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.