Subversion of Rho GTPases by WxxxE effectors of attaching and effacing pathogens

Abstract

Enteropathogenic E. coli (EPEC), Enterohaemorhagic E. coli (EHEC) and Citrobacter rodentium are constituent members of the attaching and effacing (A/E) pathogens. The A/E group of bacteria are considered to be extracellular pathogens which form characteristic lesions by intimately adhering to host enterocytes and directing the effacement intestinal brush border. EPEC and EHEC are diarrhoeal pathogens, which are a global health burden in developing and industrialised countries respectively. Citrobacter rodentium is a murine pathogen which is an excellent animal model for EPEC and EHEC infection. EPEC, EHEC and C. rodentium conserve a genomic region termed the locus of enterocyte effacement (LEE) which encodes a type 3 secretion system (T3SS), a core set of type 3 secreted effector proteins and the outer membrane adhesin intimin, which are essential for A/E lesion formation. A/E pathogens utilise their T3SSs to translocate dozens of effector proteins directly from the bacteria into host cells. Once translocated these effector proteins modulate a range of eukaryotic signalling pathways including those which regulate the host cell cytoskeleton. An example of this is the T3SS effector Tir which localises to the mammalian plasma membrane, acts as a receptor for intimin and subsequently directs the polymerisation of actin rich pedestals beneath adherent bacteria. Subversion of the eukaryotic cytoskeleton is a strategy employed by a range of bacterial pathogens. Due to the pivotal role of Rho GTPases in regulating actin dynamics they are commonly targeted by bacterial virulence factors. Recently a family of type 3 secreted effector proteins has been defined based on their homology around an invariant tryptophan and glutamic acid residue separated by three variable amino acids (WxxxE). In this study we have identified the EspM family of proteins and EspT as novel WxxxE effectors in the A/E pathogens. We demonstrate that the EspM proteins and EspT are translocated into host cells in a T3SS dependent manner. Once translocated, EspM proteins direct the nucleation of actin stress fibres, while EspT drives the formation of lamellipodia and membrane ruffles. Furthermore, we found that the cytoskeletal re-arrangements associated with the WxxxE proteins are dependent on the activation of the small Rho GTPases. EspM proteins activate RhoA while EspT induces the activity of both Rac1 and Cdc42. A more detailed structural and function analysis of the mechanism by which EspM2 activates RhoA revealed that EspM2 binds RhoA in a concentration dependent manner and subsequently promotes RhoA nucleotide exchange from a GDP to GTP bound form. We also show that EspM2 adopts a structure similar to that of the previously reported Salmonella T3SS Rho GEF SopE despite their limited sequence homology. Although the catalytic domain of SopE was not conserved in EspM2 we identified a novel loop which is essential for EspM2 RhoA GEF activity. As expression of EspT resulted in the formation of membrane ruffles which are often associated with the invasion of bacterial pathogens we investigated whether EspT dependent cytoskeletal remodelling could facilitate the internalisation of the canonically non-invasive A/E pathogens. Interestingly, we found that EPEC strains expressing EspT were significantly more invasive than those which did not and furthermore that this invasion was dependent upon the activity of Rac1 and Wave2. Additionally, we demonstrate that once internalised EPEC is maintained within a vacuole (ECV) and is capable of surviving and replicating intracellularly. We also report that EPEC translocates Tir into the vacuolar membrane where it can nucleate actin in an analogous manner to the formation of pedestals by extracellular bacteria. This is the first time an intracellular bacterial pathogen has been shown to polymerise actin tails across a vacuolar membrane. Together the results presented in this study demonstrate that the EspM and EspT families of WxxxE effector proteins are potent modulators of eukaryotic GTPase signalling cascades and as a result convey novel virulence attributes to the A/E pathogen group.