EPEC-induced activation of the Ca²⁺ transporter TRPV2 leads to pyroptotic cell death

The enteropathogenic Escherichia coli (EPEC) type III secretion system effector Tir, which mediates intimate bacterial attachment to epithelial cells, also triggers Ca²⁺ influx followed by LPS entry and caspase-4-dependent pyroptosis, which could be antagonized by the effector NleF. Here we reveal the mechanism by which EPEC induces Ca²⁺ influx. We show that in the intestinal epithelial cell line SNU-C5, Tir activates the mechano/osmosensitive cation channel TRPV2 which triggers extracellular Ca²⁺ influx. Tir-induced Ca²⁺ influx could be blocked by siRNA silencing of TRPV2, pre-treatment with the TRPV2 inhibitor SET2 or by growing cells in low osmolality medium. Pharmacological activation of TRPV2 in the absence of Tir failed to initiate caspase-4-dependent cell death, confirming the necessity of Tir. Consistent with the model implicating activation on translocation of TRPV2 from the ER to plasma membrane, inhibition of protein trafficking by either brefeldin A or the effector NleA prevented TRPV2 activation and cell death. While infection with EPECΔnleA triggered pyroptotic cell death, this could be prevented by NleF. Taken together this study shows that while integration of Tir into the plasma membrane activates TRPV2, EPEC uses NleA to inhibit TRPV2 trafficking and NleF to inhibit caspase-4 and pyroptosis.