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Deep mutational scan of a drug efflux pump reveals its structure-function landscape

ABSTRACT: Drug efflux is a common resistance mechanism found in bacteria and cancer cells. Although several structures of drug efflux pumps are available, they provide only limited functional information on the phenomenon of drug efflux. Here, we performed deep mutational scanning (DMS) on the bacterial ATP binding cassette (ABC) transporter EfrCD from Enterococcus faecalis to determine the drug efflux activity profile of more than 1400 single variants

ORGANISM(S): Enterococcus faecalis

PROVIDER: GSE189399 | GEO | 2021/11/25

REPOSITORIES: GEO

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Bile-induced biofilm formation in Bacteroides thetaiotaomicron requires magnesium efflux by an RND pump

Project description:The study is about the role of Bacteroides thetaiotaomicron in the human gut microbiota, specifically its ability to form biofilms in response to bile salts. The study found that bile induces the expression of certain efflux pumps, and inhibiting these pumps impairs biofilm formation. Among the induced pumps, the BipABC pump is crucial for biofilm formation as it is involved in the efflux of magnesium, which affects the biofilm's extracellular matrix and structure. This discovery sheds light on how intestinal chemical cues, like bile salts, regulate biofilm formation in B. thetaiotaomicron, a significant gut symbiont.

2024-03-19 | E-MTAB-13460 | biostudies-arrayexpress

The impaired quorum sensing response of MexAB-OprM efflux pump overexpressing mutants is not due to non-physiological efflux of 3-oxo-C12-HSL

Project description:Multidrug (MDR) efflux pumps are ancient and conserved molecular machineries with relevant roles in different aspects of the bacterial physiology, besides antibiotic resistance. In the case of the environmental opportunistic pathogen Pseudomonas aeruginosa, it has been shown that overexpression of different efflux pumps is linked to the impairment of the quorum sensing (QS) response. Nevertheless, the causes of such impairment are different for each analyzed efflux pump. Herein, we performed an in-depth analysis of the QS-mediated response of a P. aeruginosa antibiotic resistant mutant that overexpresses MexAB-OprM. Although previous work claimed that this efflux pump extrudes the QS signal 3-oxo-C12-HSL, we show otherwise. Our results evidence that the observed attenuation in the QS response when overexpressing this pump is related to an impaired production of alkyl quinolone QS signals, likely prompted by the reduced availability of one of their precursors, the octanoate. Together with previous studies, this indicates that, although the consequences of overexpressing efflux pumps are similar (impaired QS response), the underlying mechanisms are different. This ‘apparent redundancy' of MDR efflux systems can be understood as a P. aeruginosa strategy to keep the robustness of the QS regulatory network and modulate its output in response to different signals.

2021-10-25 | GSE153996 | GEO

A genetic platform to investigate the functions of bacterial drug efflux pumps

Project description:Efflux pumps are a significant challenge for the development of new antibacterial agents. Overcoming efflux requires an in-depth understanding of efflux pump functions, substrate specificities, and the development of inhibitors. However, the complexities of drug efflux networks have limited such studies. To address these challenges, we report the generation of Efflux KnockOut-35 (EKO-35), a highly susceptible Escherichia coli strain lacking 35 efflux pumps. We demonstrate the utility of this strain by constructing an efflux platform consisting of strains individually expressing genes encoding efflux pumps forming tripartite complexes with the outer membrane channel TolC. This platform was profiled against a curated diverse compound collection, which enabled us to define physicochemical properties that contribute to transport. We also show the E. coli drug efflux network is conditionally essential for growth, and that the platform can be used to investigate efflux pump inhibitor specificities and also efflux pump interplay. We believe EKO-35 and the efflux platform will have widespread application for the study of drug efflux.

2022-08-31 | PXD033975 | Pride

Mutations in Multidrug Efflux Pumps of Eshcerichia coli

Project description:The number and overlapping substrate repertoire of multidrug efflux pumps in the E. coli genome suggest a physiological role apart from multidrug resistance. This role was investigated using transcriptomic analyses of cDNAs labeled from E. coli AG102 mRNA (hyper drug resistant, marR1) and its isogenic major efflux pump mutants. Keywords: Mutation Analysis

2008-03-11 | GSE10757 | GEO

Deep mutational scan of a drug efflux pump reveals its structure-function landscape

Project description:Deep mutational scan of a drug efflux pump reveals its structure-function landscape

| PRJNA782909 | ENA

Genome wide analysis of genes involved in efflux function and regulation within Escherichia coli and Salmonella enterica serovar Typhimurium

