Structural and functional characterisation of the protein targets of the anti-virulence compounds, the salicylidene acylhydrazides

Beckham, Katherine S.H. (2014) Structural and functional characterisation of the protein targets of the anti-virulence compounds, the salicylidene acylhydrazides. PhD thesis, University of Glasgow.

Abstract

Escherichia coli contributes to the commensal microbiota of most mammals by producing vitamins and aiding digestion. However, several strains of E. coli have evolved as pathogens and have become highly adapted for specific niches through the acquisition of pathogenicity islands. E. coli O157:H7 is a commensal bacterium of cattle but if transferred to humans, usually by contaminated food products, it can act as a pathogen. In humans E. coli O157:H7 causes diarrhoea, haemorrhagic colitis and in some cases haemolytic uremic syndrome which can result in death. Clinical treatment of this pathogen is difficult since the antibiotics currently available have been shown to worsen the clinical outcome of infection. Therefore, the discovery of novel anti-bacterial therapies is of high importance.

A novel approach to limit pathogenesis is to target the factors used by bacteria to cause disease - their key virulence factors. In theory, such approaches should only compromise the ability of the pathogen to cause disease, rather than its ability to survive, thereby reducing the selective pressure for the development of resistance mechanisms. The type three secretion system (LEE T3SS) is a key virulence factor for E. coli O157:H7 as it facilitates tight attachment to host cells and the secretion of effector proteins. The importance of this virulence factor for the disease process makes it an attractive target for anti-microbial therapies.

The salicylidene acylhydrazides (SA) are a class of compounds that inhibit the expression of the T3SS of several Gram-negative pathogens. When this study was started, the mode of action of these compounds was completely unknown. An affinity pull-down assay to identify the binding proteins of the SA compounds was conducted. Identification of the target proteins of the compounds was the first step in determining their mechanism for decreasing the expression of the LEE T3SS in E. coli O157. The pull-down identified nineteen putative targets, none of which had previously been linked to the regulation of the LEE T3SS.

The aim of this thesis was to systematically investigate the putative targets using a combination of approaches: phenotypic studies of deletion mutants and structural and functional characterisation of the target proteins. From the nineteen putative targets seven were selected for further investigation namely Tpx, WbrA, FolX, FkpA, FklB, SurA and AdhE. Several of these proteins were shown to bind to the SA compounds however deletion of only one of the target proteins resulted in a decrease in LEE T3S. This target, AdhE, offers an exciting new lead in the search for novel targets for antibacterial therapies.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: salicylidene acylhydrazides, type three secretion system, E. coli O157, anti-virulence
• Subjects: Q Science > QR Microbiology; Q Science > QR Microbiology > QR180 Immunology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Bacteriology
• Supervisor's Name: Roe, Dr. Andrew J. and Byron, Dr. Olwyn
• Date of Award: 2014
• Depositing User: Miss Katherine S H Beckham
• Unique ID: glathesis:2014-5154
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 16 May 2014 13:33
• Last Modified: 16 May 2014 15:23
• URI: https://theses.gla.ac.uk/id/eprint/5154