Building and deploying a platform for therapeutic target validation in Staphylococcus aureus

New and effective antibacterial compounds are much needed in the wake of the antibiotic resistance crisis. Many putative antibacterial targets have been uncovered in the post-genomics era, yet there are few tools to validate their therapeutic potential prior to extensive compound screening. In addition, researchers are turning to non-traditional antibacterial strategies, such as antivirulence, in hope of developing novel therapeutics. Virulence inhibition, however, has not been robustly validated as an effective antibacterial approach. The main aim of this research was to develop a platform that facilitates the validation and characterisation of therapeutic targets against bacteria.
The platform is based on a class of artificial non-antibody binding proteins known as Affimers, which bind their target with high affinity and can inhibit protein function. To develop the platform, Affimers that bind the essential cell division protein FtsZ of Staphylococcus aureus were isolated by phage display. Affimer binders were identified that arrested bacterial growth when expressed intracellularly. In vitro biochemical analysis revealed the most potent Affimer was able to inhibit FtsZ GTPase activity, of which is essential for its cellular function. Finally, an insect model of infection was used to assess the therapeutic promise of FtsZ inhibition. Insect survival was greatly improved when bacteria were forced to express the anti-FtsZ Affimer during infection. These results highlight FtsZ as a promising therapeutic candidate and demonstrate the utility of the Affimer-mediated knockdown platform for validating antibacterial targets.
The following chapters sought to deploy the platform in attempt to characterise and validate targets of the antivirulence strategy. Each a cytosolic, surface-associated, and secreted virulence factor of S. aureus was chosen for assessment. The virulence factors selected have been proposed in the literature as strong candidates for the development of antivirulence agents. Using phage display, Affimer binders were successfully isolated against all targets. For each virulence factor, various target-specific biochemical analyses were performed to identify Affimers that inhibited their primary function. Affimers that inhibited virulence factor function in vitro were identified for two of the three candidates. However, Affimer-mediated inhibition of either the intracellular or surface-associated virulence factors in living cells was not accomplished.