Clostridium difficile toxin B induces autophagic cell death in colonocytes.

Hung Chan,Thomas N.Y Kwong,Raphael C.Y Chan,Jeffery Ho,Maggie H.T Wang,Xiaodong Liu,William K.K Wu,Lin Zhang,Wai T Wong,Sunny H Wong,Yuanyuan Tian,Czarina C.H Leung,Shan Zhao,Matthew T.V Chan,Sidney S.B Yu,Gary Tse

doi:10.1111/jcmm.13555

Abstract

Toxin B (TcdB) is a major pathogenic factor of Clostridum difficile. However, the mechanism by which TcdB exerts its cytotoxic action in host cells is still not completely known. Herein, we report for the first time that TcdB induced autophagic cell death in cultured human colonocytes. The induction of autophagy was demonstrated by the increased levels of LC3‐II, formation of LC3+ autophagosomes, accumulation of acidic vesicular organelles and reduced levels of the autophagic substrate p62/SQSTM1. TcdB‐induced autophagy was also accompanied by the repression of phosphoinositide 3‐kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) complex 1 activity. Functionally, pharmacological inhibition of autophagy by wortmannin or chloroquine or knockdown of autophagy‐related genes Beclin 1, Atg5 and Atg7 attenuated TcdB‐induced cell death in colonocytes. Genetic ablation of Atg5, a gene required for autophagosome formation, also mitigated the cytotoxic effect of TcdB. In conclusion, our study demonstrated that autophagy serves as a pro‐death mechanism mediating the cytotoxic action of TcdB in colonocytes. This discovery suggested that blockade of autophagy might be a novel therapeutic strategy for C. difficile infection.

Highlights

Clostridium difficile is an anaerobic, Gram-positive, sporulating bacterium that gives rise to a spectrum of gastrointestinal diseases, including antibiotic-associated diarrhoea, pseudomembranous colitis and toxic megacolon.[1]
The pathogenicity of C. difficile has been attributed to the 2 major exotoxins, namely Toxin A (TcdA) and Toxin B (TcdB), encoded by its genome
To further elucidate the mechanism by TcdB-mediated mechanistic target of rapamycin (mTOR) inhibition, we studied the potential involvement of the Phosphatase and tensin homologue (PTEN)-phosphoinositide 3-kinase (PI3K)-Akt pathway, which is an important signalling cascade upstream of mTOR in regulating cell survival, cell growth and initiation of autophagy

Summary

| INTRODUCTION

Clostridium difficile is an anaerobic, Gram-positive, sporulating bacterium that gives rise to a spectrum of gastrointestinal diseases, including antibiotic-associated diarrhoea, pseudomembranous colitis and toxic megacolon.[1]. The pathogenicity of C. difficile has been attributed to the 2 major exotoxins, namely Toxin A (TcdA) and Toxin B (TcdB), encoded by its genome Both exotoxins can glycosylate and thereby inhibiting small GTPases of host cells to (i) mediate cytotoxicity; (ii) disrupt actin cytoskeletons and tight junctions and impair the epithelial barrier function; and (iii) promote the release of inflammatory mediators, such as interleukin-8 and macrophage inflammatory protein 2. To this end, TcdB is 10 times more potent than TcdA in mediating the pathogenicity.[5].

| MATERIAL AND METHODS

| RESULTS

| DISCUSSION