Structural studies on actin-ADP ribosylating binary toxin from C. difficile

摘要: Clostridium difficile infection (CDI) is a serious problem within the healthcare environment where bacterium causes symptoms ranging from mild diarrhoea to life-threatening colitis. In addition its principal virulent factors, Toxin A and B, some C. strains produce binary toxin (CDT) composed of two subunits namely CDTa CDTb that are produced secreted cell as separate polypeptides. Once in gut, these fragments have potential combine form potent cytotoxin whose role pathogenesis CDI presently unclear. This thesis step towards understanding structural functional aspects by difficile. The first half this (chapter I II) provides brief introduction method structure determination proteins molecules, i. e. X-ray crystallography detailed overview three known toxins – A, B toxin. Chapter II further focuses on other related toxins. These toxins, ADP-ribosylating (ADPRTs) big family which includes Cholera, Pertussis Diphtheria capable transferring ADP-ribose part NAD/NADPH varity substrates target ultimately results death. second comprises experimental procedures were carried out during course study their results. Cloning expression methods for recombinant bacterial system followed purification described with abnormal behaviour exhibited III). We show time purified can an active CDT cytotoxic Vero cells (Chapter IV). processes yielded milligram quantities high purity led successful crystallisation IV) studies. High resolution crystal structures native (at pH 4.0, 8.5 9.0) complex ADP ribose donors -NAD NADPH been determined V). protein ‘pronounced conformational flexibility’ confined site region ‘enhanced’ disorder at low while highlight significant differences ‘ligand specificity’ compared enzymatic subunit close homologue, perfringens Iota (Ia). data provide information protein-donor substrate stabilisation may implications recognition. Crystallisation preliminary crystals. optimisation conditions underway. concludes thoughts discussion future directions research.