TaMIE · Targeting MarA to Inhibit Efflux

Targeting MarA to Inhibit Efflux

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.

Data: CORDIS, © European Union