Antimicrobial resistance is a growing threat to human health. One of the pathogens of most serious concern is Acinetobacter baumannii, which is an opportunistic pathogen in hospitals owing to its resistance to several antimicrobials, such as chlorhexidine. Resistance in A. baumannii is achieved by several mechanisms, one of which is the expression of multidrug efflux pumps, which export antimicrobial compounds out of the cell. In A. baumannii, the “Acinetobacter chlorhexidine efflux” membrane protein, designated ‘AceI’, is responsible for the increased efflux of chlorhexidine. Hundreds of homologues have since been identified, including many in pathogens, and classified as the “Proteobacterial Antimicrobial Compound Efflux”, designated "PACE", membrane transport family. In addition to biocides, members of the PACE family of proteins also transport polyamines, which may be the naturally intended substrates, across the membrane. Functional studies of PACE proteins suggest that they use the proton motive force to energise transport. Despite their importance, there are no known representative structures of the PACE family of proteins and our functional understanding is still limited.
This Thesis sets out to identify the most promising PACE homologue for expression and purification for structural and biochemical studies. Out of ten candidate proteins that were purified and characterised by SDS-PAGE and size exclusion chromatography, a promising homologue was selected due to its high levels of overexpression and stability in Escherichia coli. After unsuccessful X-ray crystallography trials, a construct of the homologue with a BRIL fusion protein was designed for use with a high-affinity anti-BRIL antibody in cryo-EM studies. Due to the heterogeneity of the sample, a 3D reconstruction of the protein structure was not possible. However, 2D classes of the protein in complex with the antibody were observed and the cryo-EM workflow described in this thesis can be used to inform future experiments. Additionally, the mechanistic roles of five residues conserved across the PACE family in the homologue were explored through mutagenesis. The mutant proteins were characterised on the basis of their thermostability, heterogeneity, substrate binding and transport properties. One of the mutant proteins showed increased thermostability and an improved heterogeneity profile, making it a suitable construct to explore in further structural studies. Taken together, the findings of this Thesis provide a strong basis for the informed exploration of further functional and structural characterisation of the PACE family of multidrug efflux proteins. Having a representative structure for the family would enable a mechanistic understanding of the transport process it catalyses and aid the development of therapies to address the contribution of the PACE transporters to antimicrobial resistance.
