Understanding the mechanism of action of E coli 0157:H7 virulence inhibitors at unprecedented resolution
Professor Olwyn Byron (School of Infection and Immunity)
There is an urgent need to develop new strategies to combat infectious disease against the background of the antimicrobial resistant (AMR) infection crisis. Our work in this area is focused on the development of new chemical entities which interfere with bacterial virulence. In contrast with traditional antibiotics, that kill or inhibit growth, anti-virulence (AV) compounds have no effect on bacterial viability, a trait that reduces the selective pressure to develop resistance. We have had a long-standing interest in understanding the mechanism of action (MOA) of a group of promising AV compounds, the salicylidene acylhydrazides (SA) which bind to AdhE, a bi-functional acetaldehyde-CoA dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) involved in central metabolism. Unusually, AdhE oligomerises in vivo and in vitro to form long (15-120 nm) filaments (called spirosomes) heterogeneous in size, that can be visualised (Fig. 1) using negative stain electron microscopy (EM). The aim of this project is to test by EM the effect on AdhE spirosomes of four different SA compounds to better understand their MOA and to decide which is the best candidate for further development and for more detailed analysis via cryo-EM.
The main project will be divided into two objectives:
1. AdhE spirosome purification and fractionation
AdhE protein from Enterohemorrhagic Escherichia coli (EHEC), fused to a histidine tag (His6-AdhE), will be purified by affinity chromatography and fractionated by size using size exclusion chromatography (SEC). The range of spirosome lengths in each SEC fraction will be determined by analytical ultracentrifugation (AUC) sedimentation velocity (SV) analysis.
2. What is the effect of the SA compounds on AdhE spirosomes?
After completion of the objective 1, we will have fractions containing big or small spirosomes, as corroborated by AUC-SV. As a control, big and small spirosomes will be visualised by negative stain EM. Then, both big and small spirosomes will be incubated with four different SA compounds at 37°C for different incubation times and the effect of the compounds will be analysed by negative stain EM.