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

Lead Research Organisation: University of Edinburgh
Department Name: The Roslin Institute

Abstract

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Technical Summary

'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.

Planned Impact

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

Publications

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Description This was a joint project with Imperial College London to explore the role of the E. coli O157:H7 type III secreted protein EspZ. The Roslin Institute component (lasting 4 months of the 36 at Imperial College) was completed in 2015. The magnitude and duration of faecal excretion of an espZ mutant of E. coli O157:H7 was compared to the parent strain and a complemented derivative following oral inoculation of calves (n=4 per strain). The results indicated modest but statistically significant attenuation of the espZ mutant in cattle, consistent with a role for EspZ in colonisation of the bovine host. Data will be included in a future publication.

Prof. Frankel and I co-authored a comprehensive review on Type III secretion and effector proteins for the second edition of a respected text on E. coli O157 and other Shiga toxin-producing E. coli (reprinted in Microbiology Spectrum).
Exploitation Route Too early to say.
Sectors Agriculture

Food and Drink

Pharmaceuticals and Medical Biotechnology

 
Description Collaboration with Professor Gad Frankel, Imperial College London. 
Organisation Imperial College London
Department Department of Bioengineering
Country United Kingdom 
Sector Academic/University 
PI Contribution This was a joint project, where Prof. Frankel was the lead Principal Investigator. Our component was to evaluate the phenotype of an espZ mutant of E. coli O157 in cattle relative to the isogenic parent strain.
Collaborator Contribution Provision of strains for testing in cattle.
Impact We anticipate that the data arising from our in vivo studies with the E. coli O157 espZ mutant will be included in a future publication.
Start Year 2011
 
Description Public lecture entitled Confronting the Microbial Menace in Our Food'. Professor Mark Stevens 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact The inaugural lecture of Professor Mark Stevens was held on 30 October 2017 on his BBSRC-funded research to identify bacterial and host factors influencing the ability of Salmonella, Campylobacter and E. coli to colonise farm animals and cause disease. It was attended by children from local schools, members of the public, students at The Roslin Institute and wider University of Edinburgh and posted online.
Year(s) Of Engagement Activity 2017,2018
URL https://media.ed.ac.uk/media/Inaugural+lectureA+Confronting+the+microbial+menace+in+our+food/1_x5k5e...