METABOLIC AND RESPIRATORY PATHWAYS CONTROLLING VIBRIO CHOLERAE COLONIZATION By Andrew John Van Alst A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Microbiology and Molecular Genetics – Doctor of Philosophy 2021 ABSTRACT METABOLIC AND RESPIRATORY PATHWAYS CONTROLLING VIBRIO CHOLERAE COLONIZATION By Andrew John Van Alst Vibrio cholerae is an enteric pathogen of the human small intestine that proliferates to high cell density during human infection. Although not typically classified as a virulence factor, metabolism is a cornerstone for fitness in the host environment. In this work, I explore the essential role of aerobic metabolism, including oxidative respiration, for successful colonization of V. cholerae in the infant mouse model. Oxidative respiration is the most efficient energy generating metabolic pathway in living organisms and supports the rapid proliferation of V. cholerae in the small intestinal environment. Despite knowledge that oxygen diffuses from the host epithelium into the gut lumen, the role of oxygen in supporting colonization and proliferation of V. cholerae had not been explored prior to the work presented here in Chapters 2 and 3. In Chapter 2, by targeting the pyruvate dehydrogenase (PDH) complex, an enzyme required to convert pyruvate to acetyl-CoA under aerobic conditions, I show that aerobic metabolism through the PDH complex is required for population expansion in the infant mouse. As the gut was predominantly considered anaerobic and exists in a state of low oxygen tension, I also examined the contribution of anaerobic metabolism to infant mouse colonization. By targeting cognate pyruvate formate-lyase (PFL) that similarly converts pyruvate to acetyl-CoA, but only under anaerobic conditions, I determined that anaerobic respiration is dispensable for colonization. In Chapter 3, I directly test the importance of aerobic and anaerobic respiration by targeting the complete set of terminal oxidases and terminal reductases encoded by V. cholerae. Using a modified Multiplex Genome Editing by Natural Transformation (MuGENT) approach, I generated strains denoted Aero7 and Ana4. Aero7 is a functionally strict anaerobe derivative of V. cholerae, lacking all four terminal oxidases (cbb3, bd-I, bd-II, and bd-III), whereas Ana4 lacked functionality in each of the four terminal reductase complexes (fumarate, trimethylamine-N-oxide, nitrate, and biotin sulfoxide reductases). Disruption in the oxidase complexes in strain Aero7 severely attenuated V. cholerae colonization in the infant mouse, however, no attenuation was observed for Ana4. These data supported our findings in Chapter 2 that aerobic, but not anaerobic metabolism was critical for V. cholerae growth in the infant mouse. Furthermore, I determined that the bd-I oxidase, and to a lesser extent the cbb3 oxidase, support oxidative respiration during infection with bd-II and bd-III oxidases being dispensable for colonization. In summation, aerobic metabolism through the PDH complex and the terminal reduction of oxygen by the bd-I oxidase are essential to V. cholerae colonization of the infant mouse. Through this work, I uncovered a role for oxidative metabolism for V. cholerae colonization. These findings expand our knowledge of V. cholerae biology and pathogenicity in the gastrointestinal tract and implicate oxygen as a critical electron acceptor that shapes the progression of enteric infections. To my family: My mother Julie, my father Ed, my sisters Kassie and Lizzie, my grandparents, aunts, uncles, and cousins. iv ACKNOWLEDGEMENTS First and foremost I would like to thank Victor DiRita, Ph.D. for providing me the opportunity to carry out research in his lab and for supporting my growth as a researcher and an individual as member of his lab family. Through every step, Vic was provided unwavering support and guidance, and for that I will be forever grateful. I would also like to thank my guidance committee, Christopher Waters, Ph.D., Shannon Manning, Ph.D., and Lee Kroos, Ph.D. The path to a Ph.D. is hardly ever linear and I am thankful for their guidance through the ups, downs, and turns that my project had taken to reach the finish line. It is not uncommon to pick up tendencies of those we hold in high esteem and I am thankful I was able to experience the kindness and joy each of these researchers brought to their labs each and every day. This has without a doubt imprinted upon me the type of mentor I wish to be one day. To all the members of the DiRita lab family, both past and present, for making the lab a home. First off, I would like to thank Rhia who was there at the start and now at the end of my journey. Rhia was a stalwart of stability throughout my Ph.D. and a reliable source of amazing baked goods I am sure to miss. Jeremiah Johnson, for dealing with me as a green, aspiring, microbiologist and giving guidance in the early stages of my project. Natalia Martin, for her passion and imparting on me the importance of saying “yes” to the things we care about and “no” when we can no longer give what we care most about our all. Ritam Sinha, for showing me that it is okay to slow down, take your time, and that life is not always a race. Lucas Demey, for his seemingly endless excitement and curiosity for science and for his contribution of the TcpA Westerns in Chapter 3 that v were his bread and butter. Beth Ottosen, for her like-mindedness both in kindness and introversion, keeping me sane, especially through the pandemic, for her help in pig mucin extraction, and for her willingness to get food whenever the situation called for it. I’d also like to thank my undergraduate mentee, Shaun Dunyak, who stuck it out through a trial period of my lab mentoring allowing me to develop as a mentor. Lastly from the lab, I also want to thank all the other undergraduates and visiting students who made the lab a fun place to be. I would also like to thank the Michigan State University Microbiology and Molecular Genetics Department. Working alongside such amazing people was an absolute pleasure during my time at MSU. The folks at the MSU Meat Lab were also wonderful people I had the pleasure of getting to know on mucus collection days, trading baked goods for pig intestines was always the bargain. I also appreciate the kindness from the folks at the MSU Histology Research Core, helping me despite my absolute chasm of histology research knowledge. To all of my friends who have been immeasurably important to my success – Thank you! This journey would not have been possible and not nearly as fun without you. Lastly, I want to thank my family. My parents Ed and Julie Van Alst who have always supported me in anything I have done. Who knew I would finally be leaving MSU nearly 9 years after you helped me move in that first day of undergrad. My sisters Kassie and Lizzie, for always being there and bringing joy to my life. And to my grandparents who vi have always looked upon my successes with bright eyes and wonder. The love and support I have had throughout my life is what made this Ph.D. possible. vii TABLE OF CONTENTS LIST OF TABLES ............................................................................................................xi LIST OF FIGURES ......................................................................................................... xii KEY TO ABBREVIATIONS ............................................................................................xv Chapter 1 – Introduction – Oxygen and Enteric Bacterial Infections ............................... 1 1.1 – Abstract .............................................................................................................. 2 1.2 – Introduction ......................................................................................................... 2 1.3 – Oxygen During Homeostasis .............................................................................. 4 1.3.1 – Maintaining Oxygen Levels in the Intestinal Tract ........................................ 4 1.3.2 – Microbe-Host Metabolic Crosstalk and Oxygen Regulation ......................... 6 1.4 – Oxygen During Dysbiosis ................................................................................... 9 1.4.1 – Antibiotic-Altered Microbiota Boosts an Oxygenated Gut ........................... 10 1.4.2 – Enteric-Altered Microbiota Boosts an Oxygenated Gut .............................. 11 1.4.3 – Host Inflammation Boosts an Oxygenated Gut .......................................... 13 1.5 – Oxygen as a Signal for Pathogens in the Host Environment ............................ 14 1.5.1 – Direct Oxygen Sensor Response Complexes ............................................ 15 1.5.2 – Indirect Oxygen Sensor Response Complexes .......................................... 16 1.5.3 – Spatiotemporal Oxygen Signaling Benefitting Pathogenicity ...................... 18 1.6 – The Influence of Oxygen on Pathogen Growth and Survival ............................ 22 1.6.1 – Gut Oxygen in Anaerobic and Aerobic Metabolism of Pathogens .............. 22 1.6.2 – Reactive Oxygen Species in Bacterial Growth and Death.......................... 26 1.7 – The Terminal Oxidases of Bacterial Pathogens ................................................ 27 1.8 – Measuring Oxygen During Infection .................................................................. 31 1.8.1 – Hypoxia Indicators ...................................................................................... 31 1.8.2 – Phosphorescent Probes ............................................................................. 32 1.8.3 – Protein Biosensors ..................................................................................... 33 1.9 – Conclusions and Future Perspectives .............................................................. 34 Chapter 2 – Aerobic Metabolism in Vibrio cholerae is Required for Population Expansion During Infection ........................................................................................... 35 2.1 – Preface ............................................................................................................. 36 2.2 – Abstract ............................................................................................................ 36 2.3 – Importance ........................................................................................................ 37 2.4 – Introduction ....................................................................................................... 37 2.5 – Materials and Methods ..................................................................................... 41 2.5.1 – Transposon Mutagenesis Library Screen ................................................... 41 2.5.2 – Porcine Small Intestinal Mucus Collection and Mucin Purification ............. 41 2.5.3 – Bacterial Strains and Growth Conditions .................................................... 42 2.5.4 – Primers ....................................................................................................... 42 2.5.5 – Plasmid Construction ................................................................................. 43 2.5.6 – Vibrio cholerae Mutant Construction .......................................................... 43 viii 2.5.7 – Growth Curves ........................................................................................... 44 2.5.8 – AKI Virulence-Inducing Conditions ............................................................. 45 2.5.9 – Cholera Toxin Quantification by ELISA ...................................................... 46 2.5.10 – RNA Isolation and Real Time Quantitative PCR (RT-qPCR).................... 46 2.5.11 – Infant Mouse Colonization Assays ........................................................... 47 2.5.12 – Statistical Methods ................................................................................... 48 2.6 – Results .............................................................................................................. 49 2.6.1 – Transposon Mutagenesis Screen Identified the Pyruvate Dehydrogenase Complex as Important for Growth on Mucin. .......................................................... 49 2.6.2 – The Pyruvate Dehydrogenase Complex Supports Aerobic Growth on Mucin. ............................................................................................................................... 49 2.6.3 – Pyruvate Formate-Lyase Supports Anaerobic Growth on Mucin. ............... 52 2.6.4 – Cholera Toxin Production in PDH Mutants is Equivalent to Wild Type in Both Standard and Anaerobic Toxin-Inducing Conditions. ..................................... 53 2.6.5 – Functional PDH Activity is not Required for Expression of toxT, ctxA, and tcpA. ....................................................................................................................... 54 2.6.6 – A Functional Pyruvate Dehydrogenase Complex is Necessary for Colonization of the Infant Mouse. ........................................................................... 56 2.6.7 – Pyruvate Formate-Lyase Provides Minor Growth Support during Infection. ............................................................................................................................... 59 2.7 – Discussion ........................................................................................................ 61 2.8 – Acknowledgements ........................................................................................... 66 Chapter 3 – Oxidative respiration through the bd-I and cbb3 oxidases is required for Vibrio cholerae pathogenicity and proliferation in vivo. ................................................. 68 3.1 – Preface ............................................................................................................. 69 3.2 – Abstract ............................................................................................................ 69 3.3 – Introduction ....................................................................................................... 70 3.4 – Materials and Methods ..................................................................................... 72 3.4.1 – Bacterial Strains and Growth Conditions .................................................... 72 3.4.2 – MuGENT Mutant Strain Construction ......................................................... 72 3.4.3 – Isogenic Deletion Mutant Strain Construction ............................................ 73 3.4.4 – V. cholerae Terminal Oxidase Strain Growth Curves ................................. 74 3.4.5 – V. cholerae Terminal Reductase Strain Growth Curves ............................. 74 3.4.6 – Wild Type Aerobic, Microaerobic, and Anaerobic RNA Isolation and Real- Time Quantitative PCR (RT-qPCR)........................................................................ 75 3.4.7 – Infant Mouse Colonization Assays ............................................................. 75 3.4.8 – CoMPAS Infant Mouse Infection and Sequencing ..................................... 76 3.4.9 – In vitro Competition Assays ........................................................................ 77 3.5 – Results .............................................................................................................. 78 3.5.1 – Constructing Terminal Electron Acceptor Mutant Strains ........................... 78 3.5.2 – In vitro Characterization of Terminal Electron Acceptor Complex Mutants 81 3.5.3 – Aero7 and Ana4 Infant Mouse Infections ................................................... 87 3.5.4 – Individual Oxidase Function During Infection ............................................. 91 3.5.5 – Determining Functionally Redundant Oxidases During Infection ............... 94 3.6 – Discussion ........................................................................................................ 96 ix Chapter 4 – Concluding Remarks ................................................................................. 98 4.1 – Conclusions and Significance ........................................................................... 99 4.1.1 – Metabolic Pathways Important for In Vivo Growth...................................... 99 4.1.2 – Respiration of V. cholerae During Infection .............................................. 100 4.2 – Future Directions ............................................................................................ 101 APPENDICES ............................................................................................................. 106 APPENDIX A Supplemental Material for Chapter 1 ................................................. 107 APPENDIX B Supplemental Material for Chapter 2 ................................................. 124 APPENDIX C Investigating the Mucin Response Network of Shiga-toxin Producing Escherichia coli (STEC) ........................................................................................... 144 REFERENCES ........................................................................................................ 156 x LIST OF TABLES Table 1.1. Enteric pathogens and terminal oxidase complexes. ................................... 30 Table A.1. Transposon mutagenesis screen results. .................................................. 110 Table A.2. Bacteria strain list.. ..................................................................................... 111 Table A.3. Primer list. .................................................................................................. 112 Table A.4. Purified porcine small intestinal mucin monosaccharide and sialic acid analysis determined by High-Performance Anion-Exchange Chromatography coupled with Pulsed Amperometric Detection (HPAEC-PAD). (GlycoAnalytics)....................... 113 Table B.1. Whole Genome Sequencing SNP analysis annotation. ............................. 129 Table B.2. CoMPAS sequencing reads. ...................................................................... 130 Table B.3. Bacteria strain list. ...................................................................................... 131 Table B.4. Primer list. .................................................................................................. 132 Table C.1. Shiga toxin-producing E. coli mucin response network. ............................. 146 Table C.2. Clinical outcomes of STEC infection. ......................................................... 155 xi LIST OF FIGURES Figure 1.1. Longitudinal and transverse oxygen gradients in the human intestinal tract. 5 Figure 1.2. Oxygen dynamics in the gut during homeostasis and dysbiosis. .................. 8 Figure 1.3. Oxygen responsive signaling in bacteria. .................................................... 14 Figure 1.4. Oxygen responsive spatiotemporal gene regulation in bacterial pathogens. .................................................................................................................... 20 Figure 1.5. Terminal oxidase complexes of enteric pathogens. .................................... 28 Figure 2.1. Growth curves of WT ΔaceE, and ΔaceF in M9 minimal media supplemented with 0.5% purified porcine small intestinal mucin. ......................................................... 51 Figure 2.2. Growth curves of WT and ΔpflA in M9 minimal media supplemented with 0.5% purified porcine small intestinal mucin.................................................................. 53 Figure 2.3. Cholera toxin (CT) production for WT, ΔaceE, ΔaceF, and ΔtoxT strains. .. 54 Figure 2.4. Relative fold change of toxT, ctxA, and tcpA transcript levels compared to wild type expression. ..................................................................................................... 55 Figure 2.5. Infant mouse colonization assays of WT, ΔaceE, and ΔaceF after 20h. ..... 57 Figure 2.6. Infant mouse colonization of WT, ΔaceE, and ΔaceF mono-associated infections in proximal, medial, and distal portions of the small intestine after 20h. ........ 58 Figure 2.7. Infant mouse colonization assays of WT and ΔpflA in the small intestine after 20h. ............................................................................................................................... 60 Figure A.1. V. cholerae C6706 El Tor wild type growth in LB, minimal 0.5% mucin, and minimal media with no added carbon source. ............................................................. 114 Figure A.2. Complementation growth curves of ΔaceE and ΔaceF in M9 0.5% glucose media. ......................................................................................................................... 115 Figure A.3. Growth curves of WT, ΔaceE, and ΔaceF in LB media grown aerobically, anaerobically, or anaerobically supplemented with 50mM fumarate. .......................... 116 Figure A.4. Growth curves of WT, ΔaceE, ΔaceF in M9 0.2% Casamino acid media grown aerobically. ....................................................................................................... 117 Figure A.5. Complementation growth curve of ΔpflA in M9 0.5% glucose 50mM fumarate media grown anaerobically. ......................................................................................... 118 xii Figure A.6. Growth curves of WT and ΔpflA in LB media grown aerobically, anaerobically, or anaerobically supplemented with 50mM fumarate. .......................... 119 Figure A.7. Cholera toxin (CT) production for WT, ΔaceE, ΔaceF, and ΔtoxT strains.. 120 Figure A.8. Infant mouse colonization assays of WT and ΔpflA in the large intestine after 20h. ............................................................................................................................. 121 Figure A.9. Growth curves of WT, ΔaceE, ΔaceF and ΔpflA in M9 0.3% acetate. ....... 122 Figure A.10. In vitro mono-culture and competition assays of WT, ΔaceE, ΔaceF, and ΔpflA in M9 0.5% glucose after 20h. ............................................................................ 123 Figure 3.1. Verification of MuGENT generated mutant strains. ..................................... 79 Figure 3.2. Terminal oxidases support aerobic growth in V. cholerae. .......................... 85 Figure 3.3. Terminal reductase mutants are reduced for anaerobic growth in the presence of cognate electron acceptor molecules. ....................................................... 87 Figure 3.4. Aerobic respiration, and not anaerobic respiration, is required for growth and colonization of the infant mouse small intestine. ........................................................... 89 Figure 3.5. Terminal oxidases are functionally redundant in supporting colonization of the infant mouse small intestine. ................................................................................... 92 Figure 3.6. bd-I oxidase alone supports wild type levels of colonization in the infant mouse small intestine with cbb3 supporting colonization to a lesser extent. .................. 