Investigation into the structure and function of an amino acid efflux pump from E. coli
Year: 2024
Type: dissertation
Abstract: Continual emergence of antimicrobial resistance has become a major global health crisis needing novel antimicrobial development. One potential approach we outline is targeting essential transporters, as these enact critical roles, particularly in preventing accumulation of toxic small molecules. Here we focus on an amino acid efflux pump in Escherichia coli called AlaE and evaluate its viability as a target by further structural and functional characterisation. For functional assessment we conducted both liquid and solid growth assays in M9 media with/without increasing concentrations of L-alanine and its derivatives and discovered that the derivatives were exponentially more inhibitory to alaE knockouts (JW2645-5), with 16 mM Ala-Ala and 8 mM Ala-Ala-Ala sufficient to be completely inhibitory. Re-introduction of the alaE gene showed recovery of growth comparable to that of the wild type (BW25113). Site directed mutagenesis alongside growth rescue assays were employed to determine residues of functional relevance in AlaE, including D14, R45, D64, D84, and D133. We developed methods of expression and purification to produce purified AlaE protein. Ligand binding was assessed via GFP-TS and DSF, with GFP-TS showing AlaE binding to L-alanine at 30 mM and 160 mM. Cysteine accessibility assay with mPEG5K determined AlaE’s cysteines to not be easily accessible. Our findings confirm that alaE is essential in the efflux of L-alanine and prevention of toxic accumulation in E. coli, as well as further current knowledge of functionally relevant residues and growth sensitivity characteristics. We present AlaE as a promising target for antimicrobial development.
Author: Cuce, Selale
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W6907386558
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