A Trip To L-Ala-L-Ala Land: the characterisation of AlaE, an amino acid efflux protein from Escherichia coli

E. coli pathogenic strains are known to cause serious diseases in both humans and animals, sometimes leading to fatal complications. The growing challenge of treating these infections stems from multidrug resistance, which is enhanced by the bacteria's genetic adaptability and the protective nature of biofilm structures. AlaE, an L-alanine efflux pump, plays a critical role in biofilms by transporting L-alanine to nutrient-deprived regions, promoting colony growth under resource-limited conditions. The alaE-deficient mutant strain JW2645-5 (ΔalaE), exhibits bacteriostatic effects under high concentrations of L-alanine and its peptides, making AlaE a promising target for developing efflux pump inhibitors (EPIs). However, AlaE's function and structure remain poorly characterised. In this study, we hypothesised the deletion of alaE to lead to impaired efflux of L-alanine and its peptides, resulting in growth inhibition under specific conditions. Furthermore, we predicted that the tripeptide L-alanyl-L-alanyl-alanine (Ala-Ala-Ala) is an additional inhibitory substrate, accumulating to toxic levels causing growth inhibition in the absence of AlaE. Micro-growth assays in defined M9 medium confirmed ΔalaE susceptibility to L-alanyl-L-alanine, displaying phenotypic delays and impaired growth at higher concentrations. Notably, ΔalaE was completely inhibited by the tripeptide L-alanyl-L-alanyl-alanine at lower concentrations. In restrictive media lacking carbon and nitrogen, ΔalaE metabolised Ala-Ala for growth, though with a 3-hour delay, while nutrient-rich LB alleviated inhibition, highlighting AlaE's inessentiality in these conditions. Using AlaE plasmid constructs, we rescued the ΔalaE phenotype and identified H10, D14, D84, and D133 as essential residues through site-directed mutagenesis. Alanine substitutions at these sites resulted in loss of AlaE function, restoring the ΔalaE phenotypic delay. These findings are consistent with a role for AlaE in the export of alanine and alanine-containing peptides, contributing to both efflux and biofilm survival. Additionally, they highlight critical residues that could serve as antimicrobial targets, advancing the development of EPIs to combat multidrug-resistant E. coli, and providing a foundation for future studies into AlaE's structural and functional characterisation.