95 Figure B.1. MuGENT spectinomycin selective marker shows no fitness defect in vitro. ............................................................................................................................ 134 Figure B.2. cbb3 deficient strains and wild type grown anaerobically do not maintain a functional cbb3 oxidase complex. ................................................................................ 135 Figure B.3. V. cholerae oxidases generally support the same pattern of growth in minimal M9 0.2% D-glucose media as seen in LB. ..................................................... 136 Figure B.4. V. cholerae oxidases generally support the same pattern of growth in combinatorial MuGENT knockout strains. ................................................................... 137 Figure B.5. Terminal reductase mutants are variable for aerobic growth in the presence of cognate electron acceptor molecules. ..................................................................... 138 Figure B.6. Functional terminal oxidases, but not alternative terminal reductases, are required for optimal colonization of the large intestine. ............................................... 139 xiii Figure B.7. Individual and combinatorial oxidase mutants colonize the large intestine more efficiently than the small intestine but reflect overall colonization patterns present in the small intestine. ................................................................................................... 140 Figure B.8. TcpA production is functional in individual oxidase deletion mutants. ....... 141 Figure B.9. Terminal oxidase complexes are not required for cell survival under hydrogen peroxide stress. ........................................................................................... 142 Figure B.10. Variant +bd-III strain (+bd-IIIV) indicates that the bd-III oxidase, when expressed, is capable of supporting aerobic respiration in V. cholerae and colonization of the infant mouse. ..................................................................................................... 143 Figure C.1. Maximum likelihood tree of the STEC sulfatase Z2210 mucin response gene. ........................................................................................................................... 147 Figure C.2. Maximum likelihood tree of STEC mucin response gene fusK. ................ 148 Figure C.3. Maximum likelihood tree of STEC mucin response gene fusR. ................ 148 Figure C.4. Maximum likelihood tree of STEC mucin response gene galR. ................ 149 Figure C.5. Maximum likelihood tree of STEC mucin response gene galK. ................ 149 Figure C.6. Maximum likelihood tree of STEC mucin response gene agaR. ............... 150 Figure C.7. Maximum likelihood tree of STEC mucin response gene nagC. ............... 150 Figure C.8. Maximum likelihood tree of STEC mucin response gene mlc. .................. 151 Figure C.9. Maximum likelihood tree of STEC mucin response gene nanR. ............... 151 Figure C.10. Maximum likelihood tree of STEC mucin response gene fucA. .............. 152 Figure C.11. Maximum likelihood tree of STEC mucin response gene nagE. ....... 152152 Figure C.12. Maximum likelihood tree of STEC mucin response gene manA. ............ 153 Figure C.13. Maximum likelihood tree of STEC mucin response gene manX. ............ 153 Figure C.14. Maximum likelihood tree of STEC mucin response gene nanA. ............. 154 xiv KEY TO ABBREVIATIONS ACS-1........................................................................................... Acetyl-CoA Synthase-1 AKGDH ................................................................... Alpha-Ketoglutarate Dehydrogenase ANCOVA ....................................................................................... Analysis of Covariance ATP .............................................................................................Adenosine Triphosphate BSO........................................................................................................... Biotin Sulfoxide CI........................................................................................................... Competitive Index CIP ..........................................................................................Calf Intestine Phosphatase CoMPAS ............................................ Comparative Multiplex PCR Amplicon Sequencing Coupled with Pulsed Amperometric Detection CT ............................................................................................................... Cholera Toxin DMSO .................................................................................................. Dimethyl Sulfoxide EAEC ......................................................................... Enteroaggregative Escherichia coli EHEC ........................................................................ Enterohemorrhagic Escherichia coli ETC ............................................................................................Electron Transport Chain FbFP ........................................................................... Flavin-Binding Fluorescent Protein Fnr ..................................................................... Fumarate and Nitrate Regulatory Protein FRET ........................................................................ Förster Resonance Energy Transfer GCV ................................................................................. Glycine Cleavage Multienzyme GlcNAc ............................................................................................. N-Acetylglucosamine HIF-1 .......................................................................................... Hypoxia Inducible Factor HPAEC-PAD ................................. High-Performance Anion-Exchange Chromatography IBD ................................................................................................ Irritable Bowel Disease xv IP .................................................................................................................Inoculum Pool Ipa ............................................................................................. Invasion Plasmid Antigen IPTG ..................................................................... Isopropyl β-D-1-Thiogalactopyranoside LAP ............................................................................................Listeria Adhesion Protein LEE ................................................................................. Locus of Enterocyte Effacement LLO ............................................................................................................. Listeriolysin O LOD ........................................................................................................ Limit of Detection MASC-PCR .................................... Multiplex Allele-Specific Polymerase Chain Reaction MDHHS .......................................... Michigan Department of Health and Human Services MiGS ..................................................................... Microbial Genome Sequencing Center MP .................................................................................................................. Mouse Pool MuGENT ........................................ Multiplex Genome Editing by Natural Transformation NAC................................................................................................... N-Acetyl-L-Cysteine PCR....................................................................................... Polymerase Chain Reaction PDH........................................................................................... Pyruvate Dehydrogenase PFL............................................................................................. Pyruvate Formate-Lyase PMNs .............................................................................. Polymorphonuclear Neutrophils PPAR-γ............................................................. Peroxisome Proliferator-Activated Receptor γ PSIM ................................................................................. Porcine Small Intestinal Mucin Q .......................................................................................................................... Quinone ROS ......................................................................................... Reactive Oxygen Species SCFA............................................................................................. Short Chain Fatty Acid SDS ............................................................................................. Sodium Dodecyl Sulfate xvi STEC................................................................... Shiga Toxin-Producing Escherichia coli Stx .................................................................................................................. Shiga Toxin T3SS ........................................................................................ Type III Secretion System T6SS ........................................................................................ Type VI Secretion System TCP ............................................................................................. Toxin-Coregulated Pilus TMAO ..........................................................................................Trimethylamine-N-Oxide TMPD ............................... N1N1N’1N’-Tetramethyl-p-Phenylene-Diamine Dihydrochloride WT..................................................................................................................... Wild Type YFP ......................................................................................... Yellow Fluorescent Protein xvii Chapter 1 – Introduction – Oxygen and Enteric Bacterial Infections 1 1.1 – Abstract Oxygen is critical in shaping the human intestinal ecosystem. Under homeostasis, the gut is in a state of physiological hypoxia that is maintained through a complex symbiosis between host cells and the microbiota. For enteric pathogens, oxygen plays a role in many facets of pathogenesis and disease progression. All enteric pathogens interact with gut oxygen, and many have evolved mechanisms to both exploit molecular oxygen for growth and subvert damage from reactive oxygen species. Periods of dysbiosis brought on by bacterial infection and other means further drive oxygen into the lumen of the intestine. Here I review oxygen as a key molecule influencing enteric bacterial pathogenesis and disease. 1.2 – Introduction The evolution of ancient microorganisms that led to the use of oxygen in metabolism marked a significant milestone in evolutionary history (1). Oxygen became expansively abundant in Earth’s atmosphere during the great oxidation event where ancient photosynthetic cyanobacteria began converting CO2 to O2 (2). Present day organisms have been evolutionarily shaped by oxygen, accruing strategies to benefit from its high energy bonds and mitigate damage from harmful oxygen radicals. Within the intestinal tract, oxygen plays a major role in shaping the intestinal microbial landscape (3). During homeostasis, resident bacterial communities are composed of diverse taxa that maintain a healthy gut ecosystem through multilayered symbioses between gut microbes and the host (4). Facultative anaerobes of the gut maintain a relatively anaerobic lumen by scavenging oxygen that diffuses from host epithelium, 2 allowing for the growth of obligate anaerobes within the gut community (5). These obligate anaerobes produce short-chain fatty acids that support barrier function of the host epithelium, and influence host cell metabolism, which limits diffusion of oxygen into the gut lumen from host tissue (6). Gut oxygen is also critical during periods of dysbiosis brought on by enteric infections. Oxygen is one of many environmental signals for pathogens that drives efficient colonization and virulence trait expression. During enteric-induced dysbiosis, the inherent balance of oxygen between the microbiota and host shifts as resident populations are displaced and host cell metabolism changes to anaerobic glycolysis leaving unused oxygen to permeate into the gut lumen (7). The influx of oxygen and host responses that generate reactive oxygen species become transient factors that pathogens either exploit or subvert in disease progression. Pathogens capable of exploiting oxygen in the intestinal tract do so through reduction of molecular oxygen by terminal oxidase complexes that support aerobic respiration, a highly efficient energy generating metabolic pathway. Terminal oxidases that support aerobic respiration also aid detoxification of reactive oxygen species alongside other ROS defenses such as catalases, peroxidases, and superoxide dismutases. The role of oxygen during enteric infection is an emerging area of intense exploration. Here we review current knowledge of how oxygen functions as a signal for bacterial pathogenesis and how it impacts pathogen growth and fitness during infection. We also discuss the function and relevance of terminal oxidases and their role in the 3 pathogenesis for several enteric pathogens. Lastly, we review technologies, recent advancements, and practical limitations in the measurement of oxygen during infection that are critical to our understanding of oxygen during disease. 1.3 – Oxygen During Homeostasis 1.3.1 – Maintaining Oxygen Levels in the Intestinal Tract Mucosal oxygenation in the human body depends on blood flow anatomy and local tissue metabolism. Within the gut, the concentration of molecular oxygen varies both radially and longitudinally throughout the length of the intestinal tract (Figure 1.1) (3, 8). Diffusion of oxygen from the capillaries of the highly vascularized intestine leads to a steep oxygen gradient extending from the epithelial crypts to the hypoxic gut lumen (9). This condition of relatively low oxygen in the intestine is referred to as “physiological hypoxia” and is required for maintaining a healthy epithelium (8). Within intestinal villi, oxygen release into the lumen is influenced by villus host cell maturation. In the small intestine, villi extend as finger-like projections into the intestinal lumen and are essential for nutrient and water absorption; they also contain replenishing stem cells at the base of the villus crypt. In the large intestine, elongated crypts are present with stem cells at the base, however, no protruding villi are present. Within both the small and large intestine, epithelial cells are replaced and replenished every four to five days with stem cells giving rise to early transient-proliferated cells that differentiate and mature as they ascend the small intestinal villi or large intestine elongated crypt (10). Epithelial cell turnover is essential for maintaining gut homeostasis and physiological hypoxia. 4 Figure 1.1. Longitudinal and transverse oxygen gradients in the human intestinal tract. Oxygen concentrations decrease throughout the length of the intestine from the upper to lower gastrointestinal tract. In both the small and large intestine, oxygen emanates from the epithelium and decreases to anoxia in the gut lumen. Aerotolerant commensal microorganisms colonize nearer to the epithelium whereas commensal anaerobes are present in the lumen associated with host mucus. Oxygenation of the epithelial tissue is influenced by blood flow to the intestine which can fluctuate as blood flow markedly increases following food consumption to aid in digestion. A blood vessel and lymph node are depicted near the bottom of the intestinal cross sections where highly oxygenated blood is delivered to the intestine in red and exits in blue following delivery of oxygen to the tissue. In healthy adults, undifferentiated proliferating colonoyctes metabolize glucose to lactate via oxygen-independent anaerobic glycolysis and fermentation, whereas mature colonocytes present further up the crypt preferentially metabolize short chain fatty acids via oxygen-dependent β-oxidation (11). This differential metabolism of villus colonocytes leads to greater oxygenation of the crypt base compared to the ascended crypt environment. This oxygen gradient shapes the microbiota as more aerotolerant 5 microbes in the phyla Proteobacteria and Actinobacteria are associated with the intestinal mucosa, whereas phyla Bacteroidetes and Firmicutes, comprised mainly of anaerobic microbes, are associated with the mucus layer away from the epithelial tissue (3, 5). The diffusive oxygen from the epithelial tissue is readily consumed by facultative anaerobes, facilitating growth of obligate anaerobes by maintaining a hypoxic gut lumen (5). This intestinal balance of oxygen promotes complementary aerobic and anaerobic metabolisms among the microbiota and maintains a healthy gut community (12). In addition to microbial-driven luminal oxygen depletion, oxygen is also depleted through the oxidation of host cell lipids. Germ-free mice were observed to maintain a deoxygenated gut lumen comparable to that of conventional mice despite the absence of residential microbial populations. However, lipid oxidation appeared to play a secondary role as oxygen depletion occurred at a much slower rate in germ-free mice compared to conventional mice with an intact microbiota (5). 1.3.2 – Microbe-Host Metabolic Crosstalk and Oxygen Regulation Just as microbial populations contribute to a gut oxygen balance, so too do microbe- host cell interactions (Figure 1.2). The maintenance of oxygen abundance in the gut is influenced by microbiota signaling within the intestinal tract, where most of our understanding comes from large bowel microbe-host relationships. In the large bowel, the primary mechanism by which oxygen levels are regulated to maintain physiological hypoxia is through metabolite exchange between the bacteria of the gut and host cells (8). Obligate anaerobes of the intestine, primarily the Firmicutes and Bacteroidetes, produce short chain fatty acids such as butyrate (6, 13), which induces β-oxidation 6 metabolism by activation of the sensor peroxisome proliferator-activated receptor γ (PPAR-γ) in colonocytes (14). This oxidative metabolism consumes oxygen being delivered through the vasculature, preventing large influxes of oxygen from reaching the lumen of the intestine. Furthermore, a recent study revealed that microbiota-derived butyrate maintained the stability of transcription factor HIF-1 (hypoxia inducible factor) through β-oxidation metabolism, which aids epithelial barrier function (15). It is well established that HIF-1 directly regulates production of mucins, antimicrobial peptides (β -Defensin-1), and the tight junction protein claudin-1, which provides protection against several pathogenic organisms (15). Comparatively less work has explored the metabolic coordination between the microbiota and epithelial cells in the small intestine (16). Phyla that produce short chain fatty acids are present, particularly in the distal ileum which is a more anaerobic environment than the duodenum and jejunum (17). In small intestinal enteroids, addition of butyrate promoted intestinal barrier function, supporting the presence of similar metabolic crosstalk as exhibited in the large intestine (18). More work, however, is required to understand how the dynamically shifting microbiota of the small intestine, which is more unstable than the large intestine, impacts host cell metabolism, community structure, and ultimately oxygen availability (19). 7 Figure 1.2. Oxygen dynamics in the gut during homeostasis and dysbiosis. Under gut homeostasis commensal anaerobes produce short chain fatty acids (SCFAs) such as butyrate that are consumed by the host epithelium through oxygen- dependent β-oxidation. Cellular hypoxia induces HIF-1 which supports epithelial barrier function through induction of proteins such as tight junction protein claudin-1. Crypt stem cell metabolism is predominantly anaerobic glycolysis allowing for oxygen to diffuse into the crypt space where oxygen becomes most available in the gut generating an oxygen gradient extending to the anoxic gut lumen. During periods of dysbiosis brought on by antibiotic treatment, enteric infection, or irritable bowel diseases, commensal anaerobe populations are reduced, decreasing SCFA production in the gut. This leads differentiated mature cells of the intestine to shift metabolism away from β-oxidation to anaerobic glycolysis, resulting in excess oxygen diffusion into the gut. Excess oxygen leads to the outgrowth of aerotolerant microorganisms as oxygen supports growth and further displaces commensal anaerobe populations. The loss of cellular hypoxia reduces HIF-1 activity and subsequent weakening of epithelial barrier by reducing production of protective proteins such as tight junction protein claudin-1. 8 1.4 – Oxygen During Dysbiosis During dysbiosis, the balance of oxygen typically associated with homeostatic conditions becomes disrupted. Dysbiotic events can lead to dramatic fluctuations in oxygen abundance through disturbances to the precise oxygen control ecosystem of the gut. In some events, dysbiosis can lead to a positive feedback loop that perpetuates oxygen dysregulation, slowing gut remediation. Oxygen can become increasingly abundant in the intestine during periods of dysbiosis brought on by antibiotic treatment (20), enteric infection (21), inflammation (22, 23), and other factors that disrupt the resident microbiota of the intestinal tract (Figure 1.2). Within the intestines, the dominant microbial phyla are Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria, Fusobacteria, and Verrucomicrobia (24). Bacteroidetes and Firmicutes comprise approximately 90% of the intestinal community with Proteobacteria, Actinobacteria, Fusobacteria, and Verrucomicrobia being less represented overall (25, 26). The Proteobacteria and Actinobacteria are widely classified as facultative anaerobes, and expansion of the Enterobacteriaceae family of the Proteobacteria phyla is assumed to be linked to increases in environmental oxygen availability. This was evident in ileostomy patients whose oxygen-exposed intestines had increased abundance of aerotolerant Lactobacilli and Enterobacteria which returned to normal community structure upon closure of the ileostomy (27). The shift in oxygen availability that leads to expansion of aerotolerant microorganisms coincides with the decrease in aerosensitive microorganisms. These latter microbes include obligate anaerobes responsible for producing the short chain fatty acid, butyrate, which 9 is consumed by intestinal epithelial cells. Human intestinal tissues lacking butyrate shift metabolism to anaerobic glycolysis, a process that does not consume oxygen (28). This leads to accumulation of oxygen in the epithelium and heightened oxygen levels diffusing into the gut lumen. To compound these effects, depletion of butyrate producing cells reduces epithelial signaling through PPAR-γ which typically limits the bioavailability of luminal oxygen in coordination with Treg proinflammatory regulation (14). Without this activation signal, oxygen dissemination into the gut becomes dysregulated, exacerbating gut dysbiosis. 