title: Structural characterisation of spirosomes and its link to Escherichia coli O157:H7 virulence
Creators: name: Azmi, Liyana
Publisher: name: University of Glasgow
Year: 2019
Subjects: subject: AdhE, SAXS, AUC, flagella, type three secretion system.

description: 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 AdhE hindered the solving of the AdhE high-resolution structure. Work was then focused on solving and characterising the individual domains of AdhE. The ab initio structure for the N-terminal alcohol dehydrogenase agrees well with the determined high-resolution structure of the protein. Furthermore, the dummy atom model for both domains of AdhE agrees with the in-solution stoichiometry for both proteins, determined via AUC. Overall, the stoichiometry of the domains in AdhE suggests a mechanism for spirosome oligomerisation. Meanwhile, the altered acetate flux derived from the absence of adhE was proposed to be the main cause for the non-motility and flagellar overexpression phenotype of DadhE. The increased acetate production and selective pressure derived from the non-motility was presumed to have induced mutations in the genome, evidenced via whole-genome sequencing. Particularly, the alanine 46 to valine mutation in FliC was suggested to have changed the flagellar morphology. At the same time, exposures of the bacteria to free acetate which has diffused into the media disrupts the proton motive force needed for flagellar rotation. Correspondingly, the restored motility of DadhE in alkaline media supports this theory. Finally, the bacterial response towards increased acetate production was shown to be strain-specific. Contrastingly to DadhE in EHEC, deletion of adhE in the non-pathogenic Escherichia coli K12 showed non-motility with very low fliC expression. Overall, this work has structurally characterised spirosome assembly and uncovered new prospects to the design of antibiotic alternatives. Moreover, the proposed molecular mechanism underlying the DadhE phenotype provides a better understanding of the mode of action for the design of drugs targeted towards AdhE.