1. Further Insight into the Pathogenesis of Enterovirulent Escherichia coli Jeremy Brandelli and Glen Armstrong Department of Microbiology, Immunology and Infectious Diseases, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada. Abstract Infection with Shiga toxin (Stx) producing bacteria can result in bloody diarrhea, hemorrhagic colitis or hemolytic-uremic syndrome (HUS). Stx is a AB 5 holotoxin and is considered the main virulence factor of enterohemorrhagic E.coli. HUS is associated with decreased renal function and an increase in pro-inflammatory cytokines, indicating the immune response plays a role in disease progression. Many of the cytokines seen at increased levels in HUS patients are produced by macrophages or neutrophils. Macrophage-like THP-1 cells were exposed to various combinations of Stx, StxA or StxB for both Stx1 and Stx2. The cytokine response elicited by these toxins was measured through Luminex multiplex analysis. It was determined that neither StxA or StxB for Stx1 or Stx2 could elicit a cytokine response on its own. This provides evidence for the ribotoxic stress response as the mechanism through which Stx elicits a cytokine response or for the necessity of both subunits for proper binding and signal transduction. Background The enterovirulent E.coli family contains 6 classes, including: enterohemorrhagic and enteropathogenic Shiga toxin producing Escherichia coli infect >250,000 in the US each year, infection can cause hemolytic uremic syndrome (HUS) HUS has a 5-10% mortality rate There is currently no treatment for HUS Antibiotic use in STEC infected individuals is related to a more severe prognosis Individuals with HUS have an increase in pro-inflammatory cytokine levels Cytokine levels have a positive correlation with HUS symptom severity HUS patients have an accumulation of macrophages and neutrophils in their kidneys Attenuation of the immune response in STEC infected individuals may present a novel target for therapeutics Identification of important immune cells and cytokines in the pathogenesis of HUS may present targets for therapeutics Aim: To determine the mechanism that links Stx2 to severe disease and inflammation and to apply this information to the development of novel therapeutics Methods Results Conclusions Acknowledgements We would like to thank the Markin USRP for their funding and support as well as Adom Bondzi-Simpson for his help with THP-1 cell culture. StxA or StxB alone from Stx1 or Stx2 didn’t elicit a cytokine response in macrophage-like THP-1 cells StxR showed similar cytotoxicity to its holotoxin counterpart StxR showed similar immunogenic activity to Stx The subunits were in their native conformation and able to reform into functional holotoxin Neither subunits alone can engage macrophage-like THP-1 cells in a manner that elicits a cytokine response StxA is unable to be endocytosed or trafficked within the cell to elicit a response when not in a complex with StxB Future Directions Future experiments should compare the immunogenic effects of hybrid Stx1 and Stx2 toxins to determine which subunit contributes more to the cytokine response. These experiments should be expanded to include other cell types, including neutrophils and monocytes. Stx Separation Stx was incubated in a 9M urea, 0.15M NaCl and 0.15M propionic acid, pH 4.0 solution for 1 hour on ice. The dissociated toxin was applied to a high performance liquid chromatography machine to separate the subunits. Separated subunits were concentrated and resuspended in isotonic, physiologic pH phosphate buffered saline. Separated subunits were assayed for purity using SDS-PAGE and a Verocytotoxicity to ensure no contaminating holotoxin was present. THP-1 Experiments THP-1 cells (ATCC TIB-202) were cultured in RPMI1640 supplemented with 10% heat-inactivated FBS, 1% sodium pyruvate, 10mM L-glutamine and 5mM β-mercaptoethanol at 37°C, 5% CO 2. Cells were differentied with 100ng/mL phorobol 12-myristate 13-acetate for 24 hours. PMA containing media was aspirated and replaced, cells were rested for 24 hours. Macrophage-like THP-1 cells were exposed to 3.125nmol of Stx or StxA or 15.625nmol of StxB, for both Stx1 and Stx2, for 6 hours. Cell culture supernatant was collected and submitted for cytokine analysis at Eve Technologies Figure 2: 1x10 6 THP-1 cells were seeded in each well of a 24-well plate and differentiated in to macrophage-like cells by exposure to 100ng/mL of PMA for 24 hours at 37 o C, 5% CO 2. Cells were then rested for 24 hours. The media was then aspirated and replaced with a minimum volume of serum-free RPMI 1640 (250μL). Cells were then exposed to 3.125nmol of Stx1, Stx2 or a vehicle control (2μL) PBS for 6 hours. Samples were submitted to Eve Technologies for multiplex analysis of cytokine levels. The values graphed are the mean±standard deviation for six trials performed in duplicate for Stx1, two trials performed in duplicate for Stx1A and Stx1B and one trial performed in duplicate for Stx1R. Cytokine concentration in the Stx-treated samples was divided by the average of the untreated cells to obtain fold change. Statistical analysis was performed using One-way ANOVA.The p-value for the comparison of Stx1 or Stx1R with Stx1A and Stx1B is indicated with: * p<0.0001. Figure 3: 1x10 6 THP-1 cells were seeded in each well of a 24-well plate and differentiated in to macrophage-like cells by exposure to 100ng/mL of PMA for 24 hours at 37 o C, 5% CO 2. Cells were then rested for 24 hours. The media was then aspirated and replaced with a minimum volume of serum-free RPMI 1640 (250μL). Cells were then exposed to 3.125nmol of Stx1, Stx2 or a vehicle control (2μL) PBS for 6 hours. Samples were submitted to Eve Technologies for multiplex analysis of cytokine levels. The values graphed are the mean±standard deviation for six trials performed in duplicate for Stx1, two trials performed in duplicate for Stx1A and Stx1B and one trial performed in duplicate for Stx1R. Cytokine concentration in the Stx-treated samples was divided by the average of the untreated cells to obtain fold change. Statistical analysis was performed using One-way ANOVA.The p-value for the comparison of Stx1 or Stx1R with Stx1A and Stx1B is indicated with: * p<0.0001. Figure 1: The Structure of Shiga toxin. A) Cartoon diagram of Stx, illustrating the 1 A subunit and 5 B subunits. B) Ribbon diagram of Stx. C) Illustration of the furin cleavage site, where StxA is activated. D) Ribbon diagram showing Gb 3 binding sites on StxB Reference: Johannes L and Romer W. Shiga toxins-from cell biology to biomedical applications. Nat Rev Microbiol 2010;8:105-116