1.4.1 – Antibiotic-Altered Microbiota Boosts an Oxygenated Gut Antibiotic treatment is one condition that leads to disturbance of resident gut microbiota. In mice treated with a cocktail of antibiotics, the redox potential of the gut, measured by microelectrode, increased after 24 hours and subsequently returned to baseline over an additional 24h time period, indicating transient fluctuations in oxygen availability in response to antibiotic treatment (29). This effect was also observed in human patients treated with ciprofloxacin where gut taxonomic richness, diversity, and evenness decreased but returned to near pre-ciprofloxacin treatment after 4 weeks (30). In another human case study, antibiotic treatment with amoxicillin-clavulanic acid causing antibiotic-associated diarrhea led to complete loss of clostridial cluster XIVa, reduction in clostridial cluster IV, and reduction in a Bacteroidetes fragilis cluster (31) which constituted the majority of butyrate-producing species (32). The loss of butyrate production in the gut due to antibiotic treatment was also observed in mice after being fed multiple antibiotics in drinking water followed by an oral dose of clindamycin which coincided with a change in the intestinal microbiota (33). The loss of SCFA producing 10 microorganisms results in elevated oxygen levels in the gut and decreased intestinal barrier function. However, not all antibiotics are created equal. Antibiotics can target small subsets of bacteria or have broad-spectrum bactericidal properties which can differentially alter microbial gut composition. Doxycycline and clarithromycin decreased specific bacterial populations in the gut whereas phenoxymethylpenicillin, nitrofurantoin, and amoxicillin had very little impact on the gut microbiome composition (34). These summarized findings suggest that not all antibiotic treatment affect gut composition and oxygen availability in the same way. 1.4.2 – Enteric-Altered Microbiota Boosts an Oxygenated Gut Disturbances in microbiota caused by enteric infection are similar to those from antibiotic driven shifts in microbiota that affect oxygen levels of the gut (35). Among infections from multiple enteric pathogens (Escherichia coli STEC O157 and non-O157, Salmonella spp., Shigella spp., and Campylobacter spp.) infected individuals showed expansion of Proteobacteria populations and decreases in Bacteroidetes and Firmicutes, which recovered after alleviation of infection (36). In the case of Salmonella enterica serovar Typhimurium infection, infected mice colonized with a humanized microbiota had increases in Enterobacteriaceae and a decreases in the relative abundance of Bacteroidetes and Firmicutes (37, 38). This is driven in part by virulence factors deployed by Salmonella Typhimurium during infection. Specifically, through the action of the Salmonella type III secretion system (T3SS), large 11 depletions in Clostridia class microorganisms in the gut were observed (7). Type III secretion system-1 effector proteins and flagellar motility of Salmonella Typhimurium increases the respiratory burst of human neutrophils which would lead to more oxygen species in the gut altering gut community composition (39). The antimicrobial elastase activity of host neutrophils, which flood the intestine during infection, kill susceptible bacteria of the gut; a shift not seen in concomitant control neutrophil depletion infections (40). Indeed, displacement of butyrate-producing bacteria would be beneficial to S. Typhimurium as butyrate limits its pathogenicity (7, 41). Host inflammatory responses and displacement of resident microbiota subsequently leads to increased oxygenation of the gut. For the bacterial pathogens Citrobacter rodentium and S. Typhimurium, an inflamed gut leads to aerobic expansion of these pathogens, furthering disease progression (7, 38, 42). Vibrio cholerae, a pathogen of the small intestine, also appears to elicit oxygen accumulation in the gut. Through the action of cholera toxin, during infection, V. cholerae shows signatures of increased TCA cycle expression and elicits an accumulation of luminal L-lactate, both indicators that host epithelial cells have shifted to anaerobic glycolysis, resulting in oxygen accumulation in the gut (43). Direct microbe-microbe interactions between pathogenic bacteria and members of the microbiome can also disrupt bacterial community composition and affect oxygen dynamics in the host. One example of a microbe-microbe interaction that affects community structure involves Type VI secretion system (T6SS) dependent killing (44). The T6SS of V. cholerae supported colonization in both the infant mouse and infant rabbit through T6SS-dependent killing of commensal populations for niche 12 establishment (45, 46). T6SS are not just limited to V. cholerae as they exist in Campylobacter jejuni, Shigella flexneri, Citrobacter rodentium, and S. Typhimurium (47– 49). The T6SS of C. rodentium, a mouse enteropathogen model for human enteropathogenic and enterohemorrhagic E. coli infections (50), supports competition with non-pathogenic E. coli in vitro (50, 51). Salmonella Typhimurium also competes with bacteria of the gut using a T6SS, but only targets specific members of the microbiota in order to invade the gastrointestinal tract (52). Deeper understanding of how T6SSs influence pathogen biology is emerging, but how they change oxygen availability through interbacterial competition remains to be thoroughly investigated. 1.4.3 – Host Inflammation Boosts an Oxygenated Gut Host inflammation is associated with overgrowth of Enterobacteriaceae, a correlate of increased oxygen in the gut (53). In mice treated with dextran sodium sulfate, a model of colitis, non-pathogenic E. coli proliferate by formate oxidation to expand in the inflamed gut (54). Changes in gut composition are prevalent in the intestines of patients with irritable bowel disease (IBD) such as ulcerative colitis and Crohn’s disease, for whom periods of intestinal inflammation are common. Crohn’s disease results in depletion of butyrate producing bacteria (55). Ulcerative colitis reduces overall diversity and is tied to increases in Proteobacteria populations (56). These gut composition changes resulted in a shift toward Enterobacteriaceae and away from commensal anaerobic populations (57, 58). Commonly associated with intestinal dysbiosis in IBD patients, oxygen is hypothesized to perpetuate lasting problems in these patients as facultative anaerobes dominate and prolong disease (59). 13 1.5 – Oxygen as a Signal for Pathogens in the Host Environment Oxygen levels serve as a signal for intestinal microorganisms (Figure 1.3) (3). The same is true for pathogens as they progress through infection (60). Oxygen is detected by enteric pathogens through oxygen sensitive regulators that respond to intracellular redox potential to control bacterial responses that promote pathogenicity and fitness during infection (61). Figure 1.3. Oxygen responsive signaling in bacteria. Iron-sulfur containing proteins, such as Fnr, respond to oxygen through the oxidation of Fe-S clusters. Fnr responds to the presence of oxygen affecting gene expression for anaerobic metabolism. Anaerobic metabolism genes are induced in anaerobiosis and repressed in aerobiosis. Two component regulators such as ArcAB respond to oxygen through indirect energy sensing through the quinone pool. Quinones (Q) are electron carriers in the electron transport chain. In anaerobiosis, through kinase activity, ArcB phosphorylates the response regulator ArcA which induces genes involved in anaerobic metabolism. In aerobiosis, electron carrying quinones (QH2) interact with ArcB which changes from a kinase to a phosphatase, dephosphorylating ArcA, which no longer induces genes involved in anaerobic metabolism. AphB responds to oxygen by formation of disulfide bridges following oxidation of coordinating cysteines. In 14 aerobiosis, disulfide bridge formation activates AphB to induce virulence gene expression. In anaerobiosis, no disulfide linkages are formed, so no virulence genes are activated. Aer and Tsr are oxygen transducers that support aerotaxis in some bacterial cells. In the presence of oxygen, Aer and Tsr drive flagellar motility in a chemotactic response whereas in the absence of oxygen, Aer and Tsr do not affect cell motility. 1.5.1 – Direct Oxygen Sensor Response Complexes Iron-sulfur clusters are one class of oxygen responsive molecular switches that react to the presence of oxygen (62). Many types of iron-sulfur clusters exist that can collectively respond to redox potentials ranging from +500mV to -500mV, a wide range in redox response that can affect a function in oxygen sensing, electron transfer, and enzyme activities (63, 64). A prominent iron-sulfur cluster-containing regulator in pathogenic and nonpathogenic E. coli as well as other prominent bacterial pathogens, is the fumarate and nitrate regulatory (Fnr) protein that responds to the presence of oxygen (65, 66). The iron-sulfur cluster in Fnr becomes oxidized in the presence of oxygen, causing release of the protein from its DNA-bound dimeric conformation. This leads to de- repression of >100 genes in E. coli (67) and modifies >300 genes in S. Typhimurium, including genes involved in virulence (68). In fact, Fnr modulates expression of virulence genes in several pathogens (69). In S. flexneri, Fnr is important for both early and later stages of infection. Oxygen mediated Fnr de-repression directs activation of the T3SS Ipa (invasion plasmid antigen) secretion into host cells upon reaching the relatively oxygenated intestinal crypts which promotes epithelium intracellular invasion (70). Oxygen consumption for luminal S. flexneri occurs through cydAB bd oxidase which is required for intracellular survival (71), which leads to further tissue colonization 15 as foci of hypoxia develop and Fnr signaling in a now oxygen-limited environment activates anaerobic metabolic pathways to support further proliferation (72, 73). Thiol-based molecular switches are another mechanism for oxygen and redox sensing within the host environment. Thiol-based switches respond to the presence of reactive oxygen species as cysteine thiolates readily react with ROS species to form or break disulfide linkages resulting in a change of conformation to direct regulatory responses (74, 75). In V. cholerae, oxygen sensing by thiol molecular switches is proposed to be important for spatiotemporal regulation of virulence gene expression (76, 77). Virulence expression under differential oxygen conditions is controlled by thiol-switch virulence regulators, AphB and OhrR, in which homodimerization through oxygen responsive cysteine residues occurs under low oxygen conditions and leads to the expression of the toxin-coregulated pilus (TCP) (Figure 1.4) (78, 79). This oxygen response leads to early production of TCP during infection, priming V. cholerae cells for microcolony formation upon reaching intestinal crypt spaces. 1.5.2 – Indirect Oxygen Sensor Response Complexes Two component regulatory systems also sense and respond to oxygen availability. One prominent example is the ArcAB two component system most thoroughly studied in E. coli and Salmonella (80, 81). Upon oxidation of redox-active cysteine residues by quinone electron carriers in aerobic conditions, ArcB becomes a phosphatase leading to ArcA dephosphorylation and inactivation (82). Oxidation through the quinone electron carriers can be considered an indirect barometer of oxygen availability as quinones readily obtain electrons during oxidative respiration that then relays this oxygen 16 availability signal to response proteins such as ArcB. ArcB itself is insensitive to molecular oxygen and reactive oxygen species, making indirect oxidized quinones the sole pathway for oxygen sensing (82). Oxygen sensing through ArcAB promotes fitness of E. coli and S. Typhimurium by modulating expression of genes necessary for resistance to reactive oxygen stress, a trait that is critical for Salmonella intracellular survival (83, 84). In V. cholerae, active ArcA (anaerobic conditions) in Classical biotypes induces expression of the master virulence regulator toxT while expression of downstream tcpA-F, encoding the toxin-coregulated pilus, is initially expressed in the more anaerobic lumen of the gut to initiate epithelial attachment (85). However, not all ArcAB response networks are necessary for colonization as arcA deletion in Salmonella enterica serovar Enteriditis was not compromised for virulence in mice (86). Another example of an oxygen responsive two component system is RacRS in the microaerophile Campylobacter jejuni, which responds to low oxygen concentrations to optimize C. jejuni growth in the presence of alternative electron acceptors nitrate and trimethylamine-N-oxide (TMAO) (87). Oxygen sensing transducers can also influence bacterial cell signaling in response to oxygen availability and increasing energy gradients. Aerotaxis is a form of chemotaxis in which bacterial cells sense energy levels through their electron transport chain (88). In E. coli, Aer and the serine chemoreceptor Tsr sense oxygen indirectly through respiratory chain electron transport and adjust E. coli cell behavior to move in the direction of a higher energy environment, typically toward higher oxygen concentrations (89, 90). Aer and Tsr proteins are highly conserved in enterohemorrhagic O157:H7 E. 17 coli and may play an important role in pathogen colonization in directing bacterial cells to the epithelium with higher relative concentrations of oxygen, although this has yet to be investigated. V. cholerae also maintains aerotactic chemoreceptors Aer1 (AerB) (91) and Aer2 (92), although the direct contribution of each chemoreceptor during colonization is unknown. Likely, aerotaxis through Aer1 and Aer2 is not required for colonization as nonchemotactic V. cholerae outcompete wild type cells in the infant mouse, indicating that chemotaxis may not be required in vivo for a pathogen of the small intestine (93). For Salmonella, aerotaxis is also present and was found to benefit fitness during infection, directing bacteria towards high energy electron acceptors such as oxygen, nitrate, and tetrathionate (94, 95). Aerotaxis may also benefit C. jejuni as it is predicted to contain two aerotaxis responsive chemoreceptors, although the current role for these receptors during infection is unknown (96, 97). 1.5.3 – Spatiotemporal Oxygen Signaling Benefitting Pathogenicity Oxygen is also involved in the spatiotemporal regulation of virulence gene expression in enteric pathogens during infection (Figure 1.4). Enterohemorrhagic E. coli (EHEC) colonization potential is enhanced under microaerobic conditions as adherence, T3SS expression, and translocation activity were considerably elevated compared to aerobic and anaerobic conditions (98). This suggests that producing virulence factors near the microaerobic epithelium optimizes colonization and pathogenicity. In addition to the above mentioned oxygen sensing complexes of ArcAB and Fnr that drive differential gene expression, EHEC strains also have four copies of the gene encoding a small RNA, DicF, which senses low oxygen environments, three copies more than commensal E. coli (99). In low oxygen environments, such as the intestine, DicF 18 releases from the ribosome binding site allowing for the translation of PchA protein which in turn promotes expression of the T3SS (99). The elevated copies of dicF may support more sensitive and nuanced oxygen regulatory control in pathogenic E. coli than is needed in non-pathogenic strains. Oxygen availability also affects Shiga toxin (Stx) production in EHEC given that microaerobic environments lead to the highest levels of Stx production (100). Suppressing virulence expression in the anaerobic lumen and driving production near the epithelium is a fitness strategy that increases the likelihood of disease progression. Enteroaggregative E. coli (EAEC) also responds to high concentrations of oxygen, in coordination with host cell contact which induces virulence expression (101). However, virulence expression was highest in aerobic conditions for EAEC even though the transition from the anaerobic lumen to the microaerobic epithelium would still be expected to play a role in signal timing and pathogenesis. 19 Figure 1.4. Oxygen responsive spatiotemporal gene regulation in bacterial pathogens. Vibrio cholerae responds to low oxygen conditions, initially activating OhrR which induces early toxin-coregulated pilus (TCP) production. In higher oxygen concentrations, AphB becomes activated to further induce TCP production. At the host epithelium where oxygen is in greatest abundance, cholera toxin (CT) production is induced to elicit disease. Enterohemorrhagic Escherichia coli does not produce virulence factors in the low oxygen environment of the intestinal lumen. As oxygen levels increase near the host epithelium, oxygen responsive gene regulation leads to the production of the type 3 secretion system (T3SS), adhesion proteins, and Shiga toxins (Stx). Listeria monocytogenes does not produce Listeriolysin O (LLO) in the anaerobic gut lumen, however, oxygen levels near the epithelium induce initial production of the LLO hemolysin that aids in host cell uptake via phagocytosis. V. cholerae also responds to spatial localization within the intestine through oxygen sensing. AphB and OhrR thiol-switches and the ArcAB two component system in the anaerobic lumen coordinate early expression of TCP which is proposed to prime microcolony formation and adherence (78, 79). Expression of tcpA, encoding the major TCP pilin subunit, is upregulated biphasically during infection in both early and later stages of infection, presumably in an initial luminal population followed by further 20 induction in the intestinal crypt (76). ctxAB, which is coregulated with tcpA by ToxT, however, is not expressed biphasically and is only expressed in coordination with tcpA in the later stage infection (76). In the Classical V. cholerae biotype, H-NS proteins appear to prevent cholera toxin (CT) production in anaerobic, but not aerobic conditions (102). Functionally, this would translate to lack of CT production in the anaerobic lumen and CT production near the host epithelium where diffusive oxygen alleviates H-NS repression. Of note, however, is that CT is produced in anaerobic virulence factor inducing conditions by El Tor V. cholerae, indicating differential regulation between the biotypes in vitro that may affect in vivo spatiotemporal expression (103). Listeria monocytogenes is an intracellular pathogen of the intestine, colonizing the host cell cytosol, which enables its cell to cell spread (104). In the absence of oxygen, virulence factors including the Listeria adhesion protein (LAP) were highly expressed (105), mirroring early-infection expression of adhesion proteins in EHEC and V. cholerae (106). For Listeriolysin O (LLO), the primary toxin of L. monocytogenes, gene expression, but not toxin production per se, was enhanced in anaerobic conditions, particularly in the presence of short chain fatty acids (SCFAs) (106, 107). This primed expression may occur in the lumen of the intestine where SCFAs are produced by the commensal microbiota, and when exposed to low levels of SCFAs in the presence of oxygen, LLO production and activity was enhanced (107). This coordinated regulation of transcription and translation of LLO may facilitate L. monocytogenes infectivity as it comes into proximity with the host epithelium, an area typically devoid of SCFA- producing commensal bacteria in the large intestine and with relatively higher oxygen 21 concentrations (107). Stimulated extracellular LLO production might then lead to perforation of host cells to stimulate internalization by the host epithelium (108). This LLO priming may also benefit the bacteria after internalization to ensure rapid lysis of the phagosomal membrane, limiting time within the compartment to gain quick access to the host cytosol (109). 