Characterization of cellular pathways and potency of Shiga toxin on endothelial cells A dissertation submitted to the Division of Graduate Studies and Research of the University of Cincinnati In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY (Ph.D.) In the Department of Molecular Genetics, Biochemistry, & Microbiology of the College of Medicine 2015 Kayleigh A. MacMaster B.S., Nazareth College of Rochester, 2008 Committee Chair: Alison A. Weiss, Ph.D. Abstract 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 ii 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. iii iv Acknowledgements First and foremost, I would like to acknowledge my advisor, Dr. Alison Weiss, for her guidance and support during my dissertation, and for her encouragement during my scientific development. Second, I would like to acknowledge the members of my dissertation committee, Dr. Bill Miller, Dr. Jay Degen, Dr. Andrew Herr and Dr. Jerry Lingrel, who have provided helpful and constructive discussion and feedback throughout my dissertation. I would also like to recognize members of the Weiss lab who have given guidance, support and input over the years including Dr. Christine Pellino, Dr. Cynthia Fuller, Dr. Scott Millen, Dr. Marsha Gaston, Dr. Sayali Karve, Dr. Suman Pradhan, Crystal Davis and Charles Talbott. Finally, I would like to thank my family and friends for their constant support and encouragement to persevere. I would especially like to thank my parents for their incredible support in everything I choose to pursue. v Table of Contents Abstract…………………………………………………………………………………………..ii Acknowledgements……………………………………………………………………………....v Table of Contents………………………………………………………………………………..vi Figures and Tables………………………………………………………………………………ix Abbreviations…………………………………………………………………………………...xii Chapter I. Introduction: Shiga toxin Review……………………...…………………………..1 I. Shiga toxin-producing Escherichia coli and Shiga toxin background 2 Shiga toxin-producing E. coli and O157:H7 2 Transmission of O157:H7 and progression of Shiga toxin disease 2 Shiga toxin 3 Genetics and regulation of Stx 7 Stx receptor and membrane aspects 7 Endocytosis and retrograde transport of Stx 10 II. Subtypes of Shiga toxin 12 Stx subtypes 12 Interaction of subtypes with receptor 13 III. Role for Shiga toxin in disease 14 Stx and HUS 14 Stx subtypes and progression to severe disease 15 Stx toxicity to endothelial cells and a role for inflammation 18 Effect of Stx on gene expression 20 Contributions of B-subunit signaling on vascular response in HUS 21 vi Animal models 22 IV. Scope of this dissertation 24 Chapter II. Potency of Stx Variants on Endothelial Cells of Different Origins……………25 Abstract 26 Introduction 27 Materials and Methods 30 Results 34 Toxicity of Stx subtypes to primary endothelial cells 34 Inhibition of protein synthesis by Stx subtypes 42 Toxin binding to primary endothelial cells 44 Gb3 content of endothelial cells 44 Discussion 47 Chapter III. Susceptibility of Brain Microvascular Endothelial Cells to Stx2a…………...53 Abstract 54 Introduction 55 Materials and Methods 57 Results 59 Toxicity of Stx2a to cerebral microvascular endothelial cells 59 Gb3 expression on cerebral cortex endothelial cells 63 Discussion 65 vii Chapter IV. siRNA Screen to Identify Novel Components of the Cell Utilized by Shiga Toxin…………………………………………………………………………………………….71 Abstract 72 Introduction 73 Materials and Methods 75 Results and Discussion 78 Transcriptional analysis of RPTECs 78 siRNA screen 78 Pathway and gene ontology enrichment of top candidate hits 82 Stx receptor expression - Glycolipid biosynthesis 82 Cell-surface signaling and endocytosis 83 Intracellular trafficking of Stx 87 Genes associated with damage due to catalytic glycosidase activity of Shiga toxin 92 Intracellular signaling: Inflammation and apoptosis 94 Summary 97 Chapter V. Conclusions and Future Directions……………………………………………...99 Conclusions 100 Future Directions 101 References……………………………………………………………………………………...106 viii Figures Chapter I. Figure 1.1. Stx1 and Stx2a holotoxin crystal structures. 5 Figure 1.2. Surface representation and sequence alignment for Stx1 and Stx2a. 6 Figure 1.3 Intracellular trafficking of Stx. 12 Chapter II. Figure 2.1. Sequence alignments and structural comparison of Stx1 and Stx2 subtypes. 29 Figure 2.2. Potency of Stx to immortalized CDC.HMEC-1 dermal microvascular 35 endothelial cells. Figure 2.3. Inhibition of metabolic activity by Stx in primary endothelial cells. 38 Figure 2.4. Upregulation of surface ICAM-1 on HUVECs following stimulation with TNF-α. 41 Figure 2.5. Inhibition of protein synthesis by Stx in primary endothelial cells. 43 Chapter III. Figure 3.1. Metabolic activity of Stx2a-treated cerebral cortex microvascular endothelial 61 cells. Figure 3.2. Upregulation of surface ICAM-1 on BMECs following stimulation with TNF-α. 62 Chapter IV. Figure 4.1. Transfection reagent INTERFERin® does not affect transcription of primary 80 RPTECs. Figure 4.2. Top candidate hits for glycolipid biosynthesis. 85 Figure 4.3. Top candidate hits for cell surface signaling and endocytosis. 86 Figure 4.4. Top candidate hits for intracellular trafficking. 91 ix Figure 4.5. Top candidate hits for damage due to catalytic glycosidase activity. 93 Figure 4.6. Top candidate hits for intracellular signaling. 96 x Tables Chapter II. Table 2.1. Plating densities for endothelial cells. 31 Table 2.2. ED50 values (µg/ml) of Stx1 and Stx2 subtypes for endothelial cells. 40 Table 2.3. Stx1 binding to primary endothelial cells. 45 Table 2.4. Stx2 subtype binding to primary endothelial cells. 45 Table 2.5. Cell surface Gb3 on endothelial cells. 46 Chapter III. Table 3.1. Plating densities for brain endothelial cells. 58 Table 3.2. Cell-surface Gb3 on cerebral cortex microvascular endothelial cells. 64 Chapter IV. Table 4.1. Top candidate hits for siRNA screen in primary renal proximal tubule 81 epithelial cells. xi Abbreviations APC allophycocyanin BBB blood brain barrier BEI Biodefense and Emerging Infectious Diseases Research Resources Repository bEnd.3 murine cerebral cortex microvascular endothelial cells BMEC human cerebral cortex microvascular endothelial cells CFU colony forming units CNS central nervous system CO2 carbon dioxide CXCR4 chemokine (C-X-C) motif receptor 4 CXCR7 chemokine (C-X-C) motif receptor