From screen to target: insights and approaches for the development of anti-virulence compounds

A detailed understanding of host-pathogen interactions provides exciting opportunities to interfere with the infection process. Anti-virulence compounds aim to modulate or pacify pathogenesis by reducing expression of critical virulence determinants. In particular, prevention of attachment by inhibiting adhesion mechanisms has been the subject of intense research. Whilst it has proven relatively straightforward to develop robust screens for potential anti-virulence compounds, understanding their precise mode of action has proven much more challenging. In this review we illustrate this challenge from our own experiences working with the salicylidene acylhydrazide group of compounds. We aim to provide a useful perspective to guide researchers interested in this field and to avoid some of the obvious pitfalls

The structure of an orthorhombic crystal form of a 'forced reduced' thiol peroxidase reveals lattice formation aided by the presence of the affinity tag

Thiol peroxidase (Tpx) is an atypical 2-Cys peroxiredoxin, which has been suggested to be important for cell survival and virulence in Gram-negative pathogens. The structure of a catalytically inactive version of this protein in an orthorhombic crystal form has been determined by molecular replacement. Structural alignments revealed that Tpx is conserved. Analysis of the crystal packing shows that the linker region of the affinity tag is important for formation of the crystal lattice

FolX from Pseudomonas aeruginosa is octameric in both crystal and solution

FolX encodes an epimerase that forms one step of the tetrahydrofolate biosynthetic pathway, which is of interest as it is an established target for important drugs. Here we report the crystal structure of FolX from the bacterial opportunistic pathogen Pseudomonas aeruginosa, as well as a detailed analysis of the protein in solution, using analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS). In combination, these techniques confirm that the protein is an octamer both in the crystal structure, and in solution

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

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

Crystal structures of WrbA, a spurious target of the salicylidene acylhydrazide inhibitors of type III secretion in Gram-negative pathogens, and verification of improved specificity of next-generation compounds

The enterohemorrhagic Escherichia coli pathotype is responsible for severe and dangerous infections in humans. Establishment of the infection requires colonization of the gastro-intestinal tract, which is dependent on the Type III Secretion System. The Type III Secretion System (T3SS) allows attachment of the pathogen to the mammalian host cell and cytoskeletal rearrangements within the host cell. Blocking the functionality of the T3SS is likely to reduce colonization and therefore limit the disease. This route offers an alternative to antibiotics, and problems with the development of antibiotics resistance. Salicylidene acylhydrazides have been shown to have an inhibitory effect on the T3SS in several pathogens. However, the main target of these compounds is still unclear. Past work has identified a number of putative protein targets of these compounds, one of which being WrbA. Whilst WrbA is considered an off-target interaction, this study presents the effect of the salicylidne acylhydrazide compounds on the activity of WrbA, along with crystal structures of WrbA from Yersinia pseudotuberculosis and Salmonella serovar Typhimurium; the latter also containing parts of the compound in the structure. We also present data showing that the original compounds were unstable in acidic conditions, and that later compounds showed improved stability