Clostridium difficile is a gram positive, anaerobic bacterium that is the leading cause of antibiotic-associated pseudomembranous colitis worldwide. C. difficile is an extremely infectious bacterium that produces spores that are highly resistant to standard disinfectant agents and can survive on surfaces for long periods of time. Both the resistance of the spores combined with multiple patients with low-immune systems has lead to an increase in hospital-acquired C. difficile infection, which has had a severe economic impact on the healthcare system. Due to the emerging antibiotic resistance problems and the common occurrence of patient relapse using the current drugs of choice, alternative therapeutic avenues are being explored. C.difficile produces two potent exotoxins; Toxin A and Toxin B that are the causative agents of infection. These toxins have multi-modular domain organisations, with each domain playing a role in cytotoxicity. Some of these domains have been characterised structurally using X-ray crystallography. In this thesis, the low resolution SAXS structure of Toxin A will be presented along with the advances made towards determining the X-ray crystallographic structure of the full-length Toxin A.
In addition to Toxins A and B, some strains of C. difficile produce a binary toxin, CDT, which is made up of two individually produced components, CDTa and CDTb. The CDTa component is the enzymatically active component, whereas CDTb is the transport component, directly involved in translocating CDTa into target cells. The precise role of CDT in pathogenesis is unclear, however there is evidence that CDT ADP-ribosylates monomeric actin in target cells, but the detailed mechanism by which this reaction takes place is unknown. Here site directed mutagenesis of key residues of the active site of CDTa was performed and the effect of these mutations on the enzyme’s cytotoxicity tested. By separately mutating three active site residues, the cytotoxic effect of CDTa can be completely eradicated, details of which will be discussed in this thesis. Additionally, the progress made towards determining the X-ray crystallographic structure of the transport component, CDTb, will be discussed.
|Date of Award||1 Jul 2014|
|Supervisor||Ravi Acharya (Supervisor)|