Efflux-linked accelerated evolution of antibiotic resistance at a population edge

Bhattacharyya, Souvik; Bhattacharyya, Madhumita; Pfannenstiel, Dylan M.; Nandi, Anjan K.; Hwang, YuneSahng; Ho, Khang; Harshey, Rasika M.

doi:10.1016/j.molcel.2022.10.024

Cited by 12 publications

(25 citation statements)

References 126 publications

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“…It has been shown that NSC 60339 can inhibit both efflux and necrosignalling in AcrA. 16 Furthermore, whilst novobiocin does not inhibit efflux, as its primary target in bacteria is DNA Gyrase, it can also inhibit necrosignalling, albeit not to the same degree as NSC 60339. 48 It is proposed that AcrA interacts with TolC via its α helical hairpin contacting the TolC pore.…”

Section: Discussionmentioning

confidence: 99%

“…Recent work has identified AcrA as a necrosignal. 15,16 Dead cells release AcrA, which binds to TolC in the outer membrane of live cells to send a signal, increasing drug efflux and the expression of other pumps. It has been shown that NSC 60339 can inhibit both efflux and necrosignalling in AcrA.…”

Section: Discussionmentioning

confidence: 99%

“…This degree of heterogeneity would complicate HDX-MS interpretation, therefore, we considered AcrA lacking the lipidation (AcrA S ), which has previously been shown to retain its functionality and has been utilised in several other studies. [16][17][18]27 AcrA S lacks its signal peptide (residues 1-24) and has its Cys25 replaced by a starting methionine (Met25). It is expressed and purified from the cytosol and lacks lipidation on its N-terminus.…”

Section: Acra Consists Of Both Structured and Disordered Regionsmentioning

confidence: 99%

“…10,14 Moreover, AcrA has recently been shown to have more diverse functions, being identified as a bacterial 'necrosignal' within E. coli swarms 15 : when a subpopulation of the swarm dies, dead cells release the necrosignal AcrA, which binds TolC on the outside of other live cells, stimulating efflux within the affected area and the upregulation of various efflux pumps. 16 Interestingly, recent work has also shown that NSC 60339 can inhibit AcrA-mediated necrosignalling ability. 16 Understanding the molecular mechanisms of AcrA inhibition is therefore important for future EPI and necrosignalling drug discovery.…”

Section: Introductionmentioning

confidence: 99%

“…16 Interestingly, recent work has also shown that NSC 60339 can inhibit AcrA-mediated necrosignalling ability. 16 Understanding the molecular mechanisms of AcrA inhibition is therefore important for future EPI and necrosignalling drug discovery. However, a mechanism of AcrA inhibition remains elusive.…”

Section: Introductionmentioning

confidence: 99%

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Conformational restriction shapes inhibition of a multidrug efflux adaptor protein

Lewis

Uddin

Moniruzzaman

et al. 2022

Preprint

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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.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Acra Consists Of Both Structured and Disordered Regionsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Conformational restriction shapes inhibition of a multidrug efflux adaptor protein

Lewis

Uddin

Moniruzzaman

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

Efflux pump gene amplifications bypass necessity of multiple target mutations for resistance against dual-targeting antibiotic

2023

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Antibiotics that have multiple cellular targets theoretically reduce the frequency of resistance evolution, but adaptive trajectories and resistance mechanisms against such antibiotics are understudied. Here we investigate these in methicillin resistant Staphylococcus aureus (MRSA) using experimental evolution upon exposure to delafloxacin (DLX), a novel fluoroquinolone that targets both DNA gyrase and topoisomerase IV. We show that selection for coding sequence mutations and genomic amplifications of the gene encoding a poorly characterized efflux pump, SdrM, leads to high DLX resistance, circumventing the requirement for mutations in both target enzymes. In the evolved populations, sdrM overexpression due to genomic amplifications containing sdrM and two adjacent genes encoding efflux pumps results in high DLX resistance, while the adjacent hitchhiking efflux pumps contribute to streptomycin cross-resistance. Further, lack of sdrM necessitates mutations in both target enzymes to evolve DLX resistance, and sdrM thus increases the frequency of resistance evolution. Finally, sdrM mutations and amplifications are similarly selected in two diverse clinical isolates, indicating the generality of this DLX resistance mechanism. Our study highlights that instead of reduced rates of resistance, evolution of resistance to multi-targeting antibiotics can involve alternate high-frequency evolutionary paths, that may cause unexpected alterations of the fitness landscape, including antibiotic cross-resistance.

show abstract

Conformational restriction shapes the inhibition of a multidrug efflux adaptor protein

et al. 2023

View full text Add to dashboard Cite

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 barrel 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.

show abstract

Efflux-linked accelerated evolution of antibiotic resistance at a population edge

Cited by 12 publications

References 126 publications

Conformational restriction shapes inhibition of a multidrug efflux adaptor protein

Conformational restriction shapes inhibition of a multidrug efflux adaptor protein

Efflux pump gene amplifications bypass necessity of multiple target mutations for resistance against dual-targeting antibiotic

Conformational restriction shapes the inhibition of a multidrug efflux adaptor protein

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