In Vitro Induction of Apoptosis in Human Lymphocytes By Stx1 From E. Coli O157:H7

Background: Shiga toxin 1 (Stx1), produced by Escherichia coli O157:H7 and related strains, is a potent cytotoxin implicated in severe clinical conditions such as hemolytic uremic syndrome (HUS), acute kidney injury, and chronic urinary tract infections. While its mechanisms of protein synthesis inhibition and apoptosis induction are well characterized in epithelial and endothelial cells, its effects on immune cells particularly human lymphocytes remain underexplored. This study aimed to investigate the apoptotic response of human peripheral blood lymphocytes to Stx1 exposure in vitro to better understand the immunomodulatory potential of the toxin. Lymphocytes were isolated from the peripheral blood of healthy donors using density gradient centrifugation. Cells were treated with 5 µL of Stx1 toxin at three concentrations (0.25 ng/mL, 0.5 ng/mL, and 1 ng/mL) and incubated with 5 µL of acridine orange/ethidium bromide staining solution at four-time intervals (0 h, 4 h, 12 h, and 24 h). A control group of untreated cells was included at each time point. Apoptotic changes were assessed via fluorescence microscopy based on nuclear staining patterns. A substantial increase in apoptosis was observed in Stx1-treated lymphocytes compared to controls. The effect was both dose- and time-dependent, with the highest apoptotic rate noted at 1 ng/mL after 24 h. Control groups showed minimal baseline apoptosis. Stx1 exerts a strong pro-apoptotic effect on human lymphocytes in vitro, suggesting a potential role in immune suppression and pathogenesis during E. coli O157:H7 infections. These findings underscore the importance of considering lymphocyte impairment in the clinical course of HUS and related conditions. KEYWORDS Biosynthesis, caspase, centrifugation, clinical, protein, time