Dissecting the role of the Tir:Nck and Tir:IRTKS/IRSp53 signalling pathways in vivo

Abstract

Attaching and effacing (A/E) lesions and actin polymerization, the hallmark of enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR) infections, are dependent on the effector Tir. Phosphorylation of TirEPEC/CR Y474/1 leads to recruitment of Nck and neural Wiskott–Aldrich syndrome protein (N-WASP) and strong actin polymerization in cultured cells. TirEPEC/CR also contains an Asn-Pro-Tyr (NPY454/1) motif, which triggers weak actin polymerization. In EHEC the NPY458 actin polymerization pathway is amplified by TccP/EspFU, which is recruited to Tir via IRSp53 and/or insulin receptor tyrosine kinase substrate (IRTKS). Here we used C. rodentium to investigate the different Tir signalling pathways in vivo. Following infection with wild-type C. rodentium IRTKS, but not IRSp53, was recruited to the bacterial attachment sites. Similar results were seen after infection of human ileal explants with EHEC. Mutating Y471 or Y451 in TirCR abolished recruitment of Nck and IRTKS respectively, but did not affect recruitment of N-WASP or A/E lesion formation. This suggests that despite their crucial role in actin polymerization in cultured cells the Tir:Nck and Tir:IRTKS pathways are not essential for N-WASP recruitment or A/E lesion formation in vivo. Importantly, wild-type C. rodentium out-competed the tir tyrosine mutants during mixed infections. These results uncouple the Tir:Nck and Tir:IRTKS pathways from A/E lesion formation in vivo but assign them an important in vivo role.

Actin Pedestal Formation by Enterohemorrhagic Escherichia coli Enhances Bacterial Host Cell Attachment and Concomitant Type III Translocation

“…We note that these experiments were performed with nonpolarized epithelial cells, with a cellular architecture very different from that of the apical surface of intestinal mucosa, so we cannot eliminate the possibility that Tir-mediated actin assembly might significantly influence the rate of type III translocation per bound bacterium during infection of the intestinal mucosa. EHEC and other A/E pathogens have evolved complex mechanisms to trigger actin pedestal formation, and several studies have associated this activity with enhanced colonization and disease (33,34). Nevertheless, despite considerable investigation, the means by which pedestal formation promotes pathology has been poorly defined.…”

“…For example, an EHEC EspF U -deficient mutant demonstrated a mild intestinal colonization defect in rabbits and generated smaller-than-wild-type bacterial aggregates on the intestinal mucosa of gnotobiotic piglets (33). Similarly, a C. rodentium strain deficient for pedestal formation was outcompeted by a wild-type strain in mixed infections (34). Finally, the ability of A/E pathogens to induce pedestals in cultured cells is correlated with their ability to induce disease in humans and in animal models (31,35).…”

Deficiency of IRTKS as an adaptor of insulin receptor leads to insulin resistance

“…However, whether IRSp53 deficiency disrupts insulin signaling and leads to insulin resistance was not evaluated. Similarly to IRSp53, IRTKS can induce the clustering of short actin bundles, cause membrane protrusions [10] and trigger pathogen-driven actin assembly [11][12][13]. Tyrosine phosphorylation of IRTKS occurred in response to insulin stimulation when ectopic IRTKS and insulin receptor (IR) β-subunit (IRβ) were co-transfected into COS-7 cells [10], which implies that IRTKS could be a component of insulin signaling.…”

Infection strategies of enteric pathogenic Escherichia coli

“…140 Interestingly, both pathways are utilized simultaneously in vitro for most non-O157 EHEC strains, EPEC O119:H6 141 and EPEC lineage 2 strains. 140 Unexpectedly neither pathway appears to be necessary for A/E lesion formation in vivo in C. rodentium infections, in EPEC and EHEC infection of human intestinal in vitro organ cultures (IVOC), 142,143 or for EHEC colonization in the infant rabbits and gnotobiotic piglets models, 144 indicating the molecular mechanisms of A/E formation and their functions are far from understood.…”