Shiga toxin activity in human intestinal organoids

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 DESCRIPTION (provided by applicant): Shiga toxin (Stx) is the primary virulence factor of Stx producing Eschrichia coli (STEC), a leading cause of food-borne illnesses. Annually, STEC, including E. coli O157:H7, cause over 100,000 cases of infectious watery and bloody diarrhea in the U.S. About 10% of the infected progress to the life-threatening kidney disorder, hemolytic uremic syndrome (HUS). The symptoms of HUS can be replicated in primates treated with purified Stx. In HUS, platelet thrombus formation in the microvasculature compromises blood flow to the kidney, and hemolytic anemia develops when red blood cells are mechanically sheared as they squeeze through the occluded vessels. For reasons that are not clear, young children are more likely to develop HUS than adults, and it is the most common cause of acute kidney failure in children. Currently, there is no treatment for the systemic manifestations of HUS. In addition, antibiotics induce STEC to produce higher quantities of Stx, and antibiotic treatment is correlated with more severe disease outcome. A major barrier in the field is the lack of tractable mammalian models that replicate human disease, and as a consequence little is known about the actions of Stx protein toxin or the STEC pathogens on healthy, differentiated human intestinal tissues. We have developed an exciting new experimental model to study the effects of Stx and STEC on the human intestinal tract. Stem cell 'induced human intestinal organoids' (iHIOs) faithfully represent differentiated human intestinal tissue. Our preliminary studies show human intestinal organoids are sensitive to Stx injected into the lumen. Furthermore, while harmless E. coli can replicate in the lumen without causing damage, pathogenic E. coli O157:H7 destroy the intestinal epithelial barrier. This proposal will use human intestinal organoids as a novel model system to study the pathogenic processes associated with Stx and STEC. We will compare infection by harmless E. coli and pathogenic O157:H7 strains, with or without the ability to produce Stx. Aim 1 will focus on the host parameters of disease and Aim 2 will focus on the relative contribution of the bacterial virulence factors: Aim 1. What is the host response to bacterial infection and Stx expression? Aim 2. What is the relative contribution of Stx versus the pathogenic O157:H7 phenotype in mediating intestinal damage?