Novel Chemical Probes of Surface-Layer Formation in Pathogenic Bacteria

The proposed research aims to develop novel chemical technologies that will enable us to understand how C. difficile generates its highly-resistant crystalline outer coating, a key mechanism by which it persists in the human body after treatment with broad-spectrum antibiotics.
In the face of increasingly wide-spread antibiotic resistant infection, there is a pressing need to improve our understanding of the basic biology of pathogenic bacteria and then build on this work to find novel mechanisms to inhibit bacterial growth. C. difficile is one of the most lethal hospital infections, with over 3800 deaths recorded in 2005 in the UK alone (c.f. 2000 due to MRSA), and the corresponding drain on resources is a significant burden on the NHS. The information generated by this work may provide a new route to target C. difficile that can circumvent its usual antibiotic resistance. Of equal significance are the proposed advances in chemical technology, which may enable comparable insights into related species (e.g. C. tetani, the bacterium that causes tetanus) and related pathogenic processes (e.g. release and processing of bacterial toxins).

Gram-positive and Gram-negative bacterial pathogens have evolved sophisticated strategies for survival within the harsh environments of their host species. In this struggle, the bacterial cell wall plays an essential role presenting proteins that mediate adhesion to host cells and evasion of the immune response. To this end, certain pathogenic bacteria such as C. difficile produce a proteinaceous coat, the S-layer, surrounding the entire cell that is immunogenic in humans and plays a role in binding to host cells. Synthesis of S-layer in C. difficile involves site-specific proteolytic cleavage of the precursor protein, SlpA, in the bacterial cell wall by an as yet unidentified bacterial protease. Many putative proteases exist in the genome of C. difficile and it is very challenging to devise experiments to test the role of each of these in the cleavage of SlpA. Working with C. difficile presents several difficulties, and many standard genetic tools are not easily applicable to this spore forming anaerobe. Novel chemical approaches that can circumvent these restrictions would therefore offer a tremendous advantage, enabling key experiments that are otherwise very difficult or impossible to perform. This grant will be used to allow Dr. Fagan to ?hop? from Prof. Fairweather?s lab to Dr. Tate?s lab where he will develop chemical Activity-Based Probes (ABPs) that he will then apply to enrich and identify the S-layer processing protease, and to observe its expression and localisation by fluorescence microscopy in live cells. ABPs have never been applied in bacteria, and proteolytic processing during S-layer formation in C. difficile represents an ideal system to develop and apply ABPs in a prokaryotic organism for the first time. Furthermore, ABPs also inhibit the protease, and have additional applications as chemical genetic tools for knocking down protease function. This work will pump-prime projects to clone and express the protease(s) for use in high-throughput assays to identify small-molecule drugs that can target S-layer formation, whilst fluorogenic ABPs will reveal for the first time the localisation and dynamics involved in processing SlpA. Furthermore, this work will provide an important proof-of-principle for the use of ABPs in prokaryotic systems with a wide variety of potential applications in protease microbiology, for example in understanding protease-mediated host-pathogen interactions and bacterial toxin production.