Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.528730
Title: Exploitation of NHERF proteins by diarrheagenic Escherichia coli
Author: Martinez, Eric
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2010
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Abstract:
Enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherichia coli cause severe disease with symptoms ranging from mild diarrhoea to haemolytic uremic syndrome. During infection of the gut, these bacteria provoke attaching and effacing lesions (A/E lesions), characterised by intimate bacterial attachment and effacement of microvilli from the surface of enterocytes. A type III secretion system (T3SS), encoded by the locus of enterocyte effacement (LEE), allows EPEC and EHEC to inject more than twenty effector proteins into the host cell cytoplasm and to manipulate a large number of cellular processes. The non-LEE encoded effector proteins EspI and NleH1 are involved in inhibition of vesicular transport and apoptosis, respectively. The effector protein Map induces filopodia formation by activating the Rho GTPase Cdc42. Persistence of filopodia on the host cell surface depends on the interaction of Map with Na+/H+ Exchanger Regulatory Factor 1 (NHERF1) and activation of RhoA. NHERF proteins are PDZ domain-containing proteins involved in addressing, recycling and regulating numerous proteins such as ion channels in epithelial cells. Using a PDZ proteomic array and biochemical interaction assays, we confirmed that Map and EspI interact with both NHERF1 and NHERF2 through their C-terminal PDZ binding motifs. In addition, we identified NleH1 as a new binding partner of NHERF2. Employing a newly generated HeLa-NHERF2 stable cell line, we found that overexpression of NHERF2 in HeLa cells affected distribution and function of these three effectors: the transit of EspI from the bacterial attachment site to the Golgi apparatus was accelerated; Map-dependent filopodia during EPEC infection were stabilised and NleH1-mediated inhibition of apoptosis was reduced. The investigation of NHERF recruitment by diarrhoeagenic E. coli showed that EPEC, EHEC and Citrobacter rodentium, the mouse model for EPEC and EHEC, recruited NHERF1 and NHERF2 at the site of bacterial adherence. We revealed that both EPEC and EHEC could recruit NHERF2 by a mechanism which required the T3SS translocated intimin receptor (Tir), but did not involve Tir-induced actin polymerisation pathways. Furthermore, EPEC recruited NHERF2 by a T3SS-independent mechanism which, in contrast to the T3SS-dependent pathway, relied on the NHERF2 ezrin-binding domain (EBD). This work demonstrates that NHERF proteins are recruited by several diarrhoeagenic Escherichia coli and are able to modulate function of at least three effector proteins from A/E pathogens. The data strongly points to a model in which NHERFs act as central hubs regulating the temporal and spatial localisation and function of effector proteins in the host cell and indicates that they might play an essential role in the process of intestinal epithelium colonisation by these pathogens.
Supervisor: Frankel, Gad Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.528730  DOI: Not available
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