The pseudo-atomic structure of an RND-type tripartite multidrug efflux pump

Du, Dijun; Voss, Jarrod E.; Wang, Zhao; Chiu, Wah; Luisi, Ben F.

doi:10.1515/hsz-2015-0118

Cited by 8 publications

(7 citation statements)

References 12 publications

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“…The sORFs selected from our randomized sequence libraries were shown to (i) localize in the cell membrane, (ii) disrupt membrane potential, (iii) consequently increase aminoglycoside resistance to the maximum amount that can be achieved by this resistance pathway, and (iv) show additive effects when combined with resistance mechanisms that are unrelated to the membrane potential (i.e., target modification). The results presented here are fully compatible with the depolarization model, but it should be pointed out that natural small proteins, such as MgtS (37), AcrZ (38, 39), and others (24), have been shown to regulate membrane-located transporters, including efflux pumps such as AcrAB (39). Thus, hypothetically Arp1-3 could confer increased resistance by upregulating an efflux pump.…”

Section: Resultssupporting

confidence: 86%

De Novo Emergence of Peptides That Confer Antibiotic Resistance

Knopp

Guðmundsdóttir

Nilsson

et al. 2019

mBio

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The origin of novel genes and beneficial functions is of fundamental interest in evolutionary biology. New genes can originate from different mechanisms, including horizontal gene transfer, duplication-divergence, and de novo from noncoding DNA sequences. Comparative genomics has generated strong evidence for de novo emergence of genes in various organisms, but experimental demonstration of this process has been limited to localized randomization in preexisting structural scaffolds. This bypasses the basic requirement of de novo gene emergence, i.e., lack of an ancestral gene. We constructed highly diverse plasmid libraries encoding randomly generated open reading frames and expressed them in Escherichia coli to identify short peptides that could confer a beneficial and selectable phenotype in vivo (in a living cell). Selections on antibiotic-containing agar plates resulted in the identification of three peptides that increased aminoglycoside resistance up to 48-fold. Combining genetic and functional analyses, we show that the peptides are highly hydrophobic, and by inserting into the membrane, they reduce membrane potential, decrease aminoglycoside uptake, and thereby confer high-level resistance. This study demonstrates that randomized DNA sequences can encode peptides that confer selective benefits and illustrates how expression of random sequences could spark the origination of new genes. In addition, our results also show that this question can be addressed experimentally by expression of highly diverse sequence libraries and subsequent selection for specific functions, such as resistance to toxic compounds, the ability to rescue auxotrophic/temperature-sensitive mutants, and growth on normally nonused carbon sources, allowing the exploration of many different phenotypes. IMPORTANCE De novo gene origination from nonfunctional DNA sequences was long assumed to be implausible. However, recent studies have shown that large fractions of genomic noncoding DNA are transcribed and translated, potentially generating new genes. Experimental validation of this process so far has been limited to comparative genomics, in vitro selections, or partial randomizations. Here, we describe selection of novel peptides in vivo using fully random synthetic expression libraries. The peptides confer aminoglycoside resistance by inserting into the bacterial membrane and thereby partly reducing membrane potential and decreasing drug uptake. Our results show that beneficial peptides can be selected from random sequence pools in vivo and support the idea that expression of noncoding sequences could spark the origination of new genes.

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Section: Resultssupporting

confidence: 86%

De Novo Emergence of Peptides That Confer Antibiotic Resistance

Knopp

Guðmundsdóttir

Nilsson

et al. 2019

mBio

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“…These tripartite efflux structures were first identified and characterized in E. coli and P. aeruginosa strains [25,26]. Advances in biochemistry and molecular biology enabled the discovery of numerous related RND pumps in nearly all clinically important bacteria [27][28][29][30].…”

Section: Efflux Pumps-based Mdr In Gram-negative Bacteriamentioning

confidence: 99%

Multidrug efflux pumps and their role in antibiotic and antiseptic resistance: a pharmacodynamic perspective