1.6 – The Influence of Oxygen on Pathogen Growth and Survival 1.6.1 – Gut Oxygen in Anaerobic and Aerobic Metabolism of Pathogens Varying oxygen concentrations in the gut likely influences the metabolism of all gut microbes, both resident and transient. Anaerobic metabolism predominantly occurs in the hypoxic lumen, away from the more oxygenated host epithelium where oxidative respiration supports growth of facultative anaerobes. Oxidative respiration is the most efficient energy-generating metabolic pathway, supporting cellular processes for growth and proliferation. Bacterial pathogens capable of oxidative respiration therefore benefit from the presence of oxygen during infection, particularly near the epithelium where oxygen is most abundant. V. cholerae is one such pathogen that benefits from both anaerobic and aerobic metabolism during infection, using oxygen as a growth factor promoting proliferation. Given the separate microenvironments of the intestine between the more anaerobic lumen and more oxygenated epithelium, maintaining both anaerobic and aerobic metabolic pathways would be beneficial for pathogen fitness in vivo. V. cholerae benefits from anaerobic nitrate respiration, especially when coordinated with fermentation in a streptomycin-treated adult mouse model (110, 111). Aerobic 22 respiration, on the other hand, was determined to be essential for V. cholerae colonization in the infant mouse where colonization was severely attenuated in the small intestine, the primary site for V. cholerae infection in the human body (103). A much more severe colonization deficiency was observed when aerobic metabolism was disrupted than when anaerobic respiration was disrupted. For both pathogenic and non-pathogenic E. coli, anaerobic respiration is important for growth and persistence in the gut, likely in the anaerobic mucus layer (112). Additionally, pathogenic and non-pathogenic strains also require aerobic respiration for colonization of the mouse, as mutant strains defective for oxidative respiration are displaced in competition with co-respiring cells (54, 113). When comparing aerobic and anaerobic metabolisms, defects in oxidative respiration through the cytochrome bd oxidase were more attenuated for competition colonization in mice compared to nitrate or fumarate reductase deletion strains, indicating aerobic metabolism is more important to E. coli replicative success in the gut of mice (112). As the human intestine is larger than the mouse gut, there is a greater volume of anaerobic space in the human colon, which may increase the relative importance of anaerobic metabolism for E. coli colonization. S. Typhimurium also benefits from both anaerobic and aerobic metabolism to proliferate in the mouse gut (7). In genetically resistant mice that do not become moribund after S. Typhimurium infection, Clostridia spp. were reduced, and aerobic respiration was required for optimal colonization (7). In the same study, anaerobic nitrate respiration 23 was also necessary for optimal colonization of the mouse gut, suggesting the dual proliferative functions of both metabolic pathways (7). Anaerobic respiration of ethanolamine was also shown to be an important driver of S. Typhimurium growth as tetrathionate becomes readily available in the gut following the respiratory burst of PMNs during disease (114, 115). C. rodentium influences its own aerobic expansion in the gut through cryptic hyperplasia via the T3SS, which influences expansion of undifferentiated proliferating (Ki67-positive) colonic epithelial cells (42). Undifferentiated Ki67 epithelial cells are incapable of butyrate oxidation and are therefore likely to lead to higher levels of oxygen in the gut. Ki67 expansion may not be the only host change that affects oxygen availability as a C. rodentium espO (hyperplasia-inducing factor) mutant had reduced Ki67 counts yet demonstrated similar levels of shedding compared to WT (116). Nonetheless, the effects of C. rodentium infection change host cell metabolism in the infection microenvironment to one of anaerobic glycolysis and lactate efflux in mice alongside reduced uptake of microbiota-derived butyrate which promotes lumen oxygenation (117). For C. rodentium, aerobic expansion is considered to be important for colonization as strains lacking a functional cydAB bd oxidase were attenuated for colonization of the adult mouse (42). For the microaerophile C. jejuni, oxygen is required for a class I ribonucleotide reductase for DNA synthesis (118). Oxygen is therefore necessary to support DNA replication and cell division. However, too much oxygen is detrimental to C. jejuni as it is 24 non-viable at atmospheric oxygen levels. In a unique form of regulatory metabolism, formate is highly metabolized by aerobic respiration in C. jejuni, but as it is used, reduces oxidase activity resulting in expression of proteins needed for alternative electron acceptor anaerobic respiration (119). Thus C. jejuni fine tunes its response to oxygen to maintain DNA replication while also consuming excess oxygen to maintain cell viability. Clostridioides difficile, an obligate anaerobe, is particularly susceptible to oxidative damage (120). C. difficile employs specialized strategies to detoxify molecular oxygen that enters the cell. One enzyme produced to limit oxygen within the cell is cysteine desulfurase (IscS2), which plays a currently undefined role in oxygen resistance (121). Additionally, two flavodiiron proteins (FdpA and FdpF) and two reverse rubrerythrins (revRbr1 and revRbr2) detoxify oxygen and function additively in oxygen tolerance to maintain cell viability (122, 123). In addition to oxygen tolerance, reverse rubrerythrins also have peroxide reductase activity, further protecting C. difficile from this reactive oxygen species (123). Reactive oxygen species are particularly harmful to C. difficile as it lacks canonical ROS defense proteins. To address this, C. difficile employs a specialized strategy to prevent oxidative stress by sensing and capturing host heme during gastroenteritis for protection against redox stressors (124). Cells lacking the heme capture system hsmRA were attenuated for mouse colonization, indicating an essential role for this novel defense strategy (124). Oxygen and ROS defenses are critical for C. difficile as oxygen becomes prevalent during antibiotic treatment, 25 predisposing the host to infection, and during pathogen-induced gastroenteritis, which increases oxygen and ROS presence in the gut. 1.6.2 – Reactive Oxygen Species in Bacterial Growth and Death An important host defense is production of reactive oxygen species, which can affect enteric pathogen survival (125). Host polymorphonuclear neutrophils (PMNs) generate reactive oxygen species by activating NADPH oxidase to limit pathogen survival (126). Superoxide (O2-), hydrogen peroxide (H2O2), hydroxyl radicals (OH·), and hypochlorous acid (HOCl) are reactive oxygen species made by PMNs and macrophages that arrest the growth of bacterial pathogens (127, 128). And pathogens have evolved multiple mechanisms to adequately survive this stress during infection. These involve production of catalase, superoxide dismutase, and peroxidase which neutralize the reactive agents (129, 130). Gene expression to resist ROS is controlled through oxygen sensors such as Fnr (131) and ArcAB (83) and, in the case of response to hypochlorite, the transcription factor YjiE (132). These resistance mechanisms, however, do not completely protect bacteria, as at high levels of ROS cells will succumb to irreparable cellular damage (133). Even after ROS stressors are removed prior to cell death, once the threshold level is met E. coli cells will progress to cell death (134). Bacteria also have the capacity to exploit subinhibitory ROS levels to their advantage. In non-pathogenic E. coli, catalases katE and katG covert H2O2 into molecular oxygen that is respired through the secondary bd-II oxidase appBCX (cyxAB) conferring a growth advantage in the inflamed gut (135). As this has been observed in non-pathogenic E. coli, it is of interest to investigate the potential for pathogenic E. coli species and other 26 facultative anaerobic pathogens to capture and use ROS for enhanced oxidative respiration. 1.7 – The Terminal Oxidases of Bacterial Pathogens Bacterial pathogens that respire oxygen encode terminal oxidase complexes for oxygen reduction (Figure 1.5). These terminal oxidases can be of the heme-copper oxidase (HCO) superfamily, which is subdivided into A, B, and C-type (136), or of the quinol oxidase family (137). In the final step of the electron transport chain (ETC), these terminal oxidase complexes catalyze the reduction of molecular oxygen to water [O 2 + 4H+ + 4e-  2H2O] which generates an electrochemical proton gradient across the cytoplasmic membrane as four positively charged protons are consumed in this reaction (138). This step is consistent among all terminal oxidase complexes, however, additional electron transfer steps in the ETC and proton translocation efficiencies based on HCO-type vary among bacterial respiratory chains and contribute to the generation of a proton gradient (138). Generally, HCO terminal reduction is coupled to proton translocation which translates to higher energy production compared to bd oxidases (138). bd oxidases are not coupled to proton translocation, and are further characterized by typically having high affinities for oxygen, being composed of two subunits, and are subdivided into either short or long Q-loop categories base on the connecting loop between transmembrane helices 6 and 7 in subunit I (137). The electrochemical proton gradient generated through the ETC is important for the functionality of ATP synthase which generates high energy ATP for supporting cellular processes and growth (139). 27 Figure 1.5. Terminal oxidase complexes of enteric pathogens. Escherichia coli and Salmonella enterica both maintain a single bo3 oxidase complex, cyoABCDE, alongside two bd oxidases cydAB and appBC (cyxAB). Each of these complexes acquires electrons from the quinone pool. Vibrio cholerae maintains a single cbb3 oxidase, ccoNOQP, and three bd oxidases, cydAB-I, cydAB-II, and appBC. The cbb3 complex receives electrons from cytochrome c4 whereas the bd oxidases receive electrons from the quinone pool. Campylobacter jejuni maintains a cbb3 oxidase, ccoNOQP, and a bd-like oxidase, cioAB. The cbb3 complex receives electrons from one of three cytochrome c’s, cccA, cccB, or cccC whereas the bd-like oxidase receives electrons from the quinone pool. Listeria monocytogenes maintains an aa3 oxidase, qoxABCD, and a bd oxidase, cydAB. The aa3 oxidase receives electrons from a cytochrome c and the bd oxidase receives electrons from the quinone pool. All terminal oxidase complexes perform the terminal reduction of oxygen to water [O2 + 4H+ +4e-  2H2O]. 28 Broadly, bacteria possess multiple terminal oxidase complexes as part of a branched respiratory pathway repertoire (138). Enteric pathogens and their associated terminal oxidase complexes are listed in Table 1. Maintaining functionally redundant terminal oxidase complexes may be attributed to the diversity of environments enteric pathogens inhabit (140). For example, E. coli contains three terminal oxidase complexes, the HCO bo3 (cyoABCDE), and two bd-type oxidases, bd-I (cydAB) and bd-II (cyxAB). The oxidases are differentially expressed, with the bo3 complex maximally expressed in highly oxygenated environments, and the bd-type oxidases maximally expressed in microaerobiosis (141). In microaerobic conditions, a combined signaling response between the ArcAB/FNR systems leads to expression of cydAB as opposed to cyoABCDE (142). This coordinated regulation leads to cydAB expression in low oxygen environments, such as the gut, while restricting cyoABCDE expression. This is particularly beneficial as bd oxidases have relatively high affinities for oxygen compared to the low affinity bo3 oxidase, which is preferable in low oxygen microenvironments of the intestine (143, 144). Indeed, the bd-I terminal oxidase has been shown to be necessary for non-pathogenic E. coli expansion during inflammation (54) and the bd-II supports aerobic respiration for E. coli following ROS detoxification by catalase enzymes (135). The bd-I oxidase was also shown to be important for pathogenic EHEC host colonization as a cydAB deletion strain is readily outcompeted by the wild type strain (113). 29 Table 1.1. Enteric pathogens and terminal oxidase complexes. Pathogen Terminal Oxidase Reference Complexes Escherichia coli bo3 (cyoABCDE) (145, 146) bd-I (cydAB) bd-II (cyxAB / appBC) Salmonella enterica bo3 (cyoABCDE) (7) serovar Typhimurium bd (cydAB) bd (cyxAB) Vibrio cholerae cbb3 (ccoNOQP) (147) bd (cydAB-I) bd (cydAB-II) bd (appBC) Listeria monocytogenes aa3 (qoxABCD) (148) bd (cydAB) Campylobacter jejuni cbb3 (ccoNOQP) (149) bd (cioAB) Clostridium difficile - (123) bd-type oxidases provide additional protective functions for bacterial cells during infection. The bd oxidase of E. coli displays high catalase activity and protects the cells from hydrogen peroxide stress (150). Additionally, compared to the bo3 oxidase, bd oxidases are less sensitive to nitric oxide, another host produced defense molecule, making the bd oxidase better suited for use in the intestinal environment (151). Finally, the bd oxidases are insensitive to sulfide, a host and microbiota produced oxidase inhibitor, allowing for growth in the intestinal environment (152). Aside from E. coli, terminal oxidases are also important in the pathogenesis of S. Typhimurium, S. flexneri, C. jejuni, L. monocytogenes, and V. cholerae. In S. Typhimurium infection, cydAB was determined to be important in host tissue, whereas cyxAB was important in gut colonization (7). However, a separate study also found cydAB to be important in the gut by fecal sample analysis (38). Cytochrome bd was 30 required for expansion of S. flexneri by generating hypoxic foci that promote proliferation during later stages of infection (71). In C. jejuni CioAB, a low-affinity bd-like oxidase, is needed for optimal microaerobic growth (149) and was upregulated in the rabbit ileal loop model (153). However, the cbb3 oxidase of C. jejuni was concluded to be more important for microaerobic growth, as the authors of this study were unable to generate the mutant strain and the cbb3 oxidase had a higher relative affinity for oxygen (149). This was supported as a C. jejuni CcoN::Cm (cbb3) mutant was unable to colonize the commensal gut of the chicken, while a mutant lacking cioAB had no colonization defect (154). L. monocytogenes maintains two terminal oxidases, a bd and aa3-type which allow growth at different oxygen levels. The bd oxidase supports growth in atmospheric oxygen and intracellularly, whereas the aa3 oxidase supports growth during the initial stages of infection (148). V. cholerae contains one cbb3 and three bd oxidases which have yet to be investigated individually for their functionality during infection and are the subject of work described in this thesis (147). 1.8 – Measuring Oxygen During Infection As oxygen is a prominent force driving bacterial infection, methods to measure oxygen in vivo can further our understanding of oxygen-driven pathogen dynamics during infection. A variety of technologies exist capable of probing oxygen concentrations in model systems, and we discuss some of the more recent tools available in this respect. 1.8.1 – Hypoxia Indicators One method by which oxygen levels have been measured in animal models is with oxygen-sensitive nitroimidazole compounds, particularly pimonidazole which is suspected to bind to hypoxic tissues at oxygen concentrations <10mmHg O2 (155). 31 Pimonidazole binds irreversibly to cellular nucleophiles under hypoxic conditions in the presence of nitroreductase enzymes (156) and can be targeted by secondary fluorescent antibodies in formalin fixed and paraffin embedded tissues to visualize hypoxia in intestinal segments (7, 157). A second hypoxia indicator that has been used is HIF-1. HIF-1 is induced in response to an array of pathogens including bacterial, viral, fungal, and protozoa and is expressed highly in foci of infection where bacterial pathogens readily consume available oxygen (158). Immunohistochemical staining of HIF-1 regulated claudin-1 and GLUT1 has been used to detect tissue hypoxia in fixed tissues of infected animal models (33, 72). These methods are useful for the indirect measurement of oxygen at host cell interfaces but lack the ability to measure oxygen directly and beyond host cell surfaces. 1.8.2 – Phosphorescent Probes Phosphorescence oximetry is another method by which oxygen levels in the gut can be assessed. Oxyphor G4 and Oxyphor R4 are two oximetry probes that have been especially synthesized for use in in vivo tissue samples (159). Oxyphor G4 was used to determine radial oxygen gradients in mouse tissue alongside a novel luminal probe mix OxyphorMicro (3). These probes work by the phosphorescent quenching method. In this method, an excitation fiber excites the probe, and a phosphorescence detector reads the decay rate over short timescales corresponding to the partial pressure of oxygen in the environment, as oxygen quenches the probe (159). Use of these types of probes is challenging in in vivo systems as it requires surgical laparotomy in animal models to position excitation and detector wirings near the intestinal wall (3). These phosphorescent probes have seen growing use in emerging microfluidic applications 32 examining host-pathogen interactions (160, 161). A novel microfluidic device has been developed using a noninvasive phosphorescent film to measure oxygen levels (162), supporting a host-bacteria interface that could be used in future studies of pathogen interactions (163). 1.8.3 – Protein Biosensors Oxygen-sensitive proteins are also used to measure oxygen levels within cells. A Förster resonance energy transfer (FRET)-based biosensor was developed where yellow fluorescent protein (YFP) and hypoxia-tolerant Flavin-binding fluorescent protein (FbFP) were linked in a biosensor called FluBO (164). Oxygen is required for the maturation of the YFP chromophore which will only receive energy to emit fluorescence from FbFP when oxygen is present (164). However, oxygen-dependent maturation of YFP is irreversible, meaning the dynamic shifts in oxygen availability likely to occur in an in vivo host environment are difficult to measure over time. Another sensor, ANA-Y, was recently developed in which a heme-based protein, linked to YFP variant Venus, responds sensitively to oxygen and can measure oxygen levels as low as 6µM (165). This probe does, however, rely on the presence of available heme and functions within a particular range of oxygen availability that may not capture all degrees of oxygen availability during infection. Much of this work has been conducted in batch cultures, but probes such as this could be valuable to measure oxygen exposure during infection, especially when paired with other imaging technologies. Previous work has taken advantage of this approach, using a HIFα luciferase fusion reporter in mice, detecting regions of hypoxia where luciferase bioluminescence was detected by IVIS (166). 33 1.9 – Conclusions and Future Perspectives Oxygen is critical in shaping enteric infections, which are a continuing concern for global human health. Investigating the role of oxygen during infection by bacterial pathogens is necessary to our understanding of disease onset and disease progression. How oxygen influences bacterial infection is only recently explored as a crucial element of pathogen fitness and pathogenicity, and we are learning that oxidative metabolism and response networks are essential for fitness within the host environment. Oxygen-directed metabolism, induced in the presence of oxygen, can be essential for pathogen establishment within the intestinal environment. Particularly for pathogens capable of oxidative respiration that maintain a terminal oxidase complex, oxygen-driven population expansion exacerbates disease and promotes dissemination. Of future interest is the dynamic relationship between resident microbes, pathogens, and host cells that influence oxygen abundance within the gut. The temporal changes in oxygen availability arising from complex host-microbe (pathogenic and commensal) interactions are important to understand for a more complete knowledge of enteric infections and how to combat them. Although recent technologies for measuring oxygen during infection have been discussed, limitations to these approaches make accurate longitudinal measurements difficult. Oxygen is instrumental in enteric infections, and its impact on progression of infectious diseases of the intestinal tract is a rich area of study for future investigators. 34 Chapter 2 – Aerobic Metabolism in Vibrio cholerae is Required for Population Expansion During Infection 35 2.1 – Preface Contents of this chapter were published in the journal mBio in 2020 (Citation: Van Alst AV and DiRita VJ. 2021. Aerobic Metabolism in Vibrio cholerae is Required for Population Expansion during Infection. mBio 11: 5 e01989-20.) Per ASM reuse of published materials guidelines: “Authors in ASM journals retain the right to republish discrete portions of his/her article in any other publication (including print, CD-ROM, and other electronic formats) of which he or she is author or editor, provided that proper credit is given to the original ASM publication. ASM authors also retain the right to reuse the full article in his/her dissertation or thesis.” 2.2 – Abstract Vibrio cholerae replicates to high cell density in the human small intestine leading to the diarrheal disease cholera. During infection, V. cholerae senses and responds to environmental signals that govern cellular responses. Spatial localization of V. cholerae within the intestine affects nutrient availability and metabolic pathways required for replicative success. Metabolic processes used by V. cholerae to reach such high cell densities are not fully known. We sought to better define the metabolic traits that contribute to high levels of V. cholerae during infection. By disrupting the pyruvate dehydrogenase (PDH) complex and pyruvate formate-lyase (PFL), we could differentiate aerobic and anaerobic metabolic pathway involvement in V. cholerae proliferation. We demonstrate that oxidative metabolism is a key contributor to the replicative success of V. cholerae in vivo using an infant mouse model in which PDH mutants were attenuated 100-fold relative to the wild type for colonization. Additionally, metabolism of host substrates, including mucin, was determined to support V. cholerae 36 growth in vitro as a sole carbon source, primarily under aerobic growth conditions. Mucin likely contributes to population expansion during human infection as it is a ubiquitous source of carbohydrates. These data highlight oxidative metabolism as important in the intestinal environment and warrant further investigation of how oxygen and other host substrates shape the intestinal landscape that ultimately influences bacterial disease. We conclude from our results that oxidative metabolism of host substrates is a key driver of V. cholerae proliferation during infection, leading to the substantial bacterial burden exhibited in cholera patients. 2.3 – Importance Vibrio cholerae remains a challenge in the developing world and incidence of the disease it causes, cholera, is anticipated to increase with rising global temperatures and with emergent, highly infectious strains. At present, the underlying metabolic processes that support V. cholerae growth during infection are less well understood than specific virulence traits such as production of a toxin or pilus. In this study we determined that oxidative metabolism of host substrates such as mucin contribute significantly to V. cholerae population expansion in vivo. Identifying metabolic pathways critical for growth can provide avenues for controlling V. cholerae infection and the knowledge may be translatable to other pathogens of the gastrointestinal tract. 