Targeting MarA to Inhibit Efflux

Project Information

TaMIE

Grant agreement ID: 839036

DOI

10.3030/839036

Project closed

EC signature date 11 April 2019

Start date 2 September 2019

End date 1 September 2022

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 337 400,64

EU contribution

€ 337 400,64

Coordinated by

THE UNIVERSITY OF BIRMINGHAM
United Kingdom

Periodic Reporting for period 1 - TaMIE (Targeting MarA to Inhibit Efflux)

Reporting period: 2019-09-02 to 2022-09-01

Summary of the context and overall objectives of the project

Antimicrobial resistance is a silent pandemic with 1.27 million deaths directly attributable to resistance in 2019, a value that ranks behind only COVID-19 and tuberculosis in terms of global deaths from infection. One of the mechanisms employed by Gram-negative bacteria to become resistant to routinely used antibiotics is the use of efflux pumps to export antimicrobials out of the bacterial cells. There are seven families of efflux pumps, with the members of the Resistance-Nodulation-Division superfamily being the most clinically associated with MDR phenotypes. AcrAB-TolC is the main RND efflux pump in Enterobacteriaceae including Escherichia coli. The regulation of the expression of AcrAB-TolC is complex and includes global regulators such as MarA, SoxS and Rob, which increase the expression of the major efflux genes. Additionally, MarA regulates other 40 genes, many of them related to the antimicrobial resistance phenotype.

A strategy to decrease the efflux of antibiotics out of the bacteria would be to target the regulators of the efflux pumps with inhibitors. Currently, there are no inhibitors for monomeric regulators belonging to the AraC/XylS family, such as MarA. In the TaMIE project, we aimed to develop efflux inhibitors that prevent transcription of acrAB by inhibiting MarA and to test their efficacy by measuring AcrAB expression and activity. We also aimed to understand how MarA binds the DNA in order to find structural elements in MarA that could be targeted by those inhibitors.

The outcomes of this project can help in the design of improved MarA inhibitors. These inhibitors could be administered as combined therapy with antibiotics to allow them to stay inside the bacteria for longer and exert their killing activity. In this way, the European science base and European consumers will benefit from potential inhibitors to be used in infections caused by antibiotic-resistant strains.

Conclusions of the action:
1. We have succeeded in identifying the N-terminal helix of MarA as a new structural element involved in the mechanism of DNA binding. Our findings showed that this helix is a new structural target against which to design MarA inhibitors.
2. We also showed that the N-terminal helix has a role in the recognition of the functional marboxes.
3. We postulate a new mechanism of MarA inhibition that does not involve the helices with direct contact with the DNA, but the pulling of the N-terminal helix of MarA to a position different to the one observed in the protein structure. This mechanism of inhibition seems to be general for the members of the AraC/XylS family of regulators (e.g. Rob). In this way, our findings not only target an important protein for the appearance of antibiotic resistance such as MarA, but contribute to the progress of the inhibition of other global transcriptional regulators.
4. By molecular docking, we found 60 molecules that can bind the N-terminal helix of MarA all of them potential MarA inhibitors. The lab work done with one of these hits was promising.
5. An N-terminal truncation of MarA seems to be able to bind the marboxes, but shows less affinity than the wild-type MarA. This truncation can compete for the binding to the marbox becoming a potential MarA inhibitor.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

During the TaMIE project, the results have been regularly presented in laboratory open mornings, internal seminars and thought posters at College-wide events. This dissemination has allowed the establishment of collaborations with other researchers at UoB. We also communicated the TaMIE outcomes by attending international and national conferences. These conferences have been crucial to disseminate the results to the wide scientific community and creating an international collaborative network.

2022
Speaker at Universitat Autònoma de Barcelona.
Speaker in the IMI Forum at the Institute of Microbiology and Infection, University of Birmingham, United Kingdom.
Speaker in the Seedcorn Project virtual Meeting, co-organised by Uppsala Universitet and Durham University).
Poster in the Antibiotic resistance and mechanisms workshop, British Society for antimicrobial chemotherapy, Birmingham, UK.
2021
EMBO Workshop: Recent Advances in Structural Biology of Membrane.
Speaker at the 43rd Annual Meeting of the Spanish Society of Biochemistry and Molecular Biology, Barcelona (online), Spain.
2020
Poster in the 17th Iberian Peptide Meeting, Madrid, Spain.
2019
Antibiotic resistance and mechanisms workshop, British Society for antimicrobial chemotherapy, Birmingham, United Kingdom.

During the lifetime of the TaMIE project, we published a review related to the efflux of antimicrobial peptides done by efflux pumps. In this review, I tried to put together my expertise in AMPs with the expertise of the host group in efflux pumps. This review was published recently in FEMS microbiology reviews (impact factor 16), with me as the corresponding author. Currently, we are finishing three papers containing the results of the TaMIE project. These papers will be submitted shortly to high-impact journals.

For an overview of the results, see the "conclusions of the action" in the previous section.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Many gram-negative bacteria have developed resistance against routinely used antibiotics, resulting in the looming threat of untreatable infectious diseases. The inhibition of efflux pumps by efflux pump inhibitors appears to be a promising strategy to revert the multidrug-resistant strains to antibiotic-susceptible ones, recovering the potentiality of the old antibiotics.

Through the TaMIE project, we have identified a new mechanism of inhibition of MarA, that involves its N-terminal helix. This mechanism of inhibition has been shown to be general for the whole AraC/XylS family of transcriptional regulators, including the two-domain protein Rob. In this way, the overcomes of this project will contribute to the progress of the inhibition of global transcriptional regulators in general.

In addition, the identification of a molecule able to bind the N-terminal helix of MarA opens the possibility to use it as a scaffold to synthesise derivatives with more affinity towards this helix. These derivatives could become improved MarA inhibitors. Since the N-terminal helix of the members of the AraC/XylS family presents a high percentage of amino acid conservation, this molecule could also serve as a scaffold to inhibit other regulators in the same family.

The outcomes of the TaMIE project will be of great applicability to the pharmaceutical industry and European consumers since the MarA inhibitors could be administered as combined therapy with antibiotics to allow them to stay inside the bacteria for longer and exert their killing activity. In this way, the European science base and the economy will also benefit greatly from the results of this project. The impact of the research will be immediate in academia by exploring a fundamental research question and also will be felt far into the future by providing state-of-the-art knowledge.

Structure of MarA and the N-terminal mutants