Role of Shiga toxin versus H7 flagellin in enterohaemorrhagic Escherichia coli signalling of human colon epithelium in vivo

Abstract

SummaryEnterohaemorrhagic Escherichia coli O157:H7 (EHEC) is a clinically important foodborne pathogen that colonizes human colon epithelium and induces acute colonic inflammation, but does not invade the epithelial cells. Whereas Shiga toxin (Stx) and bacterial flagellin have been studied for their ability to upregulate the production of proinflammatory chemokines by cultured human colon cancer cell lines, the relevance of studies in colon cancer cell lines to the production of proinflammatory signals by normal epithelial cells in EHEC-infected human colon is not known. We show herein that Stx does not bind to human colon epithelium in vivo . Moreover, globotriaosylceramide (Gb3/CD77) synthase, the enzyme required for synthesis of the Gb3/CD77 receptor for Stx, was not expressed by normal or inflamed human colon epithelium in vivo . In contrast, Toll-like receptor (TLR) 5, the receptor for bacterial flagellin, was expressed by normal human colon epithelium and by colon epithelium in human intestinal xenografts. EHEC H7 flagellin instilled in the lumen of human colon xenografts that contain an intact human epithelium upregulated the expression of epithelial cell proinflammatory chemokines, which was accompanied by a subepithelial influx of neutrophils. Isogenic mutants of EHEC that lacked flagellin did not significantly upregulate prototypic neutrophil and dendritic cell chemoattractants by model human colon epithelia, irrespective of Stx production. We conclude that EHEC H7 flagellin and not Stx is the major EHEC factor that directly upregulates proinflammatory chemokine production by human colon epithelium in vivo.

Human colorectal tumors and metastases express Gb3 and can be targeted by an intestinal pathogen-based delivery tool

“…Some occasional cells in normal tissue showed STxB labeling. These cells may correspond to the colonic myofibroblasts described by others (15), and this finding could explain, at least in part, that some Gb 3 was detected in normal intestinal tissue, as described above. Tumor cells arranged in glandular structures clearly showed STxB labeling (Fig.…”

“…Several studies showed that human intestinal epithelium (11,15) and purified epithelial cells (12,33,34) were Gb 3 -negative. In agreement with these studies, we found that normal human colonocytes were not labeled by STxB, and in our previous in vivo experiments on mice, STxB did not accumulate in these cells (30).…”

Ligand-induced differential cross-regulation of Toll-like receptors 2, 4 and 5 in intestinal epithelial cells

“…A further complication of the commonly used human polarized intestinal cell lines is that the cellular localization of TLR5 in the various cell types appears to be different. In some cells (such as T84) TLR5 appears to be expressed solely at the basolateral cell surface (Gewirtz et al, 2001), while in other cell lines (such as Caco-2) and in colon xenografts in vivo, TLR5 is also expressed at the apical membrane (Bambou et al, 2004;Cario and Podolsky, 2000;Miyamoto et al, 2006). As under natural conditions intestinal epithelial cells will be most frequently exposed to flagellin from the luminal side, we added flagellin to the apical side of mIC cl2 cells in this study, although we noted similar responses to flagellin when the flagellin was applied from the basolateral side.…”

In Vitro and In Vivo Model Systems for Studying Enteropathogenic Escherichia coli Infections

“…EHEC is able to specifically interact with human colonic xenografts, inducing A/E lesions and tissue damage dependent on a functional T3SS (Golan et al 2011). This type of model has also been used to show that H7 flagellin is a major factor involved in increases of proinflammatory chemokines in response to EHEC infection (Miyamoto et al 2006). An advantage of this model is the ability to study the immunogenic reaction of A/E pathogens in the absence of other microbial products.…”