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Visualising transport dynamics of transmembrane pumps

Periodic Reporting for period 2 - VisTrans (Visualising transport dynamics of transmembrane pumps)

Reporting period: 2019-03-01 to 2020-08-31

The resistance of bacterial infections to antibiotic treatment is a growing problem internationally. The origins of drug resistance in bacteria are multifaceted and complex, with numerous processes contributing to the
capacity of pathogenic species to evade antibiotic action during infection. One important mechanism by which drug resistance is conferred is through the intrinsic and inducible activities of multi-drug efflux
pumps. In Gram-negative bacteria, these pumps are nano-machines that span the cell envelope and use energy of electrochemical gradients or ATP hydrolysis to expel antibiotics and other toxic compounds from
the cell, and in this way contribute to resistance phenotypes. The machines are formed of three types of components: an inner membrane protein, outer membrane component that transduces energy, and a protein
residing in the periplasm that physically links the membrane proteins. An analogous machine that exports virulence factors uses paralogous components and has a similar tripartite architecture. Structural data are
available of representative efflux pumps from the applicant’s laboratory and other research groups that use proton electrochemical gradients (AcrA-AcrB-TolC) and ATP (MacA-MacB-TolC) (Du et al., 2014; Wang et
al., 2017; Fitzpatrick et al., 2018). Targeting these machines to impede their activities in pathogens such as Pseudomonas aeruginosa may be a useful strategy to treat resistant and threatening infections.

The problem is important for society due to the growing number of cases of life threatening bacterial infections that are resistant to known antibiotics. Finding compounds to inhibit the pumps in Pseudomonas and other
pathogens is challenging due to the permeability barrier presented by the formidable cell envelope, but the structural information provided from these and related studies are helping to identify alternative ways of accessing sites to inhibit the pumps.

The overall objectives of the project is to understand how the machines work in sufficient detail that it might be possible to understand how to inactivate them and enable treatments with antibiotics.
We have developed methods to prepare the pumps in an environment that mimics the natural membrane in which the proteins are embedded. This has enabled us to obtain high resolution images of the molecules and understand how the proteins can bind and push drugs through the membrane in an energy dependent process.
We hope to gain detailed structural and mechanistic understanding of how efflux pumps and related toxin export machinery in bacteria are assembled and operate in the complex cellular environment.