AB5 toxins are produced by pathogenic bacteria and consist of enzymatic A subunits that corrupt essential eukaryotic cell functions, and pentameric B subunits that mediate uptake into the target cell. AB5 toxins include the Shiga, cholera and pertussis toxins and a recently discovered fourth family, subtilase cytotoxin, which is produced by certain Shiga toxigenic strains of Escherichia coli. Here we show that the extreme cytotoxicity of this toxin for eukaryotic cells is due to a specific single-site cleavage of the essential endoplasmic reticulum chaperone BiP/GRP78. The A subunit is a subtilase-like serine protease; structural studies revealed an unusually deep active-site cleft, which accounts for its exquisite substrate specificity. A single amino-acid substitution in the BiP target site prevented cleavage, and co-expression of this resistant protein protected transfected cells against the toxin. BiP is a master regulator of endoplasmic reticulum function, and its cleavage by subtilase cytotoxin represents a previously unknown trigger for cell death.

Enterohemorrhagic Escherichia coli (EHEC) is a group of food-borne pathogens that can cause diarrhea, colitis, and the hemolytic uremic syndrome (HUS). The importance of several of the proposed EHEC virulence factors lacks experimental verification in animal models. The limitations of current animal models led us to reexamine the infant rabbit model for the study of EHEC pathogenicity. Here, we report that intragastric inoculation of a Shiga toxin 2 (Stx2)-producing E. coli O157:H7 clinical isolate into infant rabbits led to severe diarrhea and intestinal inflammation but no signs of HUS. We constructed a set of isogenic derivatives of this isolate with deletions in several putative virulence genes, including stx 2 , eae, tir, and ehxA, to investigate the contribution of individual virulence factors to EHEC pathogenicity. stx 2 increased the severity and duration of EHEC-induced diarrhea. Furthermore, although stx 2 had no role in EHEC intestinal colonization nor was it required for EHEC-induced inflammation, stx 2 altered how the host responded to EHEC infection by promoting heterophilic infiltration of the colonic epithelium and lamina propria. Intragastric inoculation of purified Stx2 also induced inflammation and diarrhea in this model. Diarrhea and intestinal inflammation were also dependent on EHEC colonization, as EHEC derivatives with deletions in eae and tir did not colonize, form attaching and effacing lesions, or develop clinical signs of disease. Our studies indicate that infant rabbits are a useful model for investigation of the intestinal stage of EHEC pathogenesis and suggest that Shiga toxin may play a critical role in causing diarrhea and inflammation in patients infected with EHEC.

Summary Background Safety of individual probiotic strains approved under Investigational New Drug (IND) policies in cirrhosis with minimal hepatic encephalopathy (MHE) is not clear. Aim The primary aim of this phase I study was to evaluate the safety, tolerability of probiotic Lactobacillus GG (LGG) compared to placebo while secondary ones were to explore its mechanism of action using cognitive, microbiome, metabolome and endotoxin analysis in MHE patients. Methods Cirrhotic patients with MHE patients were randomized 1:1 into LGG or placebo BID after being prescribed a standard diet and multi-vitamin regimen and were followed for 8 weeks. Serum, urine and stool samples were collected at baseline and study-end. Safety was assessed at weeks 4 and 8. Endotoxin and systemic inflammation, microbiome using multi-tagged pyrosequencing, serum/urine metabolome were analyzed between groups using correlation networks. Results 30 MHE patients (14 LGG and 16 placebo) completed the study without any differences in serious adverse events. However, self-limited diarrhea was more frequent in LGG patients. A standard diet was maintained and LGG batches were comparable throughout. Only in the LGG-randomized group, endotoxemia and TNF-α decreased, microbiome changed (reduced Enterobacteriaceae and increased Clostridiales Incertae Sedis XIV and Lachnospiraceae relative abundance) with changes in metabolite/microbiome correlations pertaining to amino acid, vitamin and secondary BA metabolism. No change in cognition was found. Conclusions In this phase I study, LGG is safe and well-tolerated in cirrhosis and is associated with a reduction in endotoxemia and dysbiosis.

Shiga toxins made by Shiga toxin-producing