Endocytosis, Cytotoxicity, and Translocation of Shiga Toxin-2 Are Stimulated by Infection of Human Intestinal (HCT-8) Monolayers With an Hypervirulent E. coli O157:H7 Lacking stx2 Gene.

Nicolás Garimano,Cristina Ibarra,María Marta Amaral

doi:10.3389/fcimb.2019.00396

Abstract

Shiga toxin-producing Escherichia coli (STEC) strains are responsible for multiple clinical syndromes, including hemolytic uremic syndrome (HUS). E. coli O157:H7 is the most prevalent serotype associated with HUS and produces a variety of virulence factors being Stx2 the responsible of the most HUS severe cases. After intestinal colonization by STEC, Stx2 is released into the intestinal lumen, translocated to the circulatory system and then binds to its receptor, globotriaosylceramide (Gb3), in target cells. Thus, Stx2 passage through the colonic epithelial barrier is a key step in order to produce disease, being its mechanisms still poorly understood. We have previously reported that STEC interaction with the human colonic mucosa enhanced Stx2 production. In the present work, we have demonstrated that infection with O157:H7Δstx2, a mutant unable to produce Stx2, enhanced either Stx2 cytotoxicity on an intestinal cell line (HCT-8), or translocation across HCT-8 monolayers. Moreover, we found that translocation was enhanced by both paracellular and transcellular pathways. Using specific endocytosis inhibitors, we have further demonstrated that the main mechanisms implicated on Stx2 endocytosis and translocation, either when O157:H7Δstx2 was present or not, were Gb3-dependent, but dynamin-independent. On the other hand, dynamin dependent endocytosis and macropinocytosis became more relevant only when O157:H7Δstx2 infection was present. Overall, this study highlights the effects of STEC infection on the intestinal epithelial cell host and the mechanisms underlying Stx2 endocytosis, cytotoxic activity and translocation, in the aim of finding new tools toward a therapeutic approach.

Highlights

Shiga toxin-producing Escherichia coli (STEC) strains are responsible for multiple clinical syndromes including bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) (Karmali et al, 1985)
To examine if Stx2 cytotoxicity could be modulated by O157:H7 strain 125/99 wild type (O157):H7 stx2 infection, cell viability was measured on HCT-8 cells incubated with either 100 ng/ml Stx2 alone or in presence of 4 × 108 colonyforming units per ml (CFU/ml) O157:H7 stx2 (O157:H7 stx2+Stx2) and compared with HCT-8 cells incubated with O157:H7 stx2 alone
We demonstrate that infection of human colonic epithelial (HCT-8) monolayers by O157:H7 stx2 impacts Stx2 endocytosis, cytotoxic action, and translocation across intestinal epithelial monolayers

Summary

Introduction

Shiga toxin-producing Escherichia coli (STEC) strains are responsible for multiple clinical syndromes including bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) (Karmali et al, 1985). The genes encoded in the LEE are responsible for intimate adhesion of STEC to colonic epithelial cells (McWilliams and Torres, 2014), which is followed by injection of bacterial effector proteins into the host cell through a type III secretion system (T3SS) (Jerse et al, 1990) These effector proteins produce attaching and effacing (A/E) lesions on intestinal cells and interfere with host cells in many ways, inducing a profound rearrangement of cell cytoskeleton, and a loss of tight junction and membrane integrity (Knutton et al, 1989; Holmes et al, 2010; Ugalde-Silva et al, 2016). This activity results in an inhibition of protein synthesis in eukaryotic cells and activation of a proinflammatory signaling cascade known as the ribotoxic stress response, which is involved in apoptosis induction (Smith et al, 2003)

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