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

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

PROVIDER: PRJNA782909 | ENA |

REPOSITORIES: ENA

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

Project description: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

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

Project description:Drug efflux is a common resistance mechanism found in bacteria and cancer cells, but studies providing comprehensive functional insights are scarce. In this study, we performed deep mutational scanning (DMS) on the bacterial ABC transporter EfrCD to determine the drug efflux activity profile of more than 1,430 single variants. These systematic measurements revealed that the introduction of negative charges at different locations within the large substrate binding pocket results in strongly increased efflux activity toward positively charged ethidium, whereas additional aromatic residues did not display the same effect. Data analysis in the context of an inward-facing cryogenic electron microscopy structure of EfrCD uncovered a high-affinity binding site, which releases bound drugs through a peristaltic transport mechanism as the transporter transits to its outward-facing conformation. Finally, we identified substitutions resulting in rapid Hoechst influx without affecting the efflux activity for ethidium and daunorubicin. Hence, single mutations can convert EfrCD into a drug-specific ABC importer.

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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.

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