2.4 – Introduction Vibrio cholerae causes the diarrheagenic disease cholera in humans and is particularly problematic in regions of the world with poor water sanitation. Ingesting contaminated water sources containing sufficiently high numbers of V. cholerae bacterial cells leads to infection characterized by excessive fluid loss and a substantial bacterial burden of V. 37 cholerae during the acute phase of disease. In the human gastrointestinal tract, V. cholerae can proliferate to numbers as high as 106 to 108 cells per gram of stool (167). In this study, we sought to understand the metabolic requirements for V. cholerae that support such substantial population expansion within the gut. The mucous lining of the gastrointestinal tract, which typically serves as a barrier to infection, is saturated with a variety of carbohydrates. Mucin is a glycoprotein and the primary macromolecule of mucus. Mucin consists of a protein backbone decorated with O-linked glycan chains containing sugars such as N-acetylgalactosamine, N- acetylglucosamine, galactose, fucose, and sialic acid (168). Commensal mucin- degrading bacteria, such as Bacteroides spp. and Akkermansia mucinophila, contain numerous mucinolytic enzymes capable of releasing these sugars from the mucin glycan chain to support growth (168, 169). Mucin degradation is also a feature of bacterial pathogens such as Shigella flexneri, Helicobacter pylori, and enterohemorrhagic Escherichia coli (170–173). V. cholerae contains a number of mucolytic glycosyl hydrolases that are predicted to release glycans from mucin polysaccharides (174–176). Indeed, previous studies have linked mucus carbohydrate metabolism with infection, as V. cholerae mutants defective for N-acetylglucosamine and sialic acid metabolism were attenuated for colonization in the infant mouse (175– 177). The mechanism of acquisition of these mucin carbohydrates may be through both phosphoenolpyruvate phosphostransferase-dependent and independent systems to support growth in vivo (178). Although mucin can serve as a substrate for growth, chemical reduction of intestinal mucus during infection leads to increased numbers of V. 38 cholerae, indicating that mucus also contributes to intestinal protection and clearance of the bacteria (179). V. cholerae harbors the complete enzymatic pathways for the Embden-Meyerhof- Parnas pathway (EMP/glycolysis) pathway, the Entner-Doudoroff pathway (ED) pathway, and the pentose phosphate (PP) pathway (180, 181). The EMP and ED pathways predominantly generate the energy necessary for V. cholerae growth and proliferation. Additionally, previous work has shown that these pathways promote virulence factor production, although the direct cause for this effect is unknown (181, 182). In contrast, the PP pathway does not appear to play a significant role in the growth or colonization of V. cholerae during infection (183). These pathways culminate with the formation of pyruvate, which can then be used by the bacterium to fuel either aerobic or anaerobic metabolism to generate energy for the cell. To expand our understanding of carbohydrate metabolism and its impact on V. cholerae in vivo fitness, we targeted the pyruvate dehydrogenase (PDH) complex and pyruvate formate-lyase (PFL), which both function to convert pyruvate to acetyl coenzyme A (acetyl-CoA) (184, 185). Examination of PDH and PFL mutants enables us to assess the contribution of aerobic and anaerobic metabolism to the expansion of V. cholerae during infection. The conversion of pyruvate to acetyl-CoA precedes the tricarboxylic acid (TCA) cycle, as the first step in the cycle requires acetyl-CoA to generate citrate. In previous work, V. cholerae mutants defective in the TCA cycle expressed increased levels of toxT, which encodes the major virulence gene activator in V. cholerae. This 39 finding suggested a link between acetyl-CoA and virulence expression (186). However, these TCA cycle mutants were not tested in vivo and have been investigated only in classical biotype strains, not in strains of the El Tor biotype. Classical V. cholerae predominated among epidemic isolates prior to 1961, when it was supplanted by the El Tor biotype (187). The biotypes are differentiated by numerous physiological attributes that contributed to displacement of the classical biotype by the El Tor biotype (188– 191). Some of these are encoded on genomic islands unique to the El Tor biotype that contribute to phage resistance or acquisition of substrates (192, 193). In this study, we assessed pathways of carbohydrate metabolism as they contribute to growth, virulence factor production, and colonization of V. cholerae El Tor strain C6706. By targeting the PDH complex and PFL, we are able to draw conclusions about the aerobic and anaerobic metabolic processes that facilitate population expansion of V. cholerae during infection. Our results provide evidence supporting the importance of a functional PDH complex during infection, with significantly less reliance on PFL function. This indicates that oxidative metabolism primarily drives the growth and proliferation required to amass the high bacterial cell density observed during the disease cholera. The defects in colonization observed with strains lacking a functional PDH are attributable primarily to metabolic deficiencies, as virulence factor production was unaffected by mutation in these metabolic pathways. Given what is known in regard to oxygen availability within the intestinal environment, being highest in the intestinal crypts and decreasing to near hypoxia in the lumen, we can deduce the biogeographical localization of replicative V. cholerae (3). Our work suggests that replication within 40 intestinal crypt spaces, observed by others (179), is enabled due to the higher oxygenation of this site than of the lumen. Furthermore, by using the physiologically relevant growth substrate mucin, we could closely reflect, and assess, the growth substrates typically encountered by V. cholerae during infection. The results of this study further our understanding of central metabolism and its contribution to V. cholerae infectivity and in vivo growth. 2.5 – Materials and Methods 2.5.1 – Transposon Mutagenesis Library Screen We used a non-redundant transposon mutant library collection constructed in the El Tor C6706 background (194). Using a 96-well plate replicator, the library was replica plated onto large LB + kanamycin (0.05mg/mL) agar plates and incubated overnight at 37ºC. Subsequently, this LB-grown library was replica plated onto minimal MCLMAN media (195) plates supplemented with 0.5% type III porcine gastric mucin (Sigma). Transposon insertion mutants that were qualitatively defective for growth compared to neighboring transposon insertion mutants were marked as deficient for mucin utilization for further investigation. The complete list of identified mutants is in Table A.1. 2.5.2 – Porcine Small Intestinal Mucus Collection and Mucin Purification Fresh porcine small intestinal segments were harvested from healthy adult pigs from the Michigan State University Meat Lab. Mucus was scraped from the intestinal segments and purified in a manner similar to that previously described (196). Briefly, crude mucus was solubilized and resuspended in extraction guanidine hydrochloride (extraction GuHCl) (6M guanidine hydrochloride, 5mM EDTA, 0.01M NaH2PO4, [pH 6.5]) and 41 homogenized using a Dounce homogenizer. The crude mucus was then rocked overnight at 4ºC, followed by centrifugation at 14,000rpm and 10ºC for 45min. The supernatant was removed, and samples were washed again with extraction GuHCl. Samples were washed and centrifuged a total of five times or until the supernatant appeared clear for two consecutive washes. Mucin was then solubilized using 20ml of reduction guanidine hydrochloride (Reduction GuHCl) (6M guanidine hydrochloride, 0.1M Tris, 0.5mM EDTA, [pH 8.0]) with the addition of 25mM dithiothreitol (DTT) as powder just before use and rocked for 5 hours at 37ºC. A 75mM iodoacetamide was added after incubation as powder, and samples were rotated in the dark overnight. Samples were then centrifuged at 4,000rpm for 45min at 4ºC. The supernatant was added to dialysis tubing and dialyzed in double-distilled water (ddH2O) for a total of six changes. The samples were flash-frozen using liquid nitrogen and lyophilized for purified mucin powder. 2.5.3 – Bacterial Strains and Growth Conditions Vibrio cholerae and Escherichia coli strains used in this study are listed in Table A.2. Unless otherwise specified, V. cholerae and E. coli strains were grown aerobically at 37ºC on LB agar plates or with shaking at 210rpm in LB broth. Where indicated, antibiotics were routinely added to the media at concentrations: 0.1mg /ml streptomycin, 0.1mg/ml ampicillin, and 0.05mg/ml kanamycin. LB media was prepared according to a previously reported recipe (197); however, solid media was made with a 1.5% (wt/vol) concentration of agar. 2.5.4 – Primers Primers used in this study are listed in Table A.3. 42 2.5.5 – Plasmid Construction Plasmid construct inserts were generated by PCR using Phusion high-fidelity polymerase (Thermo Scientific). Vector backbones were generated by plasmid purification using Qiagen Mini Prep Kit and subsequent restriction digest. A modified pKAS32 suicide vector was constructed to generate ΔaceE, ΔaceF, and ΔpflA strains (198). Primer sets were used to amplify 1,000-bp homologous regions upstream and downstream of the target gene. The pKAS32 vector was restriction digested using SacI and XbaI at 37ºC for 1 h, followed by an additional 30 min at 37ºC with alkaline phosphatase from calf intestine (CIP; New England Biolabs). Vector backbone and upstream and downstream segments were joined using Gibson assembly (New England Biolabs) and subsequently electroporated into electrocompetent E. coli S17 λpir and recovered on agar plates with LB ampicillin (0.1 mg/ml). A description of the contruction of complementation plasmids can be found in the supplemental methods. 2.5.6 – Vibrio cholerae Mutant Construction Wild type V. cholerae and E. coli strains were mated on LB agar plates at 37ºC overnight. The mating was then plated on LB ampicillin (0.1mg/ml) and polymyxin B (25 U/ml). Colonies were then subjected to streptomycin counterselection as described 43 previously using LB streptomycin (2.5mg/ml) (198). Colonies were screened for the deletion using primer sets upstream and downstream of the pKAS32 homology regions. A description of the generation of complementation strains can be found in the supplemental methods. 2.5.7 – Growth Curves M9 0.5% purified porcine small intestinal mucin (PSIM) was made by combining in a 1:1 mixture 2X M9 minimal media and 2X (1%) PSIM prepared as a final 20-ml volume which was then autoclaved for 20 minutes at 121°C. Strains were initially grown on LB streptomycin (0.1mg/ml) overnight at 37ºC, and a single colony isolate was used to start a fresh broth culture on LB streptomycin (0.1mg/ml) grown overnight 210rpm at 37ºC. Overnight cultures were washed twice in phosphate-buffered saline and resuspended to an optical density at 600nm (OD600) of 1.0. Aerobic growth curves For LB growth curves, a 1:1,000 dilution of a culture at an OD600 of 1.0 was used to inoculate prepared medium (either 2ml or 50ml, depending on the experiment) which was grown at 210rpm and 37ºC. For M9 plus 0.5% PSIM, 2ml of media was added to a 15-ml round-bottom tube and inoculated 1:250 with a culture at an OD600 of 1.0 and 44 grown at 210rpm and 37ºC. At each timepoint, 100µl was removed for dilution series plating. Anaerobic growth curves Anaerobiosis was achieved using a Coy anaerobic chamber. For LB growth curves, a 1:1,000 dilution of a culture at an OD600 of 1.0 was used to inoculate prepared medium and grown statically at 37ºC. WT, ΔaceE, and ΔaceF growth curves were carried out in 50ml of LB medium in a 125-ml flask, whereas later WT and ΔpflA strain LB growth curves were performed in 2ml of LB media in a 15-ml round-bottom tube. For M9 0.5% PSIM, 2ml of medium was added to a 15-ml round-bottom tube and inoculated 1:250 with a culture at an OD600 of 1.0 which was grown statically at 37ºC. At each time point, the flask and tubes were swirled or vortexed and 100µl was removed for dilution series plating. For growth curves including 50mM fumarate, sodium fumarate (Sigma) reagent was used. Complementation growth curves Methods for complementation growth curves can be found in the supplemental methods. 2.5.8 – AKI Virulence-Inducing Conditions Standard AKI Conditions Wild type, ΔaceE, ΔaceF, and ΔtoxT strains were grown statically in 50ml pre-warmed AKI medium in 50ml conical tubes for 4 h at 37ºC, followed by a transfer to 125-ml flasks and shaking at 210rpm and 37ºC (199). One milliliter of medium was removed at 45 each time point and centrifuged 14,000rpm for 1 min. Supernatant was separated from the pellet and stored at -80ºC for cholera toxin quantification. The bacterial pellet was resuspended in 1ml TRIzol (Ambion Life Technologies) and stored at -80ºC for RNA isolation. Anaerobic AKI conditions Wild type, ΔaceE, ΔaceF, and ΔtoxT strains were grown in 50ml pre-warmed oxygen- depleted AKI medium in 50-ml conical tubes statically under anaerobic conditions using a Coy anaerobic Chamber (78). One milliliter of medium was removed at each timepoint and centrifuged 14,000rpm for 1 min. The supernatant was separated from the pellet and stored at -80ºC for cholera toxin quantification. 2.5.9 – Cholera Toxin Quantification by ELISA Cholera toxin in V. cholerae supernatants from standard and anaerobic AKI conditions was quantified by GM1 enzyme-linked immunosorbent assay (ELISA) as previously described (200, 201). GM1-coated microtiter plates were incubated with a 1:20 dilution of culture supernatant and detected using primary anti-cholera toxin and secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Invitrogen). 1-Step Ultra TMB-ELISA (Thermo Scientific) reagent was added and stabilized using 2M sulfuric acid. Colorimetric measurements were read at 450nm, and the toxin concentration was determined by comparison to a standard curve using purified cholera toxin. 2.5.10 – RNA Isolation and Real Time Quantitative PCR (RT-qPCR) RNA was harvested from AKI toxin-inducing culture pellets preserved in 1ml TRIzol using an RNEasy kit (Qiagen) using on-column DNase digestion (Qiagen) followed by 46 Turbo DNase digestion (Invitrogen). RNA concentration and quality were measured with a UV/VIS Spectrophotometer and visualized on a 2% agarose gel. cDNA was generated from RNA using Superscript III reverse transcriptase (Thermo Scientific). RT-qPCRa were carried out using SYBR green master mix (Applied Biosystems) with 5ng cDNA. Primers used to detect recA, toxT, ctxA, and tcpA transcripts are listed in Table A.3. Threshold cycle (ΔΔCT) values were calculated using recA as the gene of reference (202). 2.5.11 – Infant Mouse Colonization Assays All animal experiments in this study were approved by the Institutional Animal Care and Use Committee at Michigan State University. Infant mice were infected as described previously (203). Three- to five- day old CD-1 mice (Charles River, Wilmington, MA) were orogastrically inoculated with approximately 106 bacterial cells 2 h after separation from the dam and maintained at 30ºC. Mice were euthanized approximately 20 hours after inoculation. Mouse intestinal segments were weighed and homogenized in 3ml of PBS. Intestinal homogenates were serially diluted and plated on LB streptomycin (0.1mg/ml) for monoassociated infections and LB streptomycin (0.1mg/ml) and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X- Gal)(0.08mg/ml) for competition infections. Competition infections consisted of a 1:1 mixture of target strains with a ΔlacZ strain for differentiation by blue-white screening. 47 For PDH monoassociated and competition infections, the entire intestinal tract was homogenized for bacterial enumeration. In intestinal segment measurements, approximately 1cm of intestine from each section (proximal, medial, and distal) was homogenized for bacterial enumeration between segments. For PFL monoassociated and competition infections, the intestinal tract was divided into small intestine and large intestine plus cecum. The divided intestinal portions were then homogenized for bacterial enumeration. 2.5.12 – Statistical Methods For determining the relationship between cholera toxin output versus optical density among WT, ΔaceE, and ΔaceF strains, a simple linear regression and subsequent slope and intercept analysis were performed using GraphPad Prism software, which follows a method equivalent to analysis of covariance (ANCOVA). For in vivo experiments, CFU-per-gram intestine and competitive index scores were log10 transformed and tested for normality using a Shapiro-Wilks test. Normally distributed data were then analyzed using either parametric Student’s t test or an analysis of variance (ANOVA) with post hoc Tukey’s test to test for significance. For in vivo intestinal segment data where bacterial loads were below the limit of detection, a nonparametric Kruskal-Wallis one-way analysis of variance was used with post hoc Dunn’s test to test for significance. 48 2.6 – Results 2.6.1 – Transposon Mutagenesis Screen Identified the Pyruvate Dehydrogenase Complex as Important for Growth on Mucin. We hypothesized that intestinal mucin would serve as a growth substrate for V. cholerae during colonization. In a pilot experiment, V. cholerae was observed to exhibit enhanced growth in minimal media supplemented with mucin (Figure A.1). We then performed a transposon mutant library screen of V. cholerae El Tor strain C6706 on minimal media supplemented with 0.5% mucin (Type III; Sigma). Genes encoding two of the three components of the pyruvate dehydrogenase (PDH) complex were identified in our screen, aceE (VC2414) and aceF (VC2413) (Table A.1). The third component of the PDH complex, lpdA (VC2414), was also defective for growth in our screen; however, growth of this transposon mutant was also severely attenuated for growth on LB, as this enzyme also functions in the alpha-ketoglutarate dehydrogenase (AKGDH) and glycine cleavage multienzyme (GCV) systems (204). Because of its pleiotropic growth defect, we did not further investigate an lpdA mutant. 2.6.2 – The Pyruvate Dehydrogenase Complex Supports Aerobic Growth on Mucin. As a glycoprotein, mucin is coated in glycans, contributing to the protective function of the mucous barrier (205). To study mucin from a physiologically relevant site of infection, and to avoid potential contaminants in commercially purified mucin that may impact V. cholerae growth (206), we purified mucin from the small intestine of healthy adult pigs using a guanidine hydrochloride (GuHCl) extraction procedure. The purified mucin was then analyzed by high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD; GlycoAnalytics) (207, 208). 49 Mucin obtained by this method showed high relative presence of mucin carbohydrates galactosamine, glucosamine, galactose, fucose, and sialic acids Neu5Ac and Neu5Gc compared to non-mucin monosaccharides glucose and mannose (Table A.4). When grown in M9 minimal salts medium supplemented with 0.5% purified small intestinal mucin (PSIM), isogenic ΔaceE and ΔaceF PDH mutants were defective for growth in aerobically grown cultures compared to the wild type (Figure 2.1A). This phenotype was complemented for both the ΔaceE and ΔaceF mutants using the isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible pMMB66EH vector in M9 0.5% glucose media (Figure A.2). To verify the PDH complex does not contribute to anaerobic proliferation on mucin, we measured growth under anaerobic conditions. Under these conditions, PDH mutants grew comparably to the wild type (Figure 2.1B). To determine if addition of an alternative electron acceptor would elicit a growth difference between the wild type and PDH mutants, 50mM fumarate, which enhances V. cholerae growth via anaerobic respiration (209), was added to the growth medium. No growth disparity was observed between the wild type and PDH mutants with the addition of 50mM fumarate (Figure 2.1C). The observed phenotypes indicate that the PDH complex is not required for anaerobic growth. Energy generation under anaerobic conditions is likely due to an active pyruvate formate-lyase converting pyruvate to formate and acetyl-CoA for mixed acid fermentation (210) and acetolactate synthase, which converts pyruvate to (S)-2-acetolactate in the first step of 2,3-butanediol fermentation (191). The growth disparity observed in the minimal mucin medium under aerobic conditions was less pronounced in LB medium, which contains less than 100µM 50 collective sugars and primarily supports growth through amino acid catabolism (Figure A.3) (211). As a growth defect was observed in LB medium, we wanted to test whether this growth delay was attributable solely to perturbed carbohydrate metabolism, or if growth on amino acids was also negatively impacted by a disrupted PDH complex. In M9 supplemented with 0.2% Casamino Acids under aerobic growth conditions, the ΔaceE and ΔaceF mutants did not grow at all (Figure A.4). These findings suggest that aerobic amino acid catabolism may also have contributed to the phenotype illustrated in Figure 2.1A, as mucin molecules contain, among other amino acids, proline, threonine, and serine in repeat glycan attachment moieties and have previously been shown to support V. cholerae growth in vitro (48). It is therefore unclear what component of LB is supporting growth of the ΔaceE and ΔaceF mutants. Figure 2.1. Growth curves of WT ΔaceE, and ΔaceF in M9 minimal media supplemented with 0.5% purified porcine small intestinal mucin. Growth curves of WT, ΔaceE, and ΔaceF in M9 minimal medium supplemented with 0.5% purified porcine small intestinal mucin (PSIM) grown aerobically (A), anaerobically (B), or anaerobically supplemented with 50mM fumarate (C). Data represent the averages and SEMs for three independent biological replicates. 51 2.6.3 – Pyruvate Formate-Lyase Supports Anaerobic Growth on Mucin. As pyruvate formate-lyase (PFL) also converts pyruvate to acetyl-CoA, we sought to investigate the role of PFL in the catabolism of mucin. To accomplish this, an isogenic mutant strain with a deletion of pflA (VC1869) was tested for in vitro growth on PSIM. In M9 minimal salts media supplemented with 0.5% PSIM, the ΔpflA mutant grew comparably to the wild type under aerobic growth conditions (Figure 2.2A) and poorly compared to the wild type (which did not thrive itself) when cultured anaerobically, in both the absence and presence of 50mM fumarate (Figure 2.2B and C). This growth defect was complemented for pflA using an IPTG-inducible pMMB66EH vector in M9 0.5% glucose 50mM fumarate medium (Figure A.5). The disparity in growth between wild type and ΔpflA strains under anaerobic growth conditions indicates that PFL can indeed generate energy from mucin during anaerobic growth. As PFL is expected to function primarily in the metabolism of carbohydrates, it was not surprising to see growth comparable to that of the wild type in LB medium, both aerobic and anaerobic, but it was intriguing to find ΔpflA mutant remained viable longer than wild type in LB medium without the addition of 50mM fumarate (Figure A.6). 52 Figure 2.2. Growth curves of WT and ΔpflA in M9 minimal media supplemented with 0.5% purified porcine small intestinal mucin. Growth curves of WT and ΔpflA in M9 minimal media supplemented with 0.5% purified porcine small intestinal mucin grown aerobically (A), anaerobically (B), or anaerobically supplemented with 50mM fumarate (C). Data represent the averages and SEMs for three independent biological replicates. 