IRTKS promotes Tir membrane insertion for intimate bacterial attachment and subsequent pedestal formation

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that causes bloody diarrhea and hemolytic uremic syndrome by disrupting the intestinal brush border. During infection, EHEC injects the transmembrane virulence factor Tir into enterocytes; upon insertion into the apical membrane, Tir mediates bacterial attachment and drives formation of actin-rich pedestals needed for colonization. How Tir is inserted into the host plasma membrane remains unclear. Here, we investigated the role of brush border resident IRTKS, a Tir- and membrane-binding protein, in this process. Using multiple IRTKS gain- and loss-of-function models, we analyzed pedestal organization and component localization. Whereas canonical models position IRTKS downstream of Tir as a scaffolding link to F-actin, we found that perturbing IRTKS disrupted the distribution and abundance of Tir. Moreover, ectopic IRTKS expression enhanced Tir membrane insertion in the absence of other virulence factors. We conclude that IRTKS functions early in pedestal formation to promote Tir accumulation in the plasma membrane and, in turn, facilitate bacterial attachment.