Outer Membrane Vesicles in Shiga Toxin-Mediated Inflammatory and Thrombotic Responses Leading to Systemic Disease

Enterohemorrhagic E. coli (EHEC) serotype O157:H7 is a foodborne diarrheal illness typically transmitted by contaminated beef or produce, resulting in bloody diarrhea. EHEC produce Shiga toxin (Stx), a protein synthesis inhibitor that when absorbed from the intestine into the bloodstream, can lead to the devastating illness, hemolytic uremic syndrome (HUS). HUS is characterized by fibrin-platelet thrombi in the microvasculature and inflammatory damage of affected organs such as the kidney, resulting in renal failure. Systemic exposure to LPS or other PAMPs during the diarrheal phase of infection can potentiate Stx's deleterious effects and is thought to be important in pathogenesis. EHEC are not invasive, so Stx must traverse the intestinal epithelium to enter the systemic circulation. However, free Stx is not commonly found in patients with HUS, and we lack an understanding of how and in what form Stx enters the blood and eventually reaches the kidney. One potential under-explored mechanism is via uptake in the form of Stx-containing outer membrane vesicles (OMV's), EHEC-derived microparticles that can be absorbed from the intestinal lumen into the bloodstream and contain, in addition to Stx, a plethora of PAMPs such as LPS and flagellae, as well as other EHEC-encoded toxins. In this proposal, we will assess how outer membrane vesicles (OMV's) interact with cells of renal origin in the presence of absence of macrophages, inflammatory cells that infiltrate the kidney in animal models of HUS. Because OMV's are endocytosed, LPS sensing occurs intracellularly rather than via TLR4, thus we expect to observe responses that are fundamentally different from those occurring when Stx and LPS are co-delivered exogenously. The ability to generate OMV from EHEC mutants with altered LPS (or other PAMPs or toxins) provide the opportunity to identify key features leading to damaging cellular responses. In addition, it has been hypothesized that a key event in HUS pathogenesis occurs when Stx engages circulating blood cells, resulting in the formation of Stx-containing, prothrombotic microvesicles that then intoxicate distal organs. During EHEC infection, blood components may encounter Stx, LPS, and other potentially damaging bacterially derived virulence factors in the context of OMV's, but little is known about OMV-mediated microvesicle production. We will compare the quantity of microvesicles produced upon exposure of blood cells to purified Stx or Stx-containing OMVs from wild type EHEC or EHEC deficient in PAMPs or virulence factors. OMV-associated cargoes, particularly the prothrombotic/pro-coagulant molecules will be characterized, and for their effect on the renal cells described above. These in vitro studies lay the critical groundwork for future in vivo studies in an animal model of EHEC infection. A fundamental understanding of how and in what form Stx is encountered by the host, as well as how cellular responses are influenced by the mode of Stx delivery, may inform new therapeutic modalities to prevent HUS.