Understanding multidrug efflux inhibition in gram negative b acteria

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The aim of this studentship is to understand the mechanism of efflux inhibition (EI) of compounds purported to inhibit efflux of antibiotics from bacteria. A minority of antibacterials are active against Gram-negative bacteria. This is due to the structure of the cell wall conferring impermeability and the presence of multi-drug resistance (MDR) tripartite efflux pumps, which pump out many antibiotics so conferring intrinsic MDR. An approach to circumvent efflux is to combine a licenced antibiotic with an efflux inhibitor to 'rescue' an otherwise effective drug and extend its useful life. Little is known about the mechanism of action of many compounds described in the literature as efflux pump inhibitors. Therefore, understanding how these compounds work will inform discovery of new EI-antibiotic combinations active against MDR bacteria. Milestone 1 (by 18m). Validation of EI activity of compounds in vitro: MDR efflux pumps have a wide substrate range and so the ability of (EI) compounds to inhibit the export of antibiotics will be measured directly in MDR bacteria such as E. coli, A. baumannii and P. aeruginosa. This will use the latest whole cell methodologies to detect antibiotic accumulated and effluxed from the bacterial cell including fluorescence spectrophotometry and mass spectrometry.Milestone 2 (by 36m). Investigation of the effect of EI in vivo: In the presence of an EI an efflux-susceptible antibiotic will be more effective than alone. This is not just due to higher concentrations of antibiotic remaining inside the cell, but also because inactivation or loss of function of efflux pump proteins reduces the ability of bacteria to infect the host and form a biofilm. Industry standard models of bacterial infection (including thigh and lung burden murine models) and whole animal imaging established at Evotec will be used to determine the effect EI compounds +/- antibiotic. The ability of bacteria to adhere to, and invade tissue culture models of infection and to form and eradicate a biofilm will also be measured. Milestone 3 (by 48m). Elucidation of the mechanism of EI: Our understanding of the mechanism of transport of substrates via efflux pumps is still incomplete. The current model is that substrates are moved through the transporter component of the pump (e.g. AcrB) via binding to one of two binding pockets. Therefore, EI is proposed to occur either through competitive transport or blocking of a binding pocket. The phenotype of EI can also be conferred by a reduction in amount of the transporter component. Therefore, the mechanism of EI will be investigated via (i) measurement of efflux activity in the presence of known inhibitors of RND efflux pumps and for mutants with defined mutations within the substrate binding pockets, and (ii) transcriptomics and proteomics to determine the level of efflux pump components. This project will use molecular and cellular microbiology, clinical microbiology, in vivo studies and biochemistry.