Inhibition of water absorption and selective damage to human colonic mucosa induced by Shiga toxin-2 are enhanced by Escherichia coli O157:H7 infection

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Abstract

Shiga toxin-producing Escherichia coli (STEC) strains are responsible for a variety of clinical syndromes including bloody and non-bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Although multiple serotypes of STEC have been isolated from hemorrhagic colitis cases, E. coli O157:H7 is by far the most prevalent serotype associated with HUS. Shiga toxin is the major virulence factor of E. coli O157:H7 and is responsible for the more severe symptoms of the infection. However, the mechanisms involved in the pathogenesis of diarrhea mediated by Stx2 are not well known. In this study, we have determined the effects of E. coli O157:H7 strain 125/99 wild type (wt) on the human colonic mucosa mounted in an Ussing chamber. In response to 125/99wt, an inhibition of water absorption across human colonic mucosa was observed. Histological sections showed severe necrosis with detachment of the surface epithelium, mononuclear inflammatory infiltrate and loss of goblet cells after 1 h of incubation with 125/99wt. These alterations were not observed with the isogenic mutant strain lacking stx2 or with the filter-sterilized culture supernatant from the 125/99wt strain. These results indicate that the cell damages in human colon are induced by Stx2, and that Stx2 production is increased by the interaction with bacterial cells. Identification of host cell-derived factors responsible for increasing Stx2 can lead to new strategies for modulating STEC infections.

Introduction

Shiga toxin-producing Escherichia coli (STEC) strains are responsible for a variety of clinical syndromes including bloody and non-bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) (Karmali et al., 1985). HUS develops in 5–10% of children several days after bloody diarrhea and is a systemic disease characterized by thrombotic microangiopathy, hemolytic anemia, thrombocytopenia, and acute renal failure (Gianantonio et al., 1964, Boyce et al., 1995). Although multiple serotypes of STEC have been isolated from hemorrhagic colitis cases, E. coli O157:H7 is by far the most prevalent serotype associated with HUS. Shiga toxin (Stx), an AB5 toxin, is the major virulence factor of E. coli O157:H7 and is responsible for the more severe symptoms of the infection. The A subunit (StxA) possesses N-glycosidase activity against 28S rRNA of 60S ribosomes in the cytosol, resulting in inhibition of protein synthesis in eukaryotic cells and activation of proinflammatory signaling cascade referred to as the ribotoxic stress response (Smith et al., 2003). In fact, it has been demonstrated that Stx induces both primary response genes c-jun and c-fos and activates the stress activated protein kinases, JNK/SAPK and p38, in intestinal epithelial cells. Stx enzymatic activity is also required for the referred kinase activation. The five B subunits (StxB) form a pentamer that binds to globotriaosyl ceramide (Gb3) receptors on the cell membrane (Thompson et al., 2000). E. coli O157:H7 can produce two antigenically distinct forms of Stx proteins (Stx1 and Stx2) and their variants, being Stx2 more virulent and epidemiologically more relevant than Stx1 (Palermo et al., 2009).

The genes stxAB are located in the genome of prophages that resemble the coliphage lambda (Neely and Friedman, 1998). Phage induction is critical to Stx gene expression and to the ability of STEC to cause disease (Tyler et al., 2013). The toxin genes are late-stage genes that are transcribed only during the lytic stage of the phage. The production of Stx is linked to the replication cycle of Stx phages, and the release of Stx is dependent on the lytic phase (Herold et al., 2004), which is induced under stress conditions (Zhang et al., 2000, Wagner et al., 2001). It has been proposed that differential Stx2 expression may account for differences in virulence. In this regards, STEC have been classified in pathotypes and clades according to Stx-phage type that determines Stx variant and level of Stx-production (Karmali, 2009). In particular, Stx2 overexpression is common to STEC strains from clade 8, which are highly associated with HUS (Neupane et al., 2011). While several groups have analyzed factors and conditions in the human gastrointestinal tract able to induce Stx2-phage in E. coli O157:H7 and the corresponding increase in Stx2 production (Bansal et al., 2012) others have recently shown the existence of putative eukaryotic promoter-like sequences located upstream of the genes encoding for the Stx2A and B subunits, and an eukaryotic machinery able to recognize it (Bentancor et al., 2013a) This property gives to the mammalian cells the capacity to transcribe and translate stx2 genes e.g. after uptake of Stx2 encoding phages released after bacterial lysis, adding a putative alternative source of Stx production (Bentancor et al., 2013b).

In this study we examine the physiological and morphological effects of E. coli O157:H7 expressing Stx2 on human colonic mucosa. Our results show that the interaction of E. coli O157:H7 strain 125/99wt with the human colonic mucosa caused a significant inhibition of water absorption and histological damages on the surface epithelium. These alterations were not observed with the stx2 mutant strain or with the filter-sterilized culture supernatant from the 125/99wt strain. These results indicate that the cell damages in human colon are induced by Stx2, and that Stx2 production is increased by the interaction with bacterial cells.

Section snippets

Bacterial strains and growth conditions

Properties of E. coli strains used in this study are listed in Table 1. E. coli O157:H7 strain 125/99 wild type (125/99wt) isolated from a patient with HUS has been previously described (Rivas et al., 2006b). The strain 125/99wt carries the stx2 EDL sx2 variant and the Stx encoding phage is inserted in the yehV gene (Amigo et al. unpublished results). A mutant lacking stx2 gene from the parenteral E. coli O157:H7 strain 125/99wt (125/99Δstx2) was made for the present study. A commensal

E. coli O157:H7 strain 125/99 inhibits the normal water absorption across human colonic mucosa

Under basal conditions, an absorptive Jw (0.16 ± 0.02 μl/min cm2, n = 21) was observed in the human colonic mucosa placed between two identical Ringer solutions in the Ussing chamber. The electrical parameters tested simultaneously with the net water flux showed a PD of 2.3 ± 0.3 mV, Isc of 27.4 ± 2.9 μA/cm2 and Rt of 91.7 ± 11.4 Ω cm2.

To establish the relative contribution of bacterial cells to the cytotoxic effect of Stx2, matched colonic mucosa obtained from the same patient were incubated with 200 μl

Discussion

It is generally accepted that water absorption in the human colon is associated with ionic active transport across colonic epithelium and that the resulting diarrhea is an imbalance of absorption and secretion of ions and solutes, followed by the movement of water in an attempt to restore the appropriate ion concentrations (Kunzelmann and Mall, 2002). Previous reports of our laboratory have shown that Stx2 alters the normal water absorption across human colon and causes irreversible

Acknowledgments

This work was supported by grants to C Ibarra from the Universidad de Buenos Aires (UBACYT M095), the National Research Council of Argentina (CONICET-PIP344) and the National Agency for Promotion of Science and Technology (ANPCYT- PICT 12-0777 and PICT 10-0118).

The authors thank to Roxane M.F. Piazza from Bacteriology Laboratory, Butantan Institute, São Paulo, SP, Brazil for kindly providing the monoclonal antibody 2E11 directed against the A-subunit of Stx2 and Marisa Almuzara for providing

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