Structural characterisation of spirosomes and its link to Escherichia coli O157:H7 virulence

Escherichia coli O157: H7 (EHEC) is a pathogenic strain of E. coli and the causative agent of bloody diarrhoea, severe colitis and haemolytic uremic syndrome. The ability to initiate these diseases is due to the production of Shiga toxin, which is released upon exposure to antibiotic treatment. Hence, supportive care is the only current treatment for EHEC, and highlights the demand for alternative therapeutic solutions. Coincidentally, the rise of antimicrobial-resistant bacteria also creates an urgent need for antibiotic alternatives. During the development of alternative antibiotic treatments, the high-throughput screening of salicylidine acylhydrazides (SA) compounds had previously shown to suppress the expression of the type three secretion system (T3SS) and disable the motility of EHEC. To understand the mode of action of these compounds, their cellular targets were identified. Amongst the many targets that were found and characterised, only the deletion of adhE revealed the downregulation of the T3SS expression and showed overexpression of non-functional flagella. However, the molecular basis underlying this phenotype is unknown. The high-resolution structure of AdhE was needed to gain a better understanding of the binding of SA compounds, which will enable further structure-based drug designs. Thus, this work aimed to answer two main questions: what is the high-resolution structure of AdhE and what is the molecular mechanism for the DadhE phenotype? AdhE is known to form spirosomes which are heterogeneous oligomers in vitro. Therefore, amongst the crucial aims of this project was to disrupt spirosome assembly with the goal of achieving sample homogeneity. A range of conditions was tested for spirosome assembly. Through the use of small-angle X-ray scattering (SAXS) and analytical ultracentrifugation (AUC), the hydrophobic interaction mediated by the residue F670 of AdhE was determined to be important for spirosome formation. Inevitably, the heterogeneity of A