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Real-time visualization of drug resistance acquisition by horizontal gene transfer reveals a new role for AcrAB-TolC multidrug efflux pump

ABSTRACT: In our study, we obtained evidence showing that, in the presence of tetracycline, AcrAB-TolC multidrug efflux pump was required for production of protein encoded by a conjugative plasmid acquired by bacterial conjugation. We then address the role of AcrAB-TolC multidrug efflux pump on the level of protein synthesis activity in the presence of tetracycline, which inhibits protein translation by targeting ribosomal 16S rRNA and competing with binding of the anticodon-stem loop of an A-site tRNA. We used a quantitative TMT-nanoLC-mass spectrometry approach to obtained the protein relative abundance ratio before and after incubation with tetracycline, and so in wild-type(MG1655, K12) and AcrAB-TolC mutant strains (ΔacrA, ΔacrB and ΔtolC).

INSTRUMENT(S): Q Exactive

ORGANISM(S): Escherichia Coli

TISSUE(S): Cell Culture

SUBMITTER: Frederic DELOLME

LAB HEAD: Frederic DELOLME / Christian LESTERLIN

PROVIDER: PXD011286 | Pride | 2019-07-16

REPOSITORIES: Pride

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			Action	DRS
	WT_acrA.mgf	Mgf
	WT_acrA.mzML	Mzml
	WT_acrA.mzid.gz	Mzid
	WT_acrA.pride.mgf.gz	Mgf
	WT_acrA_Fraction1-01.raw	Raw

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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.

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