METABOLOMIC CHARACTERIZATION OF THE INTESTINAL BACTERIAL OXALOBIOME By CASEY A. CHAMBERLAIN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2019 © 2019 Casey A. Chamberlain To my wife, Michelle ACKNOWLEDGMENTS Obtaining a PhD may only bestow the title of “Doctor” upon one individual, but the journey to such an achievement is often aided by, and impossible without, the loving support of family, friends, and others along the way. I am thankful for the Lord for His providence and love and for blessing me with the unique interests, talents, and gifts that gave me the opportunity to fulfill this achievement. I am eternally grateful for my wife, Michelle, for providing me with love, support, comfort, and guidance, which propagates confidence, happiness, and fulfillment into all areas of my life. Her guiding and strengthening example has molded me into the man I am today. I am blessed to have my daughter, Maci, who reminds me daily of the purpose of this life and the love the Lord feels for each of His children. I am fortunate to have received love and support from family, both immediate and extended, which helps foster a sense of wholeness in my life. I am uplifted and reinforced by the friends I have made during this season of life, both inside and outside the lab, particularly those whom I have come to know closely on a personal level. I respect, honor, and am indebted to Tim Garrett for providing me the opportunity for this journey. As a mentor, friend, and leader of the highest quality, Tim is kind, patient, intelligent, hard-working, competitive, supportive, empathetic, respectful, and, as a result of all these qualities, successful. His steadfast example as a man who knows himself, his values, and his priorities has taught me much about how to live life in a rewarding, balanced, and fulfilling manner. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF ABBREVIATIONS ........................................................................................... 11 ABSTRACT ................................................................................................................... 15 CHAPTER 1 INTRODUCTION .................................................................................................... 17 Man and the Microbiome ........................................................................................ 17 Oxalate and Oxalate Disease ................................................................................. 19 Oxalate and the Microbiome ................................................................................... 21 Microbial Metabolomics .......................................................................................... 23 Metabolomics of the Oxalobiome ............................................................................ 25 2 METABOLOMIC AND LIPIDOMIC CHARACTERIZATION OF Oxalobacter formigenes STRAINS HC1 AND OxWR ................................................................. 26 Background ............................................................................................................. 27 Materials and Methods............................................................................................ 29 Bacterial Culture, Harvest, and Lysis ............................................................... 29 Metabolite Extraction ........................................................................................ 30 Lipid Extraction ................................................................................................. 31 Analytical Instrumentation and Methodology .................................................... 32 Data Processing and Quality Control ............................................................... 33 Statistical Analysis ............................................................................................ 34 Results and Discussion........................................................................................... 34 Metabolomics Analysis ..................................................................................... 34 Lipidomics Analysis .......................................................................................... 36 Summary ................................................................................................................ 39 3 METABOLOMIC AND LIPIDOMIC CHARACTERIZATION OF OXALOTROPHIC Lactobacillus acidophilus AND Lactobacillus gasseri ................ 59 Background ............................................................................................................. 60 Materials and Methods............................................................................................ 62 Bacterial Culture, Harvest, and Lysis ............................................................... 62 Metabolite Extraction ........................................................................................ 63 Lipid Extraction ................................................................................................. 64 5 Analytical Instrumentation and Methodology .................................................... 65 Data Processing and Quality Control ............................................................... 66 Statistical Analysis ............................................................................................ 67 Determination of Oxalate Degradation by Liquid Scintillation Counting ............ 67 Results and Discussion........................................................................................... 68 Metabolomics Analysis ..................................................................................... 68 Lipidomics Analysis .......................................................................................... 71 Oxalate Degradation by L. acidophilus and L. gasseri ..................................... 74 Summary ................................................................................................................ 74 4 Oxalobacter formigenes PRODUCES BIOCHEMICAL FEATURES UNDETECTED IN OXALOTROPHIC Bifidobacterium animalis .............................. 91 Background ............................................................................................................. 92 Materials and Methods............................................................................................ 93 Bacterial Culture, Harvest, and Lysis ............................................................... 93 Metabolite Extraction ........................................................................................ 94 Analytical Instrumentation and Methodology .................................................... 95 Data Processing and Quality Control ............................................................... 96 Statistical Analysis ............................................................................................ 97 Results .................................................................................................................... 97 Discussion .............................................................................................................. 99 Summary .............................................................................................................. 102 5 Oxalobacter formigenes POTENTIALLY INDUCES METABOLOMIC ALTERATION IN THE INTESTINAL EPITHELIUM .............................................. 109 Background ........................................................................................................... 109 Material and Methods ........................................................................................... 112 Use of Animals ............................................................................................... 112 Gavaging and Tissue Harvest ........................................................................ 112 Determining Colonization Status .................................................................... 113 Pre-Extraction Sample Preparation for Metabolite Extraction......................... 114 Metabolite Extraction ...................................................................................... 114 Analytical Instrumentation and Methodology .................................................. 115 Data Processing and Quality Control ............................................................. 116 Statistical Analysis .......................................................................................... 117 Results and Discussion......................................................................................... 117 Elevated Nicotinic Acid After Gavage with O. formigenes .............................. 117 Assessing the Effect of Gavage with Spent Media ......................................... 119 Elevated Nicotinic Acid Potentially Transcends O. formigenes Colonization .. 120 Summary .............................................................................................................. 122 6 CONCLUSIONS AND FUTURE WORK ..............................................................