Molecular and functional characterisation of the adherent properties of H7 flagella

Enterohaemorrhagic Escherichia coli (EHEC) have recently emerged as significant zoonotic pathogens. O157:H7 is one of the most common EHEC serotypes associated with human disease, which is transmitted faeco-orally from a bovine reservoir. EHEC O157:H7 preferentially colonises the bovine terminal rectum (BTR). Injection of virulence factors by type-III secretion is necessary for colonisation of cattle and results in re-modelling of the host cytoskeleton. Flagella machinery is evolutionarily related to the Type III secretion apparatus and O157 strains lacking H7 flagella show reduced adherence to the BTR. Vaccination with FliC, the main component of H7 flagella, has the potential to protect cattle against E. coli O157:H7 infection. The focus of this work was to investigate the molecular basis for H7 flagella binding to the BTR, in order to understand the basis for FliCH7 being an immuno-protective antigen. H7 flagella were shown to adhere across the surface and penetrate into BTR epithelial cells. Both the FliC shaft and the FliD cap components of flagella filaments showed the capacity to adhere to BTR epithelial cells. Preliminary studies indicate that the current FliCH7 vaccination of cattle results in FliD-specific antibodies where oral challenge with O157:H7 does not. FliD is more conserved than FliCH7, which contains a predicted 88aa structural insertion, but variation occurs along the full length of the FliD protein. There was no evidence for post-translational modification of FliCH7. A number of actin binding proteins were identified as potential FliC and FliD binding partners from BTR epithelial cell lysates. From this, a panel of purified galectin-4, cofilin-1 and βγ-actin was used to compare binding of flagella from different pathogens. H7 flagella bound more to cofilin-1 than βγ-actin, whereas phase-1 and phase-2 flagella from Salmonella Typhimurium bound more to βγ-actin, than to cofilin-1. Size-exclusion chromatography indicated that cofilin-1 alters H7 flagella filament polymerisation dynamics. αβ-ctin polymerisation and depolymerisation experiments indicate that H7, phase-1 and phase-2 flagella interactions with actin affect actin dynamics.