The type III secretion system effector EspO of enterohaemorrhagic Escherichia coli inhibits apoptosis through an interaction with HAX-1.

Sharanya Chatterjee,Zuzanna Kozik,Ewa A Grzybowska,Nicolas Constantinou,Sujinna Lekmeechai,Gad Frankel,Cedric N Berger,Abigail Clements,Jyoti S Choudhary

doi:10.1111/cmi.13366

Abstract

Many enteric pathogens employ a type III secretion system (T3SS) to translocate effector proteins directly into the host cell cytoplasm, where they subvert signalling pathways of the intestinal epithelium. Here, we report that the anti-apoptotic regulator HS1-associated protein X1 (HAX-1) is an interaction partner of the T3SS effectors EspO of enterohaemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium, OspE of Shigella flexneri and Osp1STYM of Salmonella enterica serovar Typhimurium. EspO, OspE and Osp1STYM have previously been reported to interact with the focal adhesions protein integrin linked kinase (ILK). We found that EspO localizes both to the focal adhesions (ILK localisation) and mitochondria (HAX-1 localisation), and that increased expression of HAX-1 leads to enhanced mitochondrial localisation of EspO. Ectopic expression of EspO, OspE and Osp1STYM protects cells from apoptosis induced by staurosporine and tunicamycin. Depleting cells of HAX-1 indicates that the anti-apoptotic activity of EspO is HAX-1 dependent. Both HAX-1 and ILK were further confirmed as EspO1-interacting proteins during infection using T3SS-delivered EspO1. Using cell detachment as a proxy for cell death we confirmed that T3SS-delivered EspO1 could inhibit cell death induced during EPEC infection, to a similar extent as the anti-apoptotic effector NleH, or treatment with the pan caspase inhibitor z-VAD. In contrast, in cells lacking HAX-1, EspO1 was no longer able to protect against cell detachment, while NleH1 and z-VAD maintained their protective activity. Therefore, during both infection and ectopic expression EspO protects cells from cell death by interacting with HAX-1. These results suggest that despite the differences between EHEC, C. rodentium, Shigella and S. typhimurium infections, hijacking HAX-1 anti-apoptotic signalling is a common strategy to maintain the viability of infected cells.

Highlights

Shiga toxigenic Escherichia coli (STEC, aka VTEC) strains, those that carry the locus of enterocyte effacement (LEE) pathogenicity island and belong to serotype O157:H7, cause diarrhoea, haemorrhagic colitis and haemolytic uremic syndrome (HUS)
Escherichia coli (EHEC) strains colonise the human and animal intestinal mucosa via attaching/effacing (A/E) lesions. This infection strategy is shared by two other A/E pathogens, enteropathogenic E. coli (EPEC), which is an important cause of infant diarrhoea in low-income countries (reviewed in (Chen & Frankel, 2005; Garmendia, Frankel, & Crepin, 2005) and the related mouse pathogen Citrobacter rodentium (CR; reviewed in Mundy, MacDonald, Dougan, Frankel, & Wiles, 2005; Collins et al, 2014)
EHEC O157:H7 strains encode both espO1 and espO2. This suggests that the EspO effector family is an important T3SS virulence factor amongst pathogenic E. coli as well as Shigella and S. typhimurium

Summary

| INTRODUCTION

Shiga toxigenic Escherichia coli (STEC, aka VTEC) strains, those that carry the locus of enterocyte effacement (LEE) pathogenicity island and belong to serotype O157:H7 (enterohaemorrhagic E. coli—EHEC), cause diarrhoea, haemorrhagic colitis and haemolytic uremic syndrome (HUS). EHEC strains colonise the human and animal intestinal mucosa via attaching/effacing (A/E) lesions (reviewed in Frankel et al, 1998) This infection strategy is shared by two other A/E pathogens, enteropathogenic E. coli (EPEC), which is an important cause of infant diarrhoea in low-income countries (reviewed in (Chen & Frankel, 2005; Garmendia, Frankel, & Crepin, 2005) and the related mouse pathogen Citrobacter rodentium (CR; reviewed in Mundy, MacDonald, Dougan, Frankel, & Wiles, 2005; Collins et al, 2014). Several T3SS effectors expressed by A/E pathogens have been shown to induce apoptosis (EspH and EspF) Infection with these pathogens does not lead to cell death as they employ a number of T3SS effectors to inhibit both intrinsic and extrinsic apoptosis (Wong Fok Lung et al, 2014). We report a new role for the EspO effector family, which is to inhibit intrinsic apoptosis via its interaction with the antiapoptotic protein HAX-1

| RESULTS

| DISCUSSION

Findings

| EXPERIMENTAL PROCEDURE