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Title: EspJ of enteropathogenic and enterohaemorrhagic Escherichia coli inhibits receptor tyrosine phosphorylation
Author: Young, Joanna
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) are enteric pathogens that cause disease through intimate attachment and subversion of intestinal epithelial cells. This strategy depends on a type III secretion system (T3SS) to translocate effector proteins into host cells. EspJ is a T3SS effector protein that inhibits opsono-phagocytosis through an unidentified mechanism. This study aimed to determine the mechanism of action of EspJ through analysis of the protein itself and its effects on eukaryotic cell signalling. Bioinformatic analysis of EspJ revealed a predicted ADP-ribosyltransferase (ART) domain which mediates transfer of ADP-ribose from NAD on to a target protein. Through structural comparison with known ARTs, mutations were designed targeting predicted key ART domain residues. NMR analysis revealed that EspJ binds NAD and also that this interaction was disrupted by ART domain mutations. Mutation of the ART domain also disrupted inhibition of opsono-phagocytosis by EspJ. Further analysis of phagocytosis revealed that EspJ inhibits actin polymerisation and tyrosine phosphorylation during FcγR-mediated phagocytosis. During infection, EPEC and EHEC use the translocated effector Tir to initiate actin polymerisation pathways leading to the formation of actin rich pedestals on cultured cells. TirEPEC signalling is similar to that of the FcγR and so the impact of EspJ on pedestal formation was analysed. EspJ inhibited the tyrosine phosphorylation-dependent TirEPEC-mediated actin pedestal assembly but not the phosphorylation-independent TirEHEC signalling. Inhibition of EPEC pedestals correlated with reduced tyrosine phosphorylation and recruitment of the SH2-adaptor Nck. Importantly, an absence of Src family kinases (SFK) was observed in the presence of EspJ. EspJ inhibited FcγRIIa tyrosine phosphorylation suggesting that this effector acts at the initial stages of tyrosine kinase dependent actin polymerisation pathways preventing receptor phosphorylation by SFKs. Importantly, an intact ART domain was required for EspJ mediated inhibition of tyrosine phosphorylation and downstream actin polymerisation.
Supervisor: Frankel, Gad Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available