Project description:Expression of efflux pumps is a key feature of most cells which are resistant to multiple antibiotics. This study used TraDIS-Xpress, a genome wide transposon mutagenesis technology to identify genes in Escherichia coli and Salmonella Typhimurium involved in drug efflux and its regulation. We exposed mutant libraries to the canonical efflux substrate acriflavine in the presence and absence of the efflux inhibitor phenylalanine-arginine β-naphthylamide. Comparisons between conditions identified efflux specific and drug specific responses. Known efflux associated genes were easily identified including: AcrAB-TolC, MarA, RamA and SoxS confirming specificity of the response. Further genes encoding cell envelope maintenance enzymes and products involved with stringent response activation, DNA housekeeping, respiration and glutathione biosynthesis were also identified as affecting efflux activity in both species. We identified a conserved set of pathways crucial for efflux activity in these experimental conditions which expands the list of genes known to impact efflux efficacy.

2022-11-12 | E-MTAB-12353 | biostudies-arrayexpress

Conformational restriction shapes the inhibition of a multidrug efflux adaptor protein

Project description:Membrane efflux pumps play a major role in bacterial multidrug resistance. The tripartite multidrug efflux pump system from Escherichia coli, AcrAB-TolC, is a target for inhibition to lessen resistance development and restore antibiotic efficacy, with homologs in other ESKAPE pathogens. Here, we rationalize a mechanism of inhibition against the periplasmic adaptor protein, AcrA, using a combination of hydrogen/deuterium exchange mass spectrometry, cellular efflux assays, and molecular dynamics simulations. We define the structural dynamics of AcrA and find that an inhibitor can inflict long-range stabilisation across all four of its domains, whereas an interacting efflux substrate has minimal effect. Our results support a model where an inhibitor forms a molecular wedge within a cleft between the lipoyl and αβ domains of AcrA, diminishing its conformational transmission of drug-evoked signals from AcrB to TolC. This work provides molecular insights into multidrug adaptor protein function which could be valuable for developing antimicrobial therapeutics.

2023-06-01 | PXD041359 | Pride

Physiology of Pseudomonas aeruginosa cells without major RND-type multidrug efflux pumps

Project description:Purpose: In this study, we analyzed how P. aeruginosa physiology is adapted to the lack of RND-mediated efflux activities. Methods: In this study, we use PΔ6 cells to analyze how P. aeruginosa changes its physiology in response to the lack of efflux pumps and increased permeability of the cell envelope. We compared the transcriptomes of the exponentially growing and stationary PΔ6 and its parent PAO1 cells and identified the cellular functions stressed by the lack of active efflux. High quality total RNA was further processed by removing 23S and 16S rRNAs using the Illumina Ribo-Zero Plus rRNA Depletion kit. Samples were analyzed in duplicate using Illumina MiSeq. Raw data for each sample was analyzed using CLC Genomics Workbench version 12.0.1 software (QIAGEN Aarhus, Denmark). Results: P. aeruginosa PΔ6 strain lacking six best characterized RND pumps activates a specific adaptation response that involves significant changes in expression of specific subset of genes encoding e.g. several transport systems, quorum sensing or iron acquisition. Conclusion: Our results suggest that all changes we observe serve to protect the cell envelope of efflux-deficient P. aeruginosa.

2021-04-30 | GSE169036 | GEO

Co-chaperone-mediated post-translational control of efflux pump induction underlies adaptive β-lactam resistance in Caulobacter crescentus [RNA-seq]

Project description:The acquisition of multi-drug resistance (MDR) determinants jeopardizes treatment of bacterial infections with antibiotics. The tripartite efflux pump AcrAB-NodT confers adaptive MDR in the non-pathogenic α-proteobacterium Caulobacter crescentus via transcriptional induction by first-generation quinolone antibiotics. We discovered that overexpression of AcrAB-NodT by mutation or exogenous inducers confers resistance to cephalosporin and penicillin (β-lactam) antibiotics. Combining two-step mutagenesis-sequencing (Mut-Seq) and cephalosporin-resistant point mutants, we dissected how TipR targets a common operator of divergent tipR and acrAB-nodT promoter in adaptive and/or potentiated AcrAB-NodT-directed efflux. Chemical screening identified compounds that either interfere with DNA-binding by TipR or induce its ClpXP-dependent proteolytic turnover. We found that long-term induction of AcrAB-NodT disfigures the envelope and that homeostatic control by TipR includes co-induction of the DnaJ-like co-chaperone DjlA, to boost pump assembly and/or capacity in anticipation of envelope stress. Thus, the adaptive MDR regulatory circuitry reconciles drug efflux with co-chaperone function for trans-envelope assemblies and maintenance.

2023-11-09 | GSE225488 | GEO

Co-chaperone-mediated post-translational control of efflux pump induction underlies adaptive β-lactam resistance in Caulobacter crescentus [ChIP-seq]

2023-11-09 | GSE225487 | GEO

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