Comparative Functional Analysis of Secreted Autotransporter Toxin (Sat) in Uropathogenic E. coli CFT073 and probiotic E.coli Nissle 1917 in mammalian cells A Thesis Presented to the Faculty of California State Polytechnic University, Pomona In Partial Fulfillment Of the Requirements for the Degree Master of Science In Biological Sciences By Taylor Michelle Halsey 2018 SIGNATURE PAGE THESIS: COMPARATIVE FUNCTIONAL ANALYSIS OF SECRETED AUTOTRANSPORTER TOXIN (SAT) IN UROPATHOGENIC E. COLI CFT073 AND PROBIOTIC E. COLI NISSLE 1917 IN MAMMALIAN CELLS AUTHOR: Taylor Michelle Halsey DATE SUBMITTED: Fall 2018 Department of Biological Sciences Dr. Christos Stathopoulos __________________________________________ ___ Thesis Committee Chair Biological Sciences Dr. Nancy Buckley __________________________________________ ___ Biological Sciences Dr. Junjun Liu ___________________________________________ __ Biological Sciences ii Acknowledgements I would like to sincerely thank my entire support system for walking with me through this journey. To my support system at Cal Poly Pomona, a place that I have called a home for the past eight years, I say thank you for your collective belief in my academic process and for giving me an education that will allow for so many opportunities in the future; the faculty and staff at Cal Poly Pomona have provided me with a tremendous gift and I promise to use this gift to continue to help others. To the faculty that have helped me the most, including members of my thesis committee, Drs. Buckley, Liu, and Stathopoulos (thesis committee chair), as well as the coordinators for the NIH-RISE program Drs. Adler and Valdes, thank you all for investing your precious time in me as a student and a researcher; your guidance has shaped my path and I hope to be able to inspire a new generation of scientists just as you all have inspired me. Finally, to my family and friends, you each hold a different piece of my heart and I know I could not have gotten through this program without all of your love and support. Through all of the long days and nights in the lab and all of the times I thought I could not complete this task, it was you that made it easier and allowed me to keep my goals in sight; this great project could have never been completed without all of you. iii Abstract Autotransporter proteins, typically associated with virulence in pathogenic bacteria, are thought to serve a different function in probiotic bacteria. One such protein, secreted autotransporter toxin (Sat), has been shown in numerous pathogenic bacteria to cause vacuolization and cytotoxic effects on cells. Sat is also secreted in high amounts by the non-pathogenic, probiotic Escherichia coli Nissle 1917 (EcN). The use of EcN is under review as a candidate probiotic supplement in the United States and approval has been delayed in part because of genomic similarities to uropathogenic E. coli (UPEC) CFT073, which is responsible for approximately 70% of non-hospital acquired urinary tract infections nationwide. Previous generalized studies have indicated that when Sat functions collaboratively with other native proteins in EcN, its cytotoxic effects are negated, but the mechanisms behind these actions are unknown. To independently study the effects of Sat, we used a recombinant sat overexpression plasmid derived from UPEC CFT073 (pSat) transformed into the non-pathogenic E. coli strain HB101 (HB101 pSat). Concentrated cell-free supernatant was obtained from wildtype EcN and wildtype UPEC CFT073, as well as EcNDSat for assays and an SDS-PAGE and Bradford protein concentration assay were performed to confirm the expression, concentration, and secretion profiles. In vitro infection challenges using the HeLa and Vero cell lines were performed and analyzed using the MTT cytotoxicity assay. Our preliminary results showed that Sat of UPEC CFT073 decreased viability of undifferentiated epithelial cells when subjected to challenges with concentrated cell-free supernatant. When the same challenges were given to Vero cells, a greater level of toxicity was conferred on the differentiated epithelial cell line. Cytotoxic effects in Vero cells were amplified following crude purification of cell- iv free supernatant samples using ammonium sulfate precipitation. These results suggest that Sat of UPEC CFT073 confers damage to host epithelial cells and may have greater effects on relevant differentiated cell lines. It is also suggested that purified Sat protein from both EcN and UPEC CFT073 confer similar levels of cytotoxicity to Vero cells and reduce viability compared to the concentrated supernatant samples alone. Further experiments are needed to determine the mechanisms of action behind these results and target the specific factors that prevent Sat of EcN from displaying cytotoxic features in host cells. v TABLE OF CONTENTS SIGNATURE PAGE .......................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii ABSTRACT ....................................................................................................................... iv INTRODUCTION ............................................................................................................ 1 1. Escherichia coli ............................................................................................................ 1 2. E. coli Nissle 1917 ........................................................................................................ 4 3. Probiotic Bacteria ....................................................................................................... 10 4. Uropathogenic E. coli strain CFT073 ......................................................................... 13 5. Similarities between EcN and UPEC CFT073 ........................................................... 15 6. Bacterial Toxins ......................................................................................................... 16 7. Gram-negative Bacteria Secretion Mechanisms ........................................................ 19 8. Type V (Autotransporter) Protein Secretion .............................................................. 21 9. Adhesin Involved in Diffuse Adherence (AIDA-I) and Trimeric Autotransporter Adhesins (TAA) ......................................................................................................... 28 10. Serine Protease Autotransporters of Enterobactericiaea (SPATE)...……......…........29 11. Secreted Autotransporter Toxin (Sat) ......................................................................... 31 vi 12. In vitro Cell Models .................................................................................................... 35 13. Previous Stathopoulos Lab Findings .......................................................................... 36 14. Thesis Scope and Focus .............................................................................................. 40 MATERIALS AND METHODS ................................................................................... 41 1. Bacterial Strains and Plasmids ................................................................................. 41 a) Bacteria Media Preparation ................................................................................ 41 b) Preparations of Bacterial Overnight Cultures .................................................... 42 c) Preparations of Bacterial Day Cultures .............................................................. 42 2. Bacterial Protein Work ............................................................................................. 42 a) Supernatant Isolation and Concentration ........................................................... 42 b) Isolation and Partial Purification of Secreted Autotransporter Toxin ................ 43 c) Acetone Protein Precipitation ............................................................................. 43 d) SDS-PAGE/Silver Stain Analysis ...................................................................... 44 3. Cell Lines Used in Study .......................................................................................... 47 a) Cell Culture Media Preparation .......................................................................... 47 4. Oligopeptide Cleavage Assay .................................................................................. 48 5. MTT Cytotoxicity Assay .......................................................................................... 48 6. Data Analysis and Statistics ..................................................................................... 49 RESULTS ........................................................................................................................ 50 1. Concentration of Sat protein from select strains using Filtered Centrifugation ....... 50 2. Effects of concentrated Sat of UPEC CFT073 on cell viability of HeLa cell line ... 54 vii 3. Concentrated Sat of UPEC CFT073 decreases cell viability in Vero cell line ........ 60 4. Supernatants of Wildtype EcN and Wildtype UPEC CFT073 and their effects Vero cell line viability in vitro .......................................................................................... 63 5. Partially purified Sat of UPEC CFT073 effects on Vero cell line from recombinant HB101/pSat .............................................................................................................