2.6.4 – Cholera Toxin Production in PDH Mutants is Equivalent to Wild Type in Both Standard and Anaerobic Toxin-Inducing Conditions. Cholera toxin is the primary virulence determinant of V. cholerae. To determine whether the PDH complex influences production of cholera toxin, wild type and PDH mutant strains were grown under conditions referred to as “AKI” to induce virulence factor production (199). Previous findings with strains of the classical biotype demonstrate that disruption of the TCA cycle increases toxT expression and suggests a link between acetyl-CoA and virulence expression (186). As the PDH complex is the primary enzyme responsible for the production of acetyl-CoA under aerobic growth conditions, we anticipated mutants lacking it would produce cholera toxin levels below that of wild type. However, we observed no significant difference in cholera toxin produced in the WT and PDH mutant strains under either standard or anaerobic AKI conditions. For standard AKI conditions, cholera toxin levels were measured as a function of optical density, with the ΔtoxT (VC0838) included as a negative control, as ToxT stimulates cholera toxin production (Figure 2.3A) (212). Overall, the wild type produced more total cholera toxin as it reached a higher final optical density than PDH mutants, yet when determining individual cellular capacity for cholera toxin production, it was found that at similar optical densities, cholera toxin output in the PDH mutants was comparable to that in the wild type. Cholera toxin production levels at 4h, 5h, 6h, 7h, 8h, and 24h are provided in 53 Figure A.7. Cholera toxin levels were also measured after 8h and 20h of growth under anaerobic AKI conditions and were again similar between the wild type and PDH mutant strains (Figure 2.3B and Figure A.7). These findings indicate that the PDH complex does not affect cholera toxin production in El Tor C6706 V. cholerae. Figure 2.3. Cholera toxin (CT) production for WT, ΔaceE, ΔaceF, and ΔtoxT strains. (A) CT output as a function of optical density (OD600) under standard AKI toxin-inducing conditions. Data points were collected from three biological replicates, and a line of best fit with 95% confidence intervals was plotted. WT ODs higher than 0.9 were excluded to better superimpose with ΔaceE and ΔaceF OD values. A simple linear regression found no significant differences between WT, ΔaceE, and ΔaceF strain CT production. ΔtoxT control was not plotted because no toxin was detected. Statistical analysis was performed using GraphPad Prism (B) CT values relative to optical density (pg/ml/OD600) under anaerobic AKI toxin-inducing conditions at the 8h time point are reported. The optical densities for the biological replicates are displayed below the corresponding strain on the x axis in each graph. Data represent the averages and SEMs for three biological replicates. 2.6.5 – Functional PDH Activity is not Required for Expression of toxT, ctxA, and tcpA. The relative expression of the master virulence regulator toxT and primary virulence factors ctxA and tcpA was determined by real-time quantitative PCR (RT-qPCR). The 4h and 5h time points of in vitro standard AKI conditions were selected to compare relative expression profiles, as toxT expression has been observed to be high at these 54 time points (213). PDH mutant strains at 4h exhibited somewhat elevated levels of toxT and tcpA and similar ctxA expression compared to those of the wild type, with no transcripts detected in the ΔtoxT control, as expected (Figure 2.4A). At 5h, PDH mutant strains exhibited wild type toxT and ctxA expression and a 2-fold reduction in tcpA expression (Figure 2.4B). Although there is variability in the expression of these virulence genes at the given time points, we conclude that the PDH complex is not required for virulence gene expression. Figure 2.4. Relative fold change of toxT, ctxA, and tcpA transcript levels compared to wild type expression. RNA was isolated from WT, ΔaceE, ΔaceF, and ΔtoxT cultures grown under standard AKI toxin-inducing conditions at 4h and 5h. Expression data were calculated by ΔΔCT using recA as an internal control. Data represent the averages and SEMs for three independent biological replicates. 55 2.6.6 – A Functional Pyruvate Dehydrogenase Complex is Necessary for Colonization of the Infant Mouse. Based on our findings that V. cholerae PDH mutants are defective for aerobic carbohydrate metabolism, we sought to examine whether this pathway was required to support growth in vivo. The infant mouse model is used extensively to investigate intestinal colonization by V. cholerae (214). Infant mice produce a mucous layer in the intestine that can serve as a substrate for V. cholerae growth (179), although they also have reduced resident microbiota and a less developed immune system compared to adult mice (215). The reduction in resident flora increases the necessity for V. cholerae to liberate mucin glycans for substrate utilization, as the commensal population does not provide this resource as in other systems (216). In this study, we orogastrically infected CD-1 mouse neonates with ~106 CFU bacterial cells to compare colonizations by wild type and PDH mutant V. cholerae. In monoassociated infections, PDH mutants were attenuated for colonization by approximately 100-fold compared to the wild type (Figure 2.5A). We also assessed direct in vivo competition with wild type by coinfecting each mutant with a PDH+ ΔlacZ (VC2338) strain of V. cholerae. PDH mutant strains exhibited attenuation in competition with the wild type similar to that observed in monoassociated infections, with mutant recovery approximately 100-fold lower than that of the wild type after coinfection (Figure 2.5B). These data indicate a requirement for a functionally active PDH complex to support colonization of the infant mouse, suggesting that oxidative metabolism of carbohydrate substrates is critical for colonization and population expansion. 56 Figure 2.5. Infant mouse colonization assays of WT, ΔaceE, and ΔaceF after 20h. (A) Monoassociated infections of 3- to 5-day-old infant mice. (B) Competition infections of 3- to 5-day-old infant mice. Competitive index scores were calculated as ratios of output versus input [(targetOutput/ΔlacZOutput) / (targetInput/ΔlacZInput)]. WT, ΔaceE, and ΔaceF strains were co-inoculated with an aceE+/aceF+ ΔlacZ strain (PDH+) to determine the relative fitness of each test strain. Data for each experiment was obtained from eight independent mouse colonization infections in which the entire intestinal tract (small intestine, large intestine, and cecum) was extracted and homogenized for bacterial enumeration. Bars represents geometric mean. Statistical analysis was performed using GraphPad Prism where significance was tested on log- transformed data by ANOVA with post hoc Tukey’s Test. *, P<0.05. As oxygen levels (5, 8) and mucin composition (217) fluctuate along the length of the small intestine, we investigated the relative importance of PDH function across the longitudinal axis in the infant mouse intestine. One-centimeter-long pieces of intestine were harvested from the proximal, medial, and distal regions of the small intestine and assayed for recoverable CFU. Throughout the small intestine, V. cholerae PDH mutants were recovered at levels well below that of the wild type and in some cases were not detected, as counts were below our limit of detection (Figure 2.6A-C). Here we again conclude that the PDH complex promotes V. cholerae colonization and that oxidative 57 metabolism of carbohydrates is a key feature of V. cholerae growth and proliferation along the entire length of the small intestine. Additionally, bacterial loads of the PDH mutants across the individual intestinal segments do not appear to reflect the CFU-per- gram counts obtained from analyzing the entire gastrointestinal tract in the previous monoassociated infection (Figure 2.5A). This suggests that the majority of PDH mutants detected in the previous monoinfection experiment resided within either the cecum or large intestine, sites anticipated to support more anaerobic metabolism (5). This finding further supports the necessity for an active PDH, particularly at the primary site of infection in the small intestine. Figure 2.6. Infant mouse colonization of WT, ΔaceE, and ΔaceF mono- associated infections in proximal, medial, and distal portions of the small intestine after 20h. Infant mouse colonization of WT, ΔaceE, and ΔaceF monoassociated infections in proximal (A), medial (B), and distal (C) portions of the small intestine after 20h. Data for each segment was obtained from 8 independent mouse colonization infections. The bars represent the geometric means and can only be plotted for the WT strain. Statistical analysis was performed using GraphPad Prism where significance was tested on nontransformed data by Kruskal-Wallis analysis with post hoc Dunn’s test. *, P<0.05. 58 2.6.7 – Pyruvate Formate-Lyase Provides Minor Growth Support during Infection. As aerobic metabolism was determined to be beneficial to population expansion of V. cholerae, we wanted to explore the contributing effects of anaerobic metabolism to colonization. Oxygen gradation within the small intestine maintains the highest oxygen availability in the intestinal crypts and nears hypoxia at the villus tip (3, 218). To determine if anaerobic proliferation also contributes to population expansion of V. cholerae during infection, potentially in the more anoxic lumen of the small intestine, CD-1 mice were infected with ~106 CFU of the ΔpflA mutant. To accurately assess the importance of PFL during infection, recovery of V. cholerae was performed for the small intestine separately from the large intestine. As the large bowel is inherently more anoxic, where PFL would be expected to function more readily, we focused more on assessing the role of PFL in the small intestine as a more clinically relevant site for human V. cholerae infection. In monoassociated infections, the PFL mutant was attenuated for colonization by approximately 2-fold compared to the wild type (Figure 2.7A). These data suggest that PFL, and therefore anaerobic metabolism, provides a less critical level of energy production than PDH to support growth during infection. Our results are consistent with previous studies that investigated anaerobic nitrate respiration demonstrating a similar (2-fold) reduction in colonization of the infant mouse (110). However, in a competition experiment, the ΔpflA mutant colonized to levels equivalent to those of the wild type (Figure 2.7B), essentially demonstrating no colonization defect at all. The same 59 colonization pattern of the PFL mutant strain was observed in the large intestine (Figure A.8). Figure 2.7. Infant mouse colonization assays of WT and ΔpflA strains in the small intestine after 20h. (A) Monoassociated infections of 3- to 5-day-old infant mice. (B) Competition infections of 3- to 5-day-old infant mice. Competitive index scores were calculated as a ratios of output versus input [(targetOutput/ΔlacZOutput) / (targetInput/ΔlacZInput)]. WT and ΔpflA strains were co-inoculated with a pflA+ ΔlacZ strain to determine the relative fitness of each test strain. Data for each experiment was obtained from 4 or 5 independent mouse colonization infections for WT and 7 or 8 mouse infections for the ΔpflA strain. The bar represents geometric mean. Statistical analysis was performed using Graphpad Prism where significance was tested on log-transformed data by Student’s t test. *, P<0.05. One hypothesis to explain this lack of fitness defect when coinfected with the wild type is that during coinfection, the pflA+ ΔlacZ strain may produce acetate, which can be metabolized by the ΔpflA mutant to mitigate the 2-fold defect seen in monoassociated infections (219). Acetate would provide acetyl-CoA by way of acetyl-CoA synthase-1 (ACS-1), circumventing the PDH/PFL carbohydrate utilization pathways (220). To test this hypothesis, we first demonstrated that all strains are capable of growth on acetate (Figure A.9). Then, to test whether metabolic rescue of the ΔpflA strain by pflA+ ΔlacZ 60 occurs, monocultures and competition cultures were grown anaerobically in M9 minimal media with 0.5% glucose. At both the 12h and 20h time points, the PFL mutant was found to be between 2- and 10-fold reduced compared to pflA+ ΔlacZ strain for both monoculture and competition comparisons. These findings indicate that the similar output ratios detected in competition in vivo assays are unlikely related to acetate supplementation by the pflA+ ΔlacZ strain (Figure A.10). 2.7 – Discussion Oxygen-dependent metabolism is key to pathogenicity for many gastrointestinal microbes. Pathogens that actively manipulate the host environment to oxygenate the gut rapidly proliferate during infection. Although a direct link has yet to be determined, the cholera toxin of V. cholerae may increase oxygen availability in the gut through its influence on optimal TCA cycle activity during infection (43). In other gastrointestinal pathogens, oxidative metabolism is supported by inducing inflammation at the site of infection. Inflammation in response to Citrobacter rodentium or Salmonella enterica serovar Typhimurium infections promotes colonization, proliferation of the microbe, and disease as a result of increased aerobic metabolism (7, 42). While V. cholerae infection does not lead to significant changes in gross pathology of intestinal architecture and cholera is not typically characterized as a proinflammatory infection, inflammatory markers are increased in animal models and human infection (221, 222). Whether oxygen levels in the gut are elevated due to this innate immune response has yet to be determined. There is evidence to support inflammation promoting V. cholerae colonization in some circumstances. In V. cholerae strain V52, the Type VI secretion system (T6SS) increases intestinal inflammation in the infant mouse and promotes 61 increased colonization levels (223). Also, the newly emerged El Tor Haitian variant strain has higher virulence in animal models, reaching a higher bacterial cell burden than previously characterized strains and causing elevated inflammation and epithelial cell damage (224). Evolved V. cholerae strains equipped to withstand an inflamed environment could benefit from increased oxygen availability to generate more energy to support growth and proliferation. Oxygen contribution to V. cholerae pathogenicity has been examined principally in regard to the ToxR/TcpP/ToxT virulence cascade. For example, the regulators AphB and OhrR respond to reduced environmental oxygen by activating tcpP expression (79). Further, decreased oxygen levels under stationary culture conditions stimulates ToxR- TcpP interaction (225) and activation of toxT transcription (213). Translating these in vitro results to the context of oxygen distribution in vivo would imply that the anoxic lumen primes V. cholerae for virulence gene expression prior to accessing the more oxygenated host epithelium. The radial oxygen gradient within the intestine would therefore influence optimal timing of V. cholerae virulence expression. In this work, we sought to explore how oxygen-dependent and independent metabolic pathways influence population expansion during infection, but not necessarily as they relate to virulence gene expression. Oxygen availability in the crypt spaces of the intestine, along with the presence of carbohydrate-rich mucin molecules, could vastly improve growth and proliferation of V. cholerae at this site. The mucous lining of the gastrointestinal tract protects the host 62 epithelium from both resident and transient microorganisms to maintain gut homeostasis (226). V. cholerae has the capacity to bypass this host defense through motility (227) as well as to exploit it for growth substrates. However, this does not suggest that the mucous layer is inconsequential to curtailing the effects of V. cholerae pathogenicity. In mice with a chemically degraded mucus layer, V. cholerae bacterial counts exceeded that of untreated mice (179), indicating that mucus contributes to abatement of disease. Similarly, Muc2-/- mice that lack the primary secretory mucin of the intestinal tract, MUC2, exhibit inflammation in the large intestine due to commensal population interactions with the epithelium as well as exacerbated infection in Citrobacter rodentium and Salmonella Typhimurium challenge models (228–230). Thus, the mucus serves a protective function against V. cholerae yet is exploited to benefit the microbe. The sequential progression of V. cholerae pathogenicity is tightly linked to bacterial- mucin interactions. Adherence to host mucin by GbpA, an N-acetylglucosamine binding protein, is a key step in colonizing the small intestine (231). Upon reaching the host epithelium V. cholerae establishes an adherent microcolony (232). Within this microenvironment, mucin breakdown products and the presence of oxygen help drive the population expansion of V. cholerae (this work). Stimulation of the host epithelium by cholera toxin induces production and secretion of goblet cell mucin (233) in addition to other host-derived nutrients such as iron and long-chain fatty acids (43), and potentially oxygen. As the population of cells rapidly expands, mucin breakdown products stimulate motility of V. cholerae (234), a trait required for optimal colonization 63 of the proximal and medial portions of the small intestine (46, 179, 235). This interaction likely contributes to population dynamics observed for V. cholerae whereby it migrates counter to intestinal flow in the later stages of infection to populate the proximal and medial portions of the intestine (236). This motility response acts in coordination with the secreted mucolytic hemagglutinin/protease (HapA) of V. cholerae, used during cellular detachment (237, 238). HapA is stimulated both by high cell density through the activity of the regulator HapR, and directly when in the presence of mucin (239). As V. cholerae exits the host, a fraction of the population is embedded in mucin (240), which may influence hyperinfectivity of human-passaged V. cholerae (241). From initial inoculation into the human gut to the eventual passaging of the bacteria, interactions between V. cholerae and mucus substantially influence V. cholerae pathogenicity. Our work postulates that mucin metabolism enhances proliferation of V. cholerae during the course of infection. V. cholerae is a facultative anaerobe, and we sought to uncover whether aerobic or anaerobic metabolism enables it to grow to high levels during infection. We assessed the in vivo fitness of strains lacking either the aceE- or aceF- encoded components of the pyruvate dehydrogenase (PDH) complex or pflA-encoded pyruvate formate-lyase (PFL). These enzymes catalyze production of acetyl-CoA from pyruvate either aerobically (PDH) or anaerobically (PFL). In Escherichia coli, PFL is induced only during anaerobiosis, whereas the PDH complex can function in both anaerobic and anaerobic environments (242, 243). However, unlike what is observed in E. coli, in our work, V. cholerae lacking PFL was not rescued for anaerobic growth to any noticeable extent by having a functional PDH. This enabled us to differentiate the 64 contribution of aerobic and anaerobic metabolism by investigating PDH and PFL mutants. The significant loss of fitness by the ΔaceE and ΔaceF strains compared to the wild type strain suggests that V. cholerae population expansion in the small intestine is driven largely by aerobic, oxidative metabolism. This is consistent with V. cholerae preferentially localizing to the epithelial crypts (179), with greater oxygenation that enables oxidative metabolic pathways to generate energy (3). The radial distribution of oxygen in the intestine therefore biogeographically relegates replicative V. cholerae cells primarily to the epithelium, as opposed to the more anoxic lumen. Our results do not completely rule out some anaerobic growth and expansion of V. cholerae during infection, as the ΔpflA mutant colonized to levels about half those of the wild type, similar to what is observed with other anaerobic metabolism-deficient strains (110, 191, 209). Additionally, there is recent evidence to suggest that multiple anaerobic metabolic pathways function in tandem to support growth in anoxic conditions. A double mutant in ethanol fermentation and nitrate respiration showed a significant reduction in colonization, compared to single mutants that were near-wild type levels of colonization (111). While expansion of V. cholerae in vivo evidently proceeds primarily through aerobic production of acetyl-CoA using PDH as opposed to anaerobic acetyl-CoA production using PFL, how it uses its reducing equivalents to generate energy from the electron transport chain is less certain from our work. Oxygen as a terminal electron acceptor is certainly possible given the availability of oxygen within the crypt epithelium and the presence of four terminal oxidase complexes in the V. cholerae genome (147), which 65 will be the subject of future investigation. Previous in vivo transposon mutagenesis studies indicate that terminal oxidase function supports colonization, in particular a high affinity cbb3 oxidase (244, 245). V. cholerae also maintains nitrate, fumarate, TMAO, and DMSO reductases that can function as terminal electron acceptors in anaerobic respiration (147). Fumarate and TMAO support V. cholerae growth in anaerobic conditions (209), as does nitrate when in an alkaline environment (110), albeit not to the extent observed for oxidative growth. This is primarily due to the relatively low redox potentials of fumarate and TMAO relative to O2 and V. cholerae requiring alkaline pH environments for nitrate respiration, as it lacks a nitrite reductase needed to eliminate this toxic compound (110). DMSO, on the other hand, was not shown to support V. cholerae growth at all (209). However, growth in vivo with addition of the alternative electron acceptor TMAO induces high levels of cholera toxin (209). Infant mice infected with an inoculum of El Tor strain N16961 mixed with TMAO exhibited more severe signs of infection, suggesting a TMAO-dependent toxin production effect. Although anaerobic terminal reductases may not be the principal mode of V. cholerae growth and expansion in vivo, they are still likely to contribute to V. cholerae pathogenesis. Resolution of the role of different terminal reductases regarding growth and pathogenicity in vivo also awaits future examination by investigating V. cholerae terminal reductase mutants. 2.8 – Acknowledgements We thank Dr. Jeremiah Johnson for insights and encouragement in initiating this study. AJV was supported in part by SUTL and FAST Fellowships from Michigan State University, and by the Bertina Wentworth Fellowship in the Department of Microbiology 66 & Molecular Genetics at Michigan State University. This work was supported in part by the Rudolph Hugh Endowment (VJD) at Michigan State University. 67 Chapter 3 – Oxidative respiration through the bd-I and cbb3 oxidases is required for Vibrio cholerae pathogenicity and proliferation in vivo. 68 3.1 – Preface Contents of this chapter are unpublished and soon to be submitted for publication. This chapter includes one experiment conducted by a fellow graduate student in the lab of Dr. Victor DiRita, Lucas Demey. This contribution helped generate Figure B.8. 