GBP1 recruitment to actin-rich pedestals induced by extracellular Gram-negative bacteria promotes pyroptosis.

The IFN{gamma}-induced GTPase guanylate binding protein 1 (GBP1) binds to lipopolysaccharide (LPS) on cytosolic Gram-negative bacteria and promotes pyroptosis via the recruitment and activation of caspase-4 on the bacterial outer membrane. Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) are extracellular pathogens that also induce LPS- and caspase-4-dependent pyroptosis. However, whether GBP1 is involved in this process remains unknown. EPEC and EHEC adhere intimately to intestinal epithelial cells via avid interactions between the bacterial adhesin Intimin and Tir (Translocated intimin receptor), a type 3 secretion system effector protein. Intimin-mediated clustering of Tir triggers actin polymerisation, leading to pedestal-like structures at bacterial attachment sites. Here we show that GBP1 is recruited to actin pedestals in human cells infected with EPEC and EHEC in vitro and mouse colonocytes infected with the EPEC-like murine pathogen Citrobacter rodentium in vivo. GBP1-dependent caspase-4 trafficking to these sites leads to pyroptosis and IL-18 release. To dissect the mechanism of GBP1 trafficking, we engineered a chimeric receptor (Fc{gamma}R-Tir) by combining the intracellular signalling domain of Tir and the extracellular ligand-binding domain of the Fc{gamma} receptor. Clustering of Fc{gamma}R-Tir with IgG-coated beads produced sterile actin-rich pedestals that were sufficient to recruit GBP1 independently of bacteria. Our findings reveal that cytosolic GBP1 is mobilised to sites of pathogen-induced actin remodelling independently of LPS. We establish that GBP1 not only operates as a pattern-recognition receptor but also orchestrates effector-triggered immunity against pathogens that hijack the actin cytoskeleton.