Clustering of Tir during enteropathogenic E. coli infection triggers calcium influx–dependent pyroptosis in intestinal epithelial cells

by Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik, Massiel Cepeda-Molero, Luis Ángel Fernández, Avinash R. Shenoy, Chris Bakal, Gad Frankel, Jyoti S. Choudhary

Clustering of the enteropathogenic Escherichia coli (EPEC) type III secretion system (T3SS) effector translocated intimin receptor (Tir) by intimin leads to actin polymerisation and pyroptotic cell death in macrophages. The effect of Tir clustering on the viability of EPEC-infected intestinal epithelial cells (IECs) is unknown. We show that EPEC induces pyroptosis in IECs in a Tir-dependent but actin polymerisation-independent manner, which was enhanced by priming with interferon gamma (IFNγ). Mechanistically, Tir clustering triggers rapid Ca²⁺ influx, which induces lipopolysaccharide (LPS) internalisation, followed by activation of caspase-4 and pyroptosis. Knockdown of caspase-4 or gasdermin D (GSDMD), translocation of NleF, which blocks caspase-4 or chelation of extracellular Ca²⁺, inhibited EPEC-induced cell death. IEC lines with low endogenous abundance of GSDMD were resistant to Tir-induced cell death. Conversely, ATP-induced extracellular Ca²⁺ influx enhanced cell death, which confirmed the key regulatory role of Ca²⁺ in EPEC-induced pyroptosis. We reveal a novel mechanism through which infection with an extracellular pathogen leads to pyroptosis in IECs.