Mechanism and structure of the PACE family of transport proteins

Lead Research Organisation: University of Leeds
Department Name: School of Biomedical Sciences

Abstract

Background. Resistance of bacterial pathogens to drugs is a worldwide challenge to human health. Drug efflux mediated by membrane transport proteins is a significant mechanism of resistance. Recently Henderson, with Paulsen and Hassan in Australia, discovered a novel family of drug efflux pumps, called the Proteobacterial Antimicrobial Compound Efflux (PACE) family. In Leeds, Hassan working with Henderson and Goldman expressed 17 homologues from various bacteria, including serious pathogens like Acinetobacter, Klebsiella, Pseudomonas, Salmonella and Burkholderia in E. coli.
Seven PACE efflux proteins were purified in sufficient quantities for mechanistic and structural analyses, including two responsible for resistance to an antiseptic widely used in hospitals and medicines, chlorhexidine.

Objectives (1) Substrates will be identified for all expressed proteins. (2) The mechanism of active transport by each protein will be characterized. (3) Structural features of all the proteins, and individual features of each, will be established. (4) Advances will be made towards a 3D structure of at least one of the proteins.

Novelty and Timeliness No other laboratory is yet working on the mechanistic biology of PACE family proteins to our knowledge. Recognition of the artificial substrates, chlorhexidine and acriflavine, is established for only two of the proteins, and determination of the natural substrates, possibly organic amines, for each protein is a major goal. Similarly, elucidation of their energization mechanism(s) by proton or sodium gradients, or by another mechanism, will be innovative.

Experimental approaches (Leeds and Sheffield) membrane transport assays using radiolabeled and/or fluorescent substrates; (Leeds) protein expression, purification and stability trials; steady state and transient state kinetics; circular dichroism, substrate screening assays; mass spectrometry; circular dichroism. (Sheffield) production of more PACE proteins; antibiotic screens in original host organisms; determination of MIC values. (Leeds and Sheffield) Depending on progress, XRC, EM and NMR approaches to structure.

Improving understanding of this new family of efflux transport proteins, many of which effect resistance to drugs, addresses the global challenge of overcoming resistance to antibiotics. In achieving the eventual aim of determining the 3-dimensional structure of at least one PACE protein, and then modelling structures of all the homologues, we illuminate the mechanistic biology of all the related proteins in numerous proteobacteria of importance in disease and biotechnology. Thus, this is world class bioscience, underscored not just by the high number of publications from our groups but also by the high levels of citations that they obtain, highlighting their impact within the field. Of the 14 BBSRC responsive mode priorities this work addresses three (Bioenergy, Combatting Antimicrobial Resistance, Integrative Microbiome Research).

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011151/1 30/09/2015 29/09/2023
2110717 Studentship BB/M011151/1 30/09/2018 31/12/2022 Maria Nikolova