3.2 – Abstract Respiration is an energy generating process that supports growth and proliferation of many enteric pathogens. Vibrio cholerae, the bacterial pathogen that causes the disease cholera, is capable of both aerobic and anaerobic respiration. However, despite knowledge that oxygen diffuses from host tissue into the intestinal lumen, the role of aerobic respiration in supporting V. cholerae growth during infection has yet to be defined. Here, we show that V. cholerae colonization of the infant mouse requires aerobic respiration, but not anaerobic respiration, to support growth and proliferation. Using Multiplex Genome Editing by Natural Transformation (MuGENT) we created a septuple knockout strain lacking the capacity to produce any of the four terminal oxidase complexes (cbb3, bd-I, bd-II, and bd-III) where the resulting strain effectively functions as a strict anaerobic variant of V. cholerae. In infant mouse infections, this resulted in a near 106-fold reduction in colonization. Characterization of individual oxidases identified the cbb3 and bd-I oxidases, but not the bd-II or bd-III oxidases, as essential for colonizing the small intestine of the infant mouse. Unexpectedly, the bd-I oxidase was determined to be the primary oxidase in V. cholerae, only the second example of a bd-type oxidase serving as the primary oxidase supporting energy acquisition both inside and outside of a host for a bacterial pathogen. In addition to determining the oxidase requirements of V. cholerae, the results of this study further 69 implicate oxygen as a critical electron acceptor that shapes the progression of enteric infections. 3.3 – Introduction Respiration promotes growth and proliferation of bacterial cells (138). Energy acquisition through respiration relies on the metabolism of exogenously acquired substrates and the presence of terminal electron acceptor molecules to generate chemical energy, which is stored in the form of ATP to power cellular processes required for growth. Although not considered canonical virulence factors, metabolism and energy generative processes are required by pathogens to thrive during infection. Here we investigate respiration as a potent driver of replication during infection by the bacterial gastrointestinal pathogen Vibrio cholerae (246). Vibrio cholerae is a facultative anaerobe that grows in both aerobic and anaerobic environments (247). Respiration in V. cholerae is achieved aerobically through the terminal reduction of molecular oxygen or anaerobically through the terminal reduction of various alternative electron acceptors (110, 209). Recent evidence suggests there is combined contribution of both aerobic and anaerobic metabolism to V. cholerae growth in vivo (103, 111). This may be attributed to the radial and longitudinal gradients of oxygen availability in the intestinal tract enabling metabolism through both pathways in response to in vivo localization (8). We sought to investigate the relative contributions of aerobic and anaerobic respiration in vivo using an infant mouse model of colonization. V. cholerae encodes four terminal 70 oxidases and four terminal reductases that support respiration (147, 248). The terminal oxidases include one cbb3 heme-copper oxidase (249, 250) and three bd-type oxidase complexes capable of catalyzing the 4 H+/O2 reduction of oxygen to water (137). cbb3 oxidases (251, 252) and bd oxidases (253–255) have a low Km for oxygen and are typically induced under microaerobiosis, a feature particularly beneficial for pathogens colonizing near hypoxic environments of the human host (54, 135, 256). V. cholerae also carries out anaerobic respiration through four terminal reductases that use nitrate, fumarate, trimethylamine-N-oxide (TMAO) or biotin sulfoxide (BSO) (112, 209, 248, 257). Previous work found that abrogation of nitrate reductase activity reduced colonization in a streptomycin-treated adult mouse by approximately 2-fold (110) and concomitant disruption in fermentative pathways further reduced colonization, revealing a dependency between nitrate reduction and fermentation (111). Additionally, TMAO influences virulence gene expression in V. cholerae when added exogenously, however, whether reduction of TMAO is required for this response is not clear (209, 258). In this study, we looked to more thoroughly examine the complete suite of terminal electron accepting complexes encoded by V. cholerae and assess the pertinence of each terminal electron acceptor molecule to in vivo infection. By targeting terminal oxidase and terminal reductase complexes of V. cholerae, we can better understand the respiratory processes occurring during disease and identify which terminal electron accepting complexes are most critical to in vivo fitness. This work highlights that oxygen, although present at low levels diffusing from the host epithelium, is sufficient and essential to supporting V. cholerae growth in the infant mouse. These 71 findings change how we understand the host environment during V. cholerae pathogenesis and how oxygen may function in the pathogenicity of other bacterial pathogen elicited diseases. 3.4 – Materials and Methods 3.4.1 – Bacterial Strains and Growth Conditions Bacterial strains used in this study are listed in Supplemental Table 3. Vibrio cholerae El Tor C6706 was used as the wild type strain in this study and served as the strain background for all V. cholerae mutant derivatives. Strains were grown primarily on LB agar and used to inoculate 4mL LB media in preparation for subsequent assays. Addition of antibiotics when required were in given concentrations: streptomycin (100µg/ml), spectinomycin (200µg/ml), and ampicillin (100µg/ml). Strains were grown at 37°C for all growth assays. Aerobic growth assays were performed at atmospheric oxygen concentrations whereas anaerobiosis for anaerobic growth was maintained using a Coy anaerobic chamber. 3.4.2 – MuGENT Mutant Strain Construction MuGENT generated mutant strains were constructed using Enhanced Multiplex Genome Editing by Natural Transformation (259). Linear segments of V. cholerae genomic DNA were amplified using a primer with intentional base changes designed to introduce a frameshift mutation, removal of ATG start codon, insertion of 3-frame stop codons, and offsetting of the ribosomal binding site while also inserting a universal primer binding site. These fragments, along with a fragment containing an antibiotic resistance cassette in pseudogene VC1807 were transformed into a V. cholerae 72 ΔrecJΔxseA pMMB-tfoX strain. Once all mutants were integrated into the genome of carrier strains, a more traditional MuGENT approach (260) was used to amplify ~2Kb arms of homology on either side of the mutated site in each carrier strain to introduce into wild type V. cholerae by natural transformation, which maintains functional recJ and xseA. Candidate colonies were screened via colony PCR for target loci in a multiplex PCR reaction and confirmed by screening purified genomic DNA of each isolate. Strains were serially passaged in LB media to cure the pMMB-tfoX plasmid, where cured strains became sensitive to ampicillin 100µg/ml. Primers used to generate and confirm MuGENT mutant strains are listed in Supplemental Table 4. 3.4.3 – Isogenic Deletion Mutant Strain Construction Isogenic deletion strain constructs were generated using the positive allelic exchange vector pKAS32 (198). Plasmid constructs were generated by first amplifying and purifying 1Kb DNA fragments upstream and downstream of target loci that contain homology base pairing to pKAS32. The pKAS32 vector was isolated from E. coli pKAS32 cultures using a QIAprep Spin Miniprep Kit (Qiagen) and restriction digested with SacI and XbaI. DNA fragments and digested pKAS32 backbone were combined using Gibson Assembly (New England Biolabs) and transformed into E. coli ET12567 ΔdapA diaminopimelic acid auxotroph mating strain. Newly formed pKAS32 constructs were sequenced and correct vectors conjugated into V. cholerae. V. cholerae-pKAS32 strains were outgrown in LB at 37°C 210rpm and subjected to >2500µg/ml streptomycin to select for strains that have excised the plasmid from its genome. Candidate mutant strains were screened by colony PCR and confirmed by screening purified genomic 73 DNA. Primers to generate and confirm pKAS32 deletion strains are listed in Supplemental Table 4. 3.4.4 – V. cholerae Terminal Oxidase Strain Growth Curves Bacterial strains were grown either aerobically or anaerobically on LB streptomycin (100µg/ml) agar media and after 16-18h used to inoculate 4mL LB media. After 16h, bacterial strains were concentrated to a 1.0 OD600. 700µl LB media was inoculated 1:1000 (0.7µl) with the 1.0 OD600 resuspensions, vortexed, and aliquoted in triplicate 200µl volumes in a 96-well plate. Optical density was recorded every hour for the duration of the growth curve. Deoxygenated LB was used for anaerobic growth and benchtop LB used for aerobic growth. 3.4.5 – V. cholerae Terminal Reductase Strain Growth Curves Bacterial strains were grown on LB streptomycin (100µg/ml) agar media and after 16- 18h used to inoculate 4mL LB media. After 16h, bacterial strains were concentrated to a 1.0 OD600. 700µl LB media was inoculated 1:1000 (0.7µl) with the 1.0 OD600 resuspensions, vortexed, and aliquoted in triplicate 200µl volumes in a 96-well plate. Optical density was recorded every hour for the duration of the growth curve. Deoxygenated LB was used for anaerobic growth curves and benchtop LB used for aerobic growth curves. Concentrations of alternative electron acceptors supplemented to LB media were as follows: 50mM sodium fumarate (Sigma), 50mM trimethylamine-N- oxide (TMAO) (Sigma), 50mM sodium nitrate (Sigma), and 50mM dimethyl sulfoxide (DMSO) (Sigma). For strains grown in 50mM LB Nitrate media, after 3h 5µM sodium hydroxide (Fisher Chemical) final concentration was added to alkalinize the growth media to support continued nitrate respiration. 74 3.4.6 – Wild Type Aerobic, Microaerobic, and Anaerobic RNA Isolation and Real-Time Quantitative PCR (RT-qPCR) For each growth condition (aerobic / microaerobic / anaerobic) 4mL of LB media was placed in each environment at 37°C to temper the media prior to inoculation in an effort to equalize the oxygen content and to pre-warm the media. Media was inoculated 1:1000 (4µl) with a 1.0 OD600 wild type V. cholerae inoculum and grew shaking 210rpm for the aerobic culture and static for both microaerobic and anaerobic cultures. After 4h, culture tubes were centrifuged 4000rpm, 4°C, for 10min and cell pellets were resuspended in 1mL TRIzol (Invitrogen). RNA was isolated from TRIzol suspensions using an RNeasy kit (Qiagen) coupled with an on-column DNase digestion (Qiagen) and Turbo DNase digestion (Invitrogen). RNA concentrations were measured with a UV/VIS Spectrophotometer and visualized on a 2% agarose gel. cDNA was generated from RNA using Superscript III reverse transcriptase (Thermo Scientific). RT-qPCRs were performed using SYBR green master mix (Applied Biosystems) with 5ng of cDNA. Primers used to detect recA, cbb3 (VC1442), bd-I (VC1844), bd-II (VCA0872), and bd-III (VC1571) are listed in Table 4. Threshold cycle (ΔΔCt) values were calculated using recA as the gene of reference. 3.4.7 – Infant Mouse Colonization Assays All animal experiments in this study were approved by the Institutional Animal Care and Use Committee at Michigan State University. 75 Infant mice were infected as described previously (203). Briefly, three- to five-day-old mouse neonates (Charles River, Wilmington, MA) were orogastrically infected with approximately 106 bacterial cells following 2 hours of separation from dam mice and maintained at 30°C for 20h. After 20h, mice were euthanized, and intestinal segments weighed and homogenized in 4mL phosphate buffered saline (PBS). Intestinal homogenates were serially diluted and plated for CFU counts. For monoassociated infections, dilutions were plated on LB streptomycin (100 µg/mL) for growth and enumeration. For MuGENT strain competition assays, dilutions were also plated on LB spectinomycin (200 µg/ml) for differentiation from co-infected wild type V. cholerae. 3.4.8 – CoMPAS Infant Mouse Infection and Sequencing Wild type, Mucbb3, Mubd-I, Mubd-II, and Mubd-III MuGENT strains were combined in equal ratios and a total of 6 infant mice were infected with a final 0.01 OD600 inoculum, approximately ~106 CFU. Remaining inoculum volume was spun down at 4°C, 4000rpm, for 10min and where DNA of the inoculum pool (IP) was isolated using a QIAamp PowerFecal Pro DNA Kit (Qiagen). After 20h infection, small intestinal segments were homogenized and pooled from the 6 infant mice. Pooled homogenates were filtered on ice using a 70µm filter to remove residual intestinal tissue. From here, DNA of the mouse pool (MP) was isolated using a QIAamp PowerFecal Pro DNA Kit (Qiagen). 76 Recovered DNA samples were normalized to 100ng/µl and subsequently, multiplex amplicon sequencing was performed on both inoculum and mouse pools. Amplification targets included the primary subunit of each MuGENT oxidase complex (VC1442, VC1844, VCA0872, and VC1571) as well as toxT (VC0838) which is present in all input strains. Primers used are listed in Supplemental Table 4, amplification was carried out for 30 cycles. PCR products were purified using the QIAquick PCR Purification Kit (Qiagen) and quantified by Qubit dsDNA HS and normalized to 20ng/µl for MiSeq amplicon sequencing by the MSU RTSF Genomics Core (Michigan State University). Sequencing was conducted on a MiSeq Nano v2 flow cell using a 2x250bp paired end format. Sequence barcodes were trimmed and target loci read counts were quanitified using Geneious software. 3.4.9 – In vitro Competition Assays Bacterial strains were grown in 4mL LB media for 16-18h and resuspended to 1.0 OD600. Wild type and an individual target strain were combined in a 1:1 ratio and used to inoculate deoxygenated LB for anaerobic competitions or benchtop LB for aerobic competitions. Anaerobic competitions were grown at 37°C static and aerobic competitions were grown 37°C 210rpm shaking. After 20h of growth, cultures were serially diluted and plated for colony forming units. WT vs. Aero7 and WT vs. Ana4 competitions were plated on LB streptomycin (100µg/ml), and LB spectinomycin (200µg/ml) to determine strain ratios. Individual deletion strain competitions were plated on LB Str100 X-Gal 40µg/ml for blue-white screening to determine strain ratios. 77 3.5 – Results 3.5.1 – Constructing Terminal Electron Acceptor Mutant Strains Vibrio cholerae terminal electron acceptor mutants were generated using the multiplex genome editing technique MuGENT (259) and by a positive allelic exchange vector pKAS32 (198) in an El Tor C6706 V. cholerae background. Target loci and their relative chromosomal locations are depicted in Figure 3.1A. In MuGENT-generated mutant strains, target loci are disrupted by a frameshift mutation, removal of ATG start codon, insertion of 3-frame stop codons, and offseting of the ribosomal binding site. This is combined with the insertion of a universal detection sequence at each target locus and a spectinomycin cassette insert into pseudogene VC1807 for naturally competent cell selection that has no in vitro fitness cost (260) (Figure B.1). MuGENT generated strains are designated with a superscript ‘Mu’ (Mu). Mutant strains constructed via pKAS32 have the complete coding sequence for all subunits of target terminal electron acceptor complexes excised, generating isogenic deletion strains. Select strains were verified by whole genome sequencing which indicated no nucleotide polymorphisms in most strains and where present were found in hypothetical protein regions of the genome predicted to have no impact on bacterial cell fitness (Supplemental Table 1). 78 Figure 3.1. Verification of MuGENT generated mutant strains. (A) Chromosomal map of V. cholerae terminal electron reducing complex loci. (B) Multiplex allele- specific PCR (MASC-PCR) of V. cholerae terminal electron reducing complexes MuGENT mutants. Lanes are labelled with the strain name where a strain preceded by a ‘Mu+’ (Lanes 3-6) indicates it is the indicated oxidase complex as the sole remaining functional oxidase in that strain and strains preceded by a ‘Mu’ (Lanes 7-10) indicates that the specified locus is the targeted knock out. Targeted gene loci are labelled to the right of each gel image. The presence of a band indicates a targeted 79 knockout in the gene locus whereas the absence of a band indicates the wild type gene is present. V. cholerae encodes one cbb3 oxidase and three bd-type oxidase complexes (147) (Figure 3.1A). Cytochrome oxidase cbb3 is a four subunit (VC1439-VC1442) cytochrome c containing terminal oxidase of which the coding sequence for the primary subunit, CcoN (VC1442), was disrupted to generate MuGENT knockout strains. CcoN is the first open reading frame in the operon and contains the active site for reducing oxygen to water (261); its disruption was sufficient for abolishing cbb3 cytochrome c activity (Figure B.2A). For each of the three bd-type oxidases, bd-I (VC1844-43), bd-II (VCA0872-73), and bd-III (VC1570-71), both subunits of each complex were disrupted. Mutations in each target locus were confirmed by multiplex allele-specific PCR (MASC- PCR) (178) where the presence of a DNA band indicates successful genomic editing at the indicated locus via MuGENT (Figure 3.1B). To further validate the function of each oxidase, isogenic deletion strains were also generated for select terminal oxidases via pKAS32 positive allelic exchange. V. cholerae also encodes four alternative terminal reductase complexes (147) capable of reducing alternative electron acceptors that can support respiration in the absence of oxygen. The four terminal reductases include a fumarate reductase (VC2656-59), trimethylamine-N-oxide (TMAO) reductase (VC1692-94), nitrate reductase (VCA0676- 0680), and a biotin sulfoxide reductase (BSO) (VC1950-51). For each of these multi- subunit complexes, the active reducing subunit was disrupted via MuGENT and confirmed by MASC-PCR (Figure 3.1B). 80 3.5.2 – In vitro Characterization of Terminal Electron Acceptor Complex Mutants Terminal Oxidase Growth Characterization V. cholerae oxidase mutant strains were grown in LB media in aerobic and anaerobic conditions. Inocula were prepared anaerobically to ensure consistent growth of oxidase- deficient strains. After anaerobic preparation of inocula, both cbb3 and bd-I oxidase complexes were found to be required for wild type levels of growth in aerobic conditions whereas bd-II and bd-III oxidases were not (Figure 3.2A). Cultures lacking the cbb3 oxidase grew at a consistently lower optical density and never reached the peak OD600 of wild type. The bd-I oxidase was determined to be the most critical oxidase complex for supporting aerobic respiration in V. cholerae, as cells lacking it showed drastically reduced growth. This finding was unexpected as electron transport and oxygen reduction by the cbb3 oxidase is more efficient at generating a proton gradient (and therefore ATP) for the cell (262). Electrons passed to the cbb3 oxidase are shuttled through the bc1 complex, which accounts for a Δ6H+ proton gradient (263) along with translocation of two additional protons coupled to the terminal reduction of oxygen by the cbb3 complex (264). The bd-I reducing pathway generates a relatively weaker proton gradient resulting in less ATP for the cell (137, 265). Thus our observation that it serves as the primary oxidase in V. cholerae under atmospheric oxygen conditions was unanticipated. All oxidase-disrupted mutants grew comparably to wild type in anaerobic conditions (Figure 3.2B) suggesting the observed defect in aerobic growth is not due to a general growth defect imposed by the mutations. In vitro competition assays were also performed, demonstrating a competitive defect for both cbb3 and bd-I deficient strains in aerobic conditions (Figure 3.2C-D). These growth phenotypes were 81 recapitulated in M9 0.2% D-glucose media, although bd-I deficient strains were further hampered for growth aerobically and showed a minor shift in reaching exponential phase anaerobically (Figure B.3). We hypothesized that bd-I deficient strains grown anaerobically may experience a growth lag prior to expression of alternative oxidases, such as the cbb3 oxidase (Figure B.2B), accounting for the observed growth kinetics. To test this, we prepared inocula aerobically and observed the growth phenotype. In this condition, strains lacking bd-I oxidase grew to wild type optical density (Figure 3.2E) indicating that delayed expression of alternative oxidases when inocula are prepared anaerobically may account for the observed growth lag. Similar growth patterns were observed in anaerobic growth conditions for inocula of aerobically prepared oxidase mutants (Figure 3.2F). We examined expression patterns of wild type V. cholerae under anaerobic, microaerobic, and aerobic conditions, by qRT-PCR ΔΔCt analysis using recA as the gene of reference. Expression values are reported relative to bd-III expression in anaerobic conditions, which served as the baseline for comparison among the oxidases (Figure 3.2G). The cbb3 and bd-I oxidases were more highly expressed relative to bd-II and bd-III. In anaerobic conditions, bd-I oxidase was expressed nearly 10-times higher than cbb3 oxidase whereas in the presence of oxygen (microbaerobic or aerobic) cbb3 oxidase was expressed slightly higher than bd-I. We conclude that the bd-I oxidase is critical for aerobic respiration in V. cholerae during transition from an anaerobic to aerobic environment and hypothesize that expression of the cbb3 oxidase is delayed during this transition period. 82 Further characterization of the oxidase complexes was carried out using double, triple, and quadruple oxidase mutant strains. Isogenic double and triple deletion strains, as well as quadruple oxidase MuGENT mutant Aero7, were grown in aerobic and anaerobic conditions in LB from inocula grown anaerobically (Figure 3.2H-I). Strain +bd-I, harboring solely a functional bd-I oxidase, grew to near wild type levels while strains +cbb3 and +bd-III, having solely the cbb3 or bd-III oxidase complex, grew after a considerable lag phase. This lag phase, however, was reduced when inocula were adapted to an aerobic environment prior to the growth assay (Figure 3.2J-K). Strain Aero7, defective for production of all terminal oxidases encoded by V. cholerae, and strain +bd-II, containing only the bd-II oxidase, were completely deficient for aerobic growth. This was further exemplified for Aero7 by a near 107-fold attenuation in in vitro competition assays (Figure 3.2L). These observed growth phenotypes were recapitulated in M9 0.2% D-glucose media (Figure B.3). Additionally, triple oxidase MuGENT mutant growth mirrored that observed in isogenic deletion strains, although strain Mu+bd-III (encoding only bd-III) exited lag phase more rapidly (Figure B.4). From these results, we conclude that cbb3, bd-I, and bd-III oxidases support aerobic growth of V. cholerae to varying degrees while bd-II does not. Despite its low mRNA expression level in wild type cells relative to other oxidases, bd-III oxidase supported +bd-III aerobic growth, particularly when culture inocula were grown aerobically. Taken together, these findings make clear that bd-I oxidase is the primary oxidase in V. cholerae, priming a transition from anaerobic to aerobic environments and functioning as the primary oxidase in atmospheric oxygen environments. To our knowledge, V. 83 cholerae and Listeria monocytogenes (148) are the only pathogens demonstrated to preferentially use a bd-type oxidase in lieu of a heme-copper oxidase such as the bo3 oxidase of Escherichia coli (141) and Salmonella Typhimurium (7), the cbb3 oxidase of Psuedomonas aeruginosa (266), Campylobacter jejuni, and Helicobacter pylori (267), or the aa3 oxidase of Staphylococcus aureus (268) to support growth in atmospheric oxygen. 84 Figure 3.2. Terminal oxidases support aerobic growth in V. cholerae. Growth characteristics of the terminal oxidases in V. cholerae. (A-B) Single terminal oxidase mutants, both MuGENT and isogenic deletion, growth in LB. Inoculums were prepared anaerobically and subsequently grown in aerobic and anaerobic conditions, respectively. (C-D) Single terminal oxidase isogenic deletion strain in vitro LB competition assays in both aerobic and anaerobic conditions, respectively. Competitive index scores were calculated as a ratio of output versus input [(TargetOutput/ΔlacZOutput) / (TargetInput/ΔlacZInput)], where a ΔlacZ strain served as a psuedo-wild type to determine relative fitness via blue-white screening. Red dots indicate the limit of detection where no CFUs were recovered for these trials. (E-F) 85 Single terminal oxidase isogenic deletion mutant growth in LB where inoculums were prepared aerobically and subsequently grown in aerobic and anaerobic conditions, respectively. (G) In vitro expression of terminal oxidases in anaerobic, microaerobic, and aerobic growth conditions.(H-I) Combinatorial terminal oxidase deletion mutant growth in LB. Inoculums were prepared anaerobically and subsequently grown in aerobic and anaerobic conditions, respectively. Triple deletion mutant strains have a ‘+’ with an oxidase name (e.g. +cbb3), indicating the sole remaining oxidase, with the other three oxidases disrupted by mutation. (J-K) Combinatorial terminal oxidase deletion mutant growth in LB. Inoculums were prepared aerobically and subsequently grown in aerobic and anaerobic conditions, respectively. (L) In vitro aerobic and anaerobic competition assay between Aero7 and Wild Type V. cholerae with competitive index scores calculated as [(Aero7Output/WTOutput) / (Aero7Input/WTInput)]. Growth curves are an average of three biological replicates where error bars represent the standard error of the mean. Bars for in vitro competitions and expression data represent the arithmetic mean where error bars represent the standard error of the mean. Growth of Mutants Lacking Terminal Reductases MuGENT terminal reductase V. cholerae mutants were prepared aerobically and used to inoculate fresh LB with and without alternative electron acceptors in both aerobic and anaerobic conditions. Individual reductase MuGENT-derived mutant strains were made lacking the active subunit of fumarate reductase (VC2656), TMAO reductase (VC1692), nitrate reductase (VCA0678), and BSO reductase (VC1950). And a combinatorial mutant was also constructed, denoted Ana4, in which all reductases were disrupted. All terminal reductase mutants, including Ana4, grew similarly to wild type in LB in both aerobic and anaerobic growth conditions. However, when grown anaerobically in the presence of the alternative electron acceptors fumarate, TMAO, nitrate, or DMSO, mutant strains were defective for growth compared to wild type (Figure 3.3A-D). This indicated that the MuGENT-generated reductase mutants were defective for reductase function. As Ana4 was reduced for growth in the presence of all alternative electron 86 acceptors, we concluded it adequately represented a strain incapable of utlizing these molecules to support growth and included it in our in vivo analysis described below. The presence of these alternative electron acceptors under aerobic conditions led to varying growth responses by wild type V. cholerae. With fumarate or DMSO, growth was boosted, however, with either nitrate or TMAO growth was reduced (Figure B.5A-D). Figure 3.3. Terminal reductase mutants are reduced for anaerobic growth in the presence of cognate electron acceptor molecules. Growth characteristics of terminal reductases of V. cholerae. MuGENT generated terminal reductase mutants grown in LB in the presence and absence of alternative electron acceptors (A) 50mM fumarate, (B) 50mM trimethylamine-N-oxide (TMAO), (C) 50mM nitrate, and (D) 50mM dimethyl sulfoxide (DMSO). Inoculums were prepared aerobically subsequently grown in anaerobic conditions. Growth curves are an average of three biological replicates where error bars represent the standard error of the mean. 3.5.3 – Aero7 and Ana4 Infant Mouse Infections To examine the importance of aerobic and anaerobic respiration during infection, V. cholerae Aero7 and Ana4 MuGENT strains were tested for their ability to colonize the 87 infant mouse intestinal tract. Single strain and competition infections in neonatal mice were performed for both strains. Aero7 was severely attenuated for colonization of the small intestine, in both single strain and competition infections (Figure 3.4A-B). In single strain infections, wild type V. cholerae was recovered near 108 CFU/g intestine whereas Aero7 was recovered near 103 CFU/g intestine, a 5-log decrease in colonization. This reduction was also observed in the competition infections where the competitive index (CI) score of Aero7, calculated as [[Aero7Output/WTOutput] / [Aero7Input/WTInput]] was approximately 10-5. Aero7 competed better In the large intestine, although was still at a fitness disadvantage, with a CI of 10 - 3. Its greater fitness in the large intestine may be due to the more anaerobic environment of this site, which has a more complex microbiota (269) (Figure B.6A-B). That Aero7 is considerably less fit in the infant mouse small intestine is relevant as this is the site of infection in humans. We conclude from these findings that maintaining functional terminal oxidases, and therefore aerobic respiration, is critical for V. cholerae to establish infection and proliferate. 88 Figure 3.4. Aerobic respiration, and not anaerobic respiration, is required for growth and colonization of the infant mouse small intestine. Aero7 and Ana4 small intestine colonization in monoassociated and competition infections. (A) Monoassociated infection of strain Aero7. (B) Competition infection of strain Aero7. (C) Monoassociated infection of strain Ana4. (D) Competition infection of strain Ana4. Bars represent the geometric mean. Horizontal dashed lines indicate the limit of detection (LOD) and red dots indicate recovered CFUs were below the LOD. Competitive index scores were calculated as [(MutantOutput/WTOutput) / (MutantInput/WTInput)]. Statistical analysis was performed using GraphPad PRISM. *, P < 0.05. A Mann-Whitney U-test was used in the determination of significance between WT and Aero7. A Student’s T-test was performed on log transformed data in the determination of signifance between WT and Ana4. 89 In contrast to a strain lacking all terminal oxidases, the Ana4 mutant lacking all terminal reductases, which is deficient for anaerobic growth with specific alternative electron acceptors (Figure 3.3A-D), colonized both the small and large intestines to wild type levels in both single strain and competition infections (Figure 3.4C-D and Figure B.6C- D). The lack of an observable phenotype in our experiments differs from an earlier study demonstrating a two-fold reduction in colonization by a mutant strain of V. cholerae lacking nitrate reductase (napA; VCA0678) in the adult streptomycin-treated mouse model (110). This two-fold reduction may be observed in adult mice, but not infant, as the adult gut has greater anaerobic luminal volume compared to the limited luminal space in the infant. Nitrate availability has also been shown to increase following streptomycin treatment, which may contribute to the observed colonization discrepancy between wild type and napA V. cholerae (270), as lacking a functional nitrate reductase may incur a greater fitness cost in the streptomycin-treated adult mouse. Overall, our data suggests that anaerobic respiration using alternative electron acceptors is not a prominent feature of V. cholerae growth during infection of the infant mouse. The slight growth defect reported with a nitrate reductase mutant in the streptomycin-treated adult mouse (110) suggests that there may be a limited role for anaerobic respiration although more work is required to ascertain the effects of a mature, non-disturbed microbiota on these questions. 90 3.5.4 – Individual Oxidase Function During Infection Comparative Multiplex PCR Amplification Sequencing (CoMPAS) To examine the requirements of the terminal oxidases of V. cholerae in vivo, we took a novel approach that combines elements of insertion-site sequencing (Tn-Seq) with targeted amplification of MuGENT generated oxidase mutations (Comparative Multiplex PCR Amplicon Sequencing (CoMPAS)). Individual terminal oxidase MuGENT strains were pooled along with a wild type strain in equal proportions, and the pool was used to infect infant mice. Small intestinal segments were pooled from 6 mice and genomic DNA extracted along with the input inoculum for CoMPAS analysis. Mutant allele abundances were determined for each oxidase complex and relative sequence abundances were normalized to toxT gene amplification (Figure 3.5A). Sequence coverage for each pool are presented in Supplemental Table 2. Comparative index scores are reported for each of the primary oxidase subunits VC1442 (cbb3), VC1844 (bd-I), VCA0872 (bd-II), and VC1571 (bd-III). Comparative index scores were calculated as [(Output PoolTarget Reads / Output PooltoxT Reads) / (Input PoolTarget Reads / Input PooltoxT Reads)]. In this experiment, Mubd-I oxidase knockout strain was underrepresented in the output sequencing pool approximately 10-fold relative to the input. Conversely, all other MuGENT oxidase mutant strains were within a 2-fold change relative to the input. Overall, the bd-I oxidase was determined to be the most important oxidase complex supporting growth in vivo. 91 Figure 3.5. Terminal oxidases are functionally redundant in supporting colonization of the infant mouse small intestine. Single oxidase in vivo colonization dynamics in the small intestine. (A) Comparative Multiplex PCR Amplification Sequencing (CoMPAS) sequence analysis. Comparative index scores were calculated as [(Output PoolTarget Reads / Output PooltoxT Reads) / (Input PoolTarget Reads / Input PooltoxT Reads)]. Vertical red dashed lines indicate a 2-fold change in output to input sequence ratios. Sequence coverage for each input pool (IP) and associated mouse output pool (MP) are shown in the bar plots. (B) Individual oxidase deletion monoassociated infections. Bars represent the geometric mean. Horizontal dashed lines indicate the limit of detection (LOD) and red dots indicate recovered CFUs were below the LOD. Statistical analysis was performed using GraphPad PRISM. *, P < 0.05. A Mann-Whitney U-test was used in the determination of significance between WT and Aero7 whereas an Analysis of Variance with post-hoc Dunnett’s multiple comparisons test was conducted on log transformed CFU/g intestine for all other strain comparisons. Single Oxidase Complex Deletion Infections Isogenic terminal oxidase deletion strains were also examined for colonization levels in single strain infections of the infant mouse. Loss of any one of the terminal oxidases did not result in a colonization defect in the small or large intestine of the infant mouse (Figure 3.5B and Figure B.7A). Wild type and mutant strains colonized to approximately 108 CFU/g intestine. These findings indicate that in single strain infections, functional redundancy exists among the oxidases that support aerobic respiration during infection. As multiple oxidases were shown to support aerobic growth of V. cholerae in our growth assays, we hypothesized that the cbb3, bd-I, and potentially the bd-III oxidase could all 92 be supporting growth in vivo. This hypothesis also reflects data present in a large Tn- Seq dataset where transposon insertions into the cbb3, bd-I, and bd-III oxidases showed a reduced capacity for colonization (244). Counter to the observed defect in a Mubd-I knockout strain in our CoMPAS analysis, no colonization defect was present in monoassociated infections. We reasoned that a bd-I oxidase deficient strain was capable of colonizing the infant mouse, however, as cultures prepared anaerobically were delayed for aerobic growth as in Figure 3.2A, this growth delay resulted in the bd-I deficient strain being outcompeted during the pooled mouse infection. For all oxidase mutants, one concern is that disruption to oxidase function may impact other requirements for colonization such as virulence factor production or protection against reactive oxygen species (ROS). To address whether mutations in the oxidases alter production of virulence factors required for colonization, TcpA protein levels in the mutants were examined as described in Supplemental Materials and Methods and were equivalent to wild type production (Figure B.8). As oxidative stress can also prevent bacterial growth in vivo and the bd oxidases of Escherichia coli exhibit low levels of catalase activity (150), we tested the minimum inhibitory concentraion of hydrogen peroxide on V. cholerae oxidase mutants, observing no growth defects for any mutant strain (Figure B.9). These findings support our conclusions that observed colonization defects can be attributed to a reduction in respiratory energy generation and not related 93 to virulence factor production or increased ROS sensitivity that could have also limited colonization efficiency. 3.5.5 – Determining Functionally Redundant Oxidases During Infection To identify which oxidases primarily support growth in vivo, triple oxidase isogenic deletion strains were used to colonize the infant mouse. By infecting with triple mutants, we could determine the importance of the single remaining oxidase. Strain +bd-I expressing solely the bd-I oxidase colonized comparable to wild type and strain +cbb3 with a functional cbb3 oxidase colonized at a ~1.5-fold reduction compared to wild type (Figure 3.6A). This finding further supports bd-I oxidase as the primary oxidase of V. cholerae. Strains containing solely the bd-II or bd-III oxidase were unable to colonize (Figure 3.6A). This pattern of colonization was also reflected in the large intestine, however with higher levels of recovered CFU/g intestine, again likely due to the more anaerobic environment (Figure B.7B). As the cbb3 oxidase was determined to be less expressed and inactive in anaerobic conditions (Figure 3.2G and Figure B.2B), and mouse inocula were prepared anaerobically prior to infection, we investigated whether preparation in aerobic conditions could prime expression and activation of the cbb3 oxidase to better support growth in vivo. Despite preparing +cbb3 oxidase cultures aerobically, we still observed a significant ~1-log reduction in colonization (Figure 3.6B). However, the colonization efficiency of aerobically grown +cbb3 cultures was improved compared to anaerobically prepared +cbb3 cultures but was not enough to support wild type levels of colonization. No significant difference was detected in the large intestine, which was also previously observed in the anaerobically prepared culture infections (Figure B.7C). 94 Figure 3.6. bd-I oxidase alone supports wild type levels of colonization in the infant mouse small intestine with cbb3 supporting colonization to a lesser extent. (A) Combinatorial oxidase deletion in vivo colonization dynamics in the small intestine. (B) Colonization of aerobically prepared wild type and +cbb3 oxidase inoculums. Triple deletion mutant strains have a ‘+’ with an oxidase name (e.g. +cbb3), indicating the sole remaining oxidase, with the other three oxidases disrupted by mutation. Bars represent the geometric mean. Horizontal dashed lines indicate the limit of detection (LOD) and red dots indicate recovered CFUs were below the LOD. Statistical analysis was performed using GraphPad PRISM. *, P < 0.05. A Mann- Whitney U-test was used in the determination of significance between WT and +bd-II, +bd-III, and Δcbb3Δbd-I whereas an Analysis of Variance with post-hoc Dunnett’s multiple comparisons test was conducted on log transformed CFU/g intestine for all other strain comparisons. As the +bd-III oxidase strain could grow in vitro in aerobic LB conditions following aerobic overnight preparation while lacking both cbb3 and bd-I oxidases, we looked to determine whether wild type expression levels of bd-III oxidase could support aerobic growth in this strain. By ΔΔCt qRT-PCR analysis, +bd-III aerobic growth was supported by increased expression of bd-III transcript, minimally 40x higher than wild type. We hypothesized that this increased expression may be due to selective pressure for 95 mutations that increase bd-III expression. A variant strain, +bd-IIIV was isolated that supported high levels of aerobic growth and exhibited increased expression of the bd-III oxidase (Figure B.10B-D). We determined the genome sequence of this variant, identifying a mutation in chrR (VC2301), encoding an anti-sigma factor for SigmaE, which controls bd-III expression (Supplemental Table 1) (271). In infant mouse infections, +bd-IIIV colonized near one order of magnitude lower than wild type in the small intestine of the infant mouse and was comparable to wild type in the large intestine (Figure B.10E-F). Comparatively in infant mouse infections, the +bd-IIIV strain performed significantly better than +bd-III in the initial colonization infections (Figure 3.6A). In wild type V. cholerae, we found that the bd-III oxidase is not typically expressed and does not contribute to in vivo colonization, however, in scenarios where the bd-III oxidase is the sole remaining oxidase and is expressed, it can support aerobic growth and in vivo colonization. 3.6 – Discussion Oxidative respiration is required for population expansion of V. cholerae in the infant mouse where the cbb3 and bd-I oxidases function as the terminal reducing complexes during infection. This is also the first instance where a bd oxidase was determined to support growth of a bacterial pathogen in aerobic conditions and is also necessary for the transition from an anaerobic to aerobic environment. The finding that the cbb3 and bd-I oxidases of V. cholerae are necessary for aerobic respiration in the low oxygen environment of the small intestine aligns well with the typically low Km observed for each of these classes of oxidases (54, 135, 256, 272–274). This work highlights that the low 96 oxygen level in the small intestine is sufficient and essential for V. cholerae growth and implicates oxygen as a key electron acceptor for bacterial pathogenesis in the gut. 97 Chapter 4 – Concluding Remarks 98 4.1 – Conclusions and Significance Prior to this work, the influence of oxygen on the pathogenicity of V. cholerae in vivo was an unexplored area of research. It has been well established that preparation of laboratory grown V. cholerae can be carried out aerobically (275) and that toxin inducing conditions required aeration (199), but the role of oxygen as it supports growth in vivo during infection was first examined in the work presented in Chapters 2 and 3. 4.1.1 – Metabolic Pathways Important for In Vivo Growth Through the investigation of V. cholerae metabolism via the pyruvate dehydrogenase (PDH) complex, we concluded that aerobic metabolism is a key driver of V. cholerae population expansion during infection (103). Under aerobic conditions, the PDH complex converts pyruvate to acetyl-CoA to feed into the TCA cycle, providing reducing molecules for the electron transport chain (ETC), supporting aerobic respiration. We first established that V. cholerae was capable of aerobic growth on mucin when the PDH complex was intact. We then sought to further investigate the role of the PDH complex and, therefore, aerobic metabolism in vivo. As the intestine contains many substrates in addition to mucin, we were unable to determine whether mucin-specific metabolism is essential for proliferation in vivo. V. cholerae colonization is elevated in mice treated with the mucolytic agent N-acetyl-L-cysteine (NAC), indicating that mucus primarily serves as a barrier to colonization and may only secondarily act as a substrate for growth (179). From our results, we were able to conclude that aerobic metabolism as a whole is essential to colonization, a new finding in the field of V. cholerae research. We further examined the relationship between aerobic and anaerobic metabolism by investigating a pyruvate formate-lyase (PFL) mutant which also converts pyruvate to 99 acetyl-CoA, but only under anaerobic conditions. As no significant defect in colonization was observed, we concluded that anaerobic respiration plays only a minor role in colonization of the infant mouse. Anaerobic metabolism of V. cholerae during infection has been investigated by another group, however our data suggest that aerobic metabolism is substantially more important for efficient colonization (111). 4.1.2 – Respiration of V. cholerae During Infection After determining that aerobic metabolism is key to the fitness of V. cholerae during infection, we wanted to further define how oxidative respiration controls proliferation during infection. The PDH complex, in addition to its role in supporting aerobic respiration, leads to the generation of other metabolic intermediates that can promote cell viability. To directly examine oxidative respiration, we targeted the terminal oxidase complexes of V. cholerae. By generating individual and combinatorial oxidase mutant strains, both through modified multiplex genome editing through natural transformation (MuGENT) and pKAS positive allelic exchange vector approaches, we were able to show conclusively the necessity of oxidative respiration during V. cholerae infection. Of the four terminal oxidase complexes of V. cholerae, the cbb3 and bd-I oxidases support growth in the infant mouse intestine. Between these two, the bd-I oxidase is dominant in supporting aerobic growth, both in atmospheric and in vivo conditions. Typically, bd-type oxidases are induced under microaerobic conditions characteristic of the host environment, which is consistent with the importance of this oxidase in vivo. Identifying bd-I as the primary oxidase active in atmospheric oxygen conditions was unexpected and offers insight into the oxygen response pathways of V. cholerae both inside and outside of the host environment. 100 Overall, results presented in this thesis support a model of V. cholerae pathogenesis in which oxygen is a key metabolic factor that promotes growth and proliferation during infection. The implications of this work further our understanding of V. cholerae biology and present a new view of V. cholerae disease progression reliant on oxidative respiration and growth. 4.2 – Future Directions The influence of oxygen on the pathogenicity of V. cholerae had been under-explored prior to this work. An avenue for future work is to explore how oxygen is generated in the intestine during V. