Novel in vivo therapeutic approaches to Escherichia coli O157:H7 infection

Sammanfattning: Shiga toxin (Stx), the unique virulence factor released by enterohemorrhagic Escherichia coli (EHEC), associated with gastrointestinal infection and in severe cases hemolytic uremic syndrome (HUS). Up until now, no effective therapies have been developed to control disease progression. In this thesis, four novel treatment strategies have been investigated targeting different aspects of EHEC pathogenesis. An established mouse model was used in which mice were infected with EHEC intragastrically. During EHEC infection, extracellular vesicles (EVs) are involved in the transport of Stx from the gut to the kidney. High levels of prothrombotic EVs that expose phosphatidylserine and tissue factor have been detected in patients with EHEC-associated HUS.In the first study, treatment with annexin A5 (anxA5) induced an increase in phagocytic uptake of EVs in vitro. Administration of anxA5 to EHEC-infected mice resulted in lower levels of circulating platelet-derived EVs and delayed disease development.In the second study, Stx triggered adenosine triphosphate (ATP) release from HeLa cells and similar results were obtained in a mouse model. ATP signals via purinergic receptors. Inhibition of purinergic P2X receptors with NF449 or suramin inhibited Stx-induced calcium influx and EV release. NF449 protected cells from Stx-induced toxicity and suramin decreased EV release even in vivo in mice.The third study targeted extracellular ATP by using apyrase in EHEC-infected mice. Apyrase cleaves extracellular ATP and adenosine diphosphate (ADP). Mice were injected with apyrase intraperitoneally. Treatment protected the mouse intestines from damage and delayed the onset of disease symptoms. Apyrase decreased bacterial release of Stx2 by reducing RecA involved in the SOS response and bacteriophage activation. In addition, apyrase lowered platelet aggregation when platelets were co-incubated with Stx2 and E. coli O157:H7 lipopolysaccharide in the presence of collagen. Thus, apyrase had a dual protective effect on both the bacterial release of toxin and host cell activation and injury.The fourth study focused on the effect of immunoglobulin G (IgG) on EHEC infection. Intraperitoneal administration of murine IgG to EHEC-infected mice had a clear beneficial effect on bacterial colonization, survival and intestinal and renal pathology. In vitro studies utilized both mouse and human IgG and showed that the Fc domain bound to the EHEC virulence factor, EspP, E. coli secreted serine protease. EspP is involved in bacterial adherence to the intestine, and intestinal injury during EHEC infection. It has potent enzymatic activity that was inhibited by the interaction with IgG. This indicates that the protective effects of IgG administration in EHEC-infected mice could be due to the interaction between IgG and EspP.In summary, this thesis investigated novel treatment strategies targeting different aspects of EHEC pathogenesis such as bacterial colonization, release of Stx from EHEC, phagocytic clearance of Stx-containing and prothrombotic EVs in the circulation and protection against the cytotoxic and prothrombotic effects of Stx using both in vitro and in vivo experimental set-ups. The results show that treatments such as anxA5, apyrase and IgG, when administered at an early stage of infection, may have therapeutic potential in EHEC infection.