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

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

doi:10.1371/journal.pbio.3000986

Abstract

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.

Highlights

The extracellular pathogen enteropathogenic Escherichia coli (EPEC) causes persistent infantile diarrhoea [1] and has been found to preferentially colonise colorectal cancer tissue in adult patients [2]
The increased expression of guanylate-binding protein 2 (GBP2), an interferon-induced gene, in SNU-C5 primed with interferon gamma (IFNγ) for 24 h was validated by real-time quantitative PCR (S1A Fig), confirming that SNU-C5 cells respond to IFNγ
We show that similar to macrophages [25], infection of SNU-C5 cells induced propidium iodide (PI) uptake that was dependent on translocated intimin receptor (Tir), with the magnitude of cell death directly correlated with the extent of Tir translocation

Summary

Introduction

The extracellular pathogen enteropathogenic Escherichia coli (EPEC) causes persistent infantile diarrhoea [1] and has been found to preferentially colonise colorectal cancer tissue in adult patients [2]. EPEC infection of the intestinal mucosa is mediated by a type III secretion system (T3SS) [3], a molecular syringe that injects 21 effectors directly into the host cell cytosol where. EPEC Tir triggers calcium influx-dependent pyroptosis the CRUK Centre grant (C309/A25144). "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript"

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Conclusion