Markus Wendler - Institutionen för kliniska vetenskaper, Lund

Title: Disease mechanisms and treatment strategies in E. coli O157:H7 infection

Main supervisor: Diana Karpman

Reviewers:

Associate professor Fredrik Kahn, Dept of Infection Medicine, Clinical Sciences Lund

Associate professor Manoj Puthia, Dept of Dermatology and Venereology, Clinical Sciences Lund

Abstract

Background

Enterohemorrhagic E. coli (EHEC) bacteria are ingested by oral intake of contaminated food or water. Large foodborne outbreaks of gastrointestinal infections occur worldwide. EHEC strains are associated with gastroenteritis, hemorrhagic colitis, and in the most severe forms with hemolytic uremic syndrome (HUS) occurring more in young children and the elderly. HUS manifests as non–immune hemolytic anemia, thrombocytopenia and acute kidney injury. The most severe cases will lead to death. EHEC are non-invasive bacteria, thus the bacteria do not circulate or cause bacteremia. Virulence factors are secreted in the gut. Shiga toxin is a major virulence factor of EHEC causing extensive cellular injury by intracellular inhibition of protein synthesis. It is released from bacteria in the intestine in free form or within outer membrane vesicles and transported to target organs by blood cells and extracellular vesicles. There is currently no treatment for EHEC infection and antibiotic therapy during the prodromal phase, before HUS develops, may increase the risk of developing HUS due to excess release of toxin.

Research questions

• Do extracellular vesicles carrying Shiga toxin induce disease in vivo and is the disease dependent on the Shiga toxin Gb3 receptor?
• Does EHEC infection induce disease in Gb3-negative mice?
• Is a fusion protein combining human annexin V and apyrase an effective treatment for EHEC infection?
• Can aspects of EHEC infection be reproduced in vitro using a glomerulus on a chip?

Preliminary results

Shiga toxin 2 delivered in extracellular vesicles in a murine model was more potent and caused more death and kidney injury than free toxin. The effects of toxin delivered in vesicles were dependent on the Shiga toxin Gb3 receptor and mice lacking this receptor were protected. Building on those data, a follow-up study will be conducted using an established murine model of EHEC infection in Gb3-negative mice. Possible therapeutics aimed at modifying the onset and progression of EHEC infection are of significant clinical interest. In the third study, we evaluated the efficacy of two such candidates, human apyrase and annexin V, separately and in fusion, using a murine model of EHEC infection. In vivo experiments demonstrated that administration of the apyrase-annexin fusion protein conferred a protective effect against EHEC-induced pathology in mice. A glomerulus on a chip has been developed for future studies of EHEC virulence factors.

Significance

The present project provides a better understanding of the disease mechanisms of EHEC and potential therapeutic targets.

Manuscript to be submitted

Wendler M, Gerogianni A, Hiram Betancourt Nunez L, Önnerfjord P, Arvidsson I, Karpman D. Circulating extracellular vesicles deliver lethal effects of Shiga toxin in a globotriaosylceramide-dependent manner