Alibert

Diarra

Hernandez

et al. 2016

Expert Opinion on Drug Metabolism & Toxicology

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Introduction: Worrying levels of bacterial resistance have been reported worldwide involving the failure of many available antibiotic treatments. Multidrug resistance (MDR) in Gram-negative bacteria is often ascribed to the presence of multiple and different resistance mechanisms in the same strain. RND efflux pumps play a major role and are an attractive target to discover new antibacterial drugs. Areas covered: This review discusses the prevalence of efflux pumps, their overexpression in clinical scenarios, their polyselectivity, their effect on the intracellular concentrations of various antibiotics associated with the alteration of the membrane permeability and their involvement in pathogenicity are discussed. Expert opinion: Efflux pumps are new targets for the development of adjuvant in antibiotic treatments by of efflux pump inhibition. They may allow us to rejuvenate old antibiotics acting on their concen-tration inside the bacteria and thus potentiating their activity while blocking the release of virulence factors. It is a pharmacodynamic challenge to finalize new combined therapy. KEYWORDSEfflux pump; Gram-negative bacteria; antibiotic; multidrug resistance; inhibitor Article highlights• Efflux pumps play a major role in multidrug resistance in Gram-negative bacteria • The pharmacodynamic role of RND efflux pumps in antibiotic resis-tance opens up new perspectives to alternative therapeutics • Targeting efflux pumps consists in solving the problems of polyse-lectivity, drug accumulation, membrane permeability and pathogeni-city related to bacterial resistance mechanisms • How to validate efflux pumps as target for new combined therapy?• Efflux pump inhibitor could be used as adjuvant in antibiotic treatments

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“…The best-studied RND efflux system is AcrAB-TolC, formed by the AcrB RND efflux pump, the TolC outer membrane protein, and the AcrA periplasmic adapter protein. This system is the most abundant RND efflux complex in the Enterobacteriaceae family (9). …”

Section: Introductionmentioning

confidence: 99%

“…AcrAB-TolC of Escherichia coli has been the main focus of study for structural studies, giving important information about the stoichiometry of the system, assembly and disassembly, substrate (drug) binding pockets and molecular interactions, and the proton translocation pathway (8–12). However, to investigate the relationship between MDR efflux and virulence, tractable infection models are required.…”

Section: Introductionmentioning

confidence: 99%

Lack of AcrB Efflux Function Confers Loss of Virulence on Salmonella enterica Serovar Typhimurium

Wang-Kan

Blair

Chirullo

et al. 2017

mBio

101

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AcrAB-TolC is the paradigm resistance-nodulation-division (RND) multidrug resistance efflux system in Gram-negative bacteria, with AcrB being the pump protein in this complex. We constructed a nonfunctional AcrB mutant by replacing D408, a highly conserved residue essential for proton translocation. Western blotting confirmed that the AcrB D408A mutant had the same native level of expression of AcrB as the parental strain. The mutant had no growth deficiencies in rich or minimal medium. However, compared with wild-type SL1344, the mutant had increased accumulation of Hoechst 33342 dye and decreased efflux of ethidium bromide and was multidrug hypersusceptible. The D408A mutant was attenuated in vivo in mouse and Galleria mellonella models and showed significantly reduced invasion into intestinal epithelial cells and macrophages in vitro. A dose-dependent inhibition of invasion was also observed when two different efflux pump inhibitors were added to the wild-type strain during infection of epithelial cells. RNA sequencing (RNA-seq) revealed downregulation of bacterial factors necessary for infection, including those in the Salmonella pathogenicity islands 1, 2, and 4; quorum sensing genes; and phoPQ. Several general stress response genes were upregulated, probably due to retention of noxious molecules inside the bacterium. Unlike loss of AcrB protein, loss of efflux function did not induce overexpression of other RND efflux pumps. Our data suggest that gene deletion mutants are unsuitable for studying membrane transporters and, importantly, that inhibitors of AcrB efflux function will not induce expression of other RND pumps.

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The pseudo-atomic structure of an RND-type tripartite multidrug efflux pump

Cited by 8 publications

References 12 publications

De Novo Emergence of Peptides That Confer Antibiotic Resistance

De Novo Emergence of Peptides That Confer Antibiotic Resistance

Multidrug efflux pumps and their role in antibiotic and antiseptic resistance: a pharmacodynamic perspective

Lack of AcrB Efflux Function Confers Loss of Virulence on Salmonella enterica Serovar Typhimurium

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