Heat-Labile Enterotoxin-Induced PERK-CHOP Pathway Activation Causes Intestinal Epithelial Cell Apoptosis.

Xi Lu,Yonghong Xie,Congcong Li,Pengcheng Li,Enqing Fu,Chunmeng Li,Faguang Jin

doi:10.3389/fcimb.2017.00244

Abstract

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea among children and travelers in developing countries, and heat-labile enterotoxin (LT) is one of the most important virulence factors. The pathogenesis of and virulence factors associated with ETEC have been well-characterized; however, the extent to which ETEC damages host cells remains unclear. In this study, we found that LT could induce decreases in intestinal epithelial cell viability and induce apoptosis in a dose- and time- dependent manner in both HCT-8 and Caco-2 cells. We analyzed the expression profiles of apoptosis-related proteins via protein array technology and found that Bax, p-p53(S46), cleaved caspase-3, and TNFRI/TNFRSF1A expression levels were significantly up-regulated in wild-type ETEC- but not in ΔLT ETEC-infected HCT-8 cells. Bax is essential for endoplasmic reticulum (ER) stress-triggered apoptosis, and our RNAi experiments showed that the PERK-eIF2-CHOP pathway and reactive oxygen species (ROS) are also main participants in this process. LT-induced ROS generation was decreased in CHOP-knockdown HCT-8 cells compared to that in control cells. Moreover, pretreatment with the ROS inhibitor NAC down-regulated GRP78, CHOP, Bim, and cleaved caspase-3 expression, resulting in a reduction in the apoptosis rate from 36.2 to 20.3% in LT-treated HCT-8 cells. Furthermore, ROS inhibition also attenuated LT-induced apoptosis in the small intestinal mucosa in the ETEC-inoculation mouse model.

Highlights

Enterotoxigenic Escherichia coli (ETEC) is an important pathogen that causes human and porcine morbidity and mortality (Crossman et al, 2010)
To determine whether labile enterotoxin (LT) has additional pathological effects on intestinal cells in addition to causing electrolyte loss, we investigated the HCT-8 cell line, which is derived from human ileocecal colorectal adenocarcinoma and is frequently used to study both ETEC and Vibrio cholerae
The HCT-8 cells were initially exposed to various concentrations (0–500 ng/ml) of LT for 2–24 h, after which the cytotoxic effects of LT were evaluated by Cell Counting Kit8 (CCK-8) assay (Figure 1A)

Summary

Introduction

Enterotoxigenic Escherichia coli (ETEC) is an important pathogen that causes human and porcine morbidity and mortality (Crossman et al, 2010). ETEC produces several virulence factors, including colonization factors (CFs) that are responsible for facilitating cell adhesion to the host small intestinal epithelium, and heat-stable (ST), and heat-labile enterotoxins (LTs) that induce a net secretory state leading to profuse watery diarrhea. Research on the pathogenic mechanism underlying the effects of the enterotoxin on the host has deepened the understanding of the processes by which the enterotoxin interacts with the host. These studies have shown that LT can subvert innate immune responses by blocking host NF-κB activation (Wang and Hardwidge, 2012) and enhance ETEC adherence by activating the MAPK pathway in intestinal epithelial cells (Johnson et al, 2009; Wang et al, 2012)

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Results

Conclusion