTANCE This study characterized the function of antimicrobial-resistant phenotypes attributed to plasmid-encoded guanidinium-selective small multidrug resistance (Gdm/SugE) efflux pumps. These sequences are frequently monitored as biocide resistance markers in antimicrobial resistance surveillance studies. Our findings reveal that enterobacterial gdm sequences transmitted on plasmids possess a guanidine II riboswitch, which restricts transcript translation in the presence of guanidinium. Cloning and overexpression of this gdm sequence revealed that it confers higher resistance to quaternary ammonium compound (QAC) disinfectants (which possess guanidium moieties) when grown as biofilms. Since biofilms are commonly eradicated with QAC-containing compounds, the presence of this gene on plasmids and its biofilm-specific resistance are a growing concern for clinical and food safety prevention measures.

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