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Shiga toxin-producing E. coli (STEC) are a major cause of food-borne illness in the United States and worldwide. Most STEC cases resolve without complication, however approximately 10% progress to severe disease including hemolytic uremic syndrome (HUS). Shiga toxin (Stx), the main virulence factor of STEC, is an AB5 toxin. The enzymatic A-subunit cleaves the 28S rRNA, inhibiting protein synthesis, while the homopentameric B-subunit binds Stx to the cellular receptor, globotriaosylceramide. Stx has two major antigenic forms, Stx1 and Stx2, and minor subtypes Stx1c and d and Stx2a-h. Epidemiologic studies have found that Stx2 subtype a (Stx2a) is associated with more severe disease than Stx1, and other Stx2 subtypes, although closely related to Stx2a, exhibit discrepancies in disease severity. Despite the fact that infection with Stx2a producing STEC are correlated with more severe disease, there is currently no predictive indicator of which cases will progress to HUS. Endothelial cells are suggested to play a role in HUS; therefore, we investigated whether there is a difference in susceptibility of endothelial cells from different vascular beds to Stx1 and Stx2 subtypes that affects progression to severe disease. Human umbilical vein endothelial cells (HUVECs), glomerular microvascular endothelial cells (GMECs) and cerebral cortex microvascular endothelial cells (BMECs) were fairly insensitive (ED50 > 0.2 µg/ml) to metabolic inhibition by Stx1, Stx2a, Stx2b, Stx2c and Stx2d. Human dermal microvascular endothelial cells (dHMECs) were quite sensitive (ED50 = 1.9 x 10-1 µg/ml) to all toxins except Stx2b. Susceptibility to Stx correlated with the ability of the toxins to bind each cell type which was influenced by expression level of the receptor. In addition to affecting the kidney, 20-30% of HUS cases involve central nervous system dysfunction. The role of Stx in leading to neurological complications is not well understood. Stx must either damage brain endothelial cells or pass through the blood brain barrier (BBB) to access susceptible cells. To determine if Stx accesses susceptible cells in the brain by damaging endothelial cells of the BBB, we utilized primary and immortalized cell lines. Neither immortalized microvascular endothelial cells from the cerebral cortex of mice (bEnd.3) nor primary human BMECs were susceptible to Stx2a, suggesting that direct toxicity to endothelial cells is not how Stx weakens the BBB. It is instead likely that inflammation plays a significant role in loss of BBB integrity.In order for Stx to exert toxicity on cells it must bind, undergo endocytosis and retrograde transport to the endoplasmic reticulum and then reach the cytosol. While multiple studies on Stx transport have been reported, most utilize cell lines without direct involvement in human disease. Little is known about trafficking in primary cells pertinent to disease and if it differs from that in cell lines. We used a genome wide siRNA screen to investigate the trafficking pathway of Stx2a in primary renal proximal tubule epithelial cells (RPTECs). The screen both confirmed previously reported cellular components involved in Stx trafficking and identified novel factors. Since there is currently no treatment for HUS, these results provide possible targets for future therapeutics. |
