The Type III secretion system 'translocation stop' activity of EspZ

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'Attaching & effacing' (AE) E. coli exert a substantial burden on human and animal health and use Type III secretion to colonise intestinal epithelia and induce pathology. The locus of enterocyte effacement (LEE)-encoded Type III secretion system of such pathogens mediates injection of tens of bacterial effectors into enterocytes. These orchestrate cellular pathways in a manner requiring exquisite temporal and spatial control. Our data indicate that the LEE-encoded effector EspZ is modified upon entry into host cells and is integrated into the host cell plasma membrane where it functions as an inhibitor of further effector translocation. Ectopic expression of EspZ in eukaryotic cells renders them refractory to infection, and cells infected with AE pathogens are resistant to super-infection in an EspZ-dependent manner. Bacteria lacking EspZ cause cell damage, likely owing to accumulation of effectors to toxic levels. Our yeast-2-hybrid analysis has indicated that EspZ may interact with a constituent of the translocation pore (EspD), however the molecular mechanism by which it arrests Type III secretion is not known. We propose to define the kinetics of EspZ translocation, its insertion into the host cell plasma membrane and the onset of 'translocation-stop' activity. We will map the protein-protein interaction sites of EspZ and its partner proteins, and determine the role of the partner proteins and interacting residues in its 'translocation-stop' activity. The electrophoretic mobility of EspZ shifts on entry into cells and we propose to define the nature and consequences of post-translational modification. We will also evaluate the impact of mutation and over-espression of espZ on intestinal persistence of enterohaemorrhagic E. coli in calves and Citrobacter rodentium in mice. Unravelling the mode of action of EspZ will have major implications for the design of novel strategies to control infections by pathogenic E. coli.

Please see Impact Summary and Pathway to Impact submitted by lead applicant (joint ref. M1517309).