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Title: Subversion of the host cell by enteropathogenic Escherichia coli through effector interplay
Author: Wong, Alexander Ray Chong
ISNI:       0000 0004 2723 9652
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2012
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Enteropathogenic Escherichia coli (EPEC) strains are diarrheagenic pathogens that colonize the gut mucosa via attaching-and-effacing (A/E) lesion formation. EPEC utilize a type III secretion system (T3SS) to translocate effector proteins, including Tir and EspH that subvert host cell signalling to sustain colonization and multiplication. In vitro, EPEC induces the formation of an actin-rich ‘pseudopod’-like structure, termed pedestals. My research focussed on EspH, which is implicated in the elongation of actin pedestals, and inactivation of Rho GTPase signalling. In the first study, I showed that EspH promotes Tir-dependent actin assembly at the bacterial attachment sites independently of the Tir tyrosine residues Y454 and Y474, which are implicated in actin pedestal formation. Moreover, EspH promotes the recruitment of N-WASP and the Arp2/3 complex to the bacterial attachment sites. EspH-mediated N-WASP recruitment is dependent on the N-WASP binding protein WIP. Expression of WIP and EspH during EPEC infection induces extensive actin cytoskeletal rearrangements at the bacterial attachment site, forming a protrusive actin patch that is associated with EPEC microcolonies, termed “macro-pedestals”. This study implicates the critical role of WIP in EPEC-mediated actin polymerization, and represents the first instance whereby N-WASP is efficiently activated at the EPEC attachment sites independently of the canonical Tir:Nck and Tir:IRTKS/IRSp53 pathways. In my second study, I investigated the interplay of EspH, a eukaryotic RhoGEF inhibitor, and T3SS bacterial RhoGEFs during EPEC infection. EspH does not inhibit the E. coli RhoGEFs Map, EspT, and EspM, as well as the Salmonella RhoGEF SopE. Expression of EspH induces cell rounding, detachment, caspase-3 activation and death. Importantly, EPEC EspT and EspM2, and Salmonella SopE are able to block EspH-induced cell detachment and caspase-3 activation. In addition, the bacterial RhoGEFs blocked staurosporine-induced caspase-3 activation, and can functionally compensate for the anti-apoptotic T3SS effector NleH. Therefore, this study expands the role of bacterial RhoGEFs to include cell adhesion and survival, and demonstrates that A/E pathogens silence eukaryotic DH-PH RhoGEFs while translocating their own RhoGEFs to hijack Rho GTPase signalling for the exclusive benefit of the pathogen. Highlighted in the two studies is also the concept of effector interplay, where T3SS effectors function in concert with other effectors during the subversion of host cell signaling pathways. Understanding the functional interactions between effectors enables us to gain further insight into the sophisticated nature of their biological relevance beyond that of their biochemical functions.
Supervisor: Frankel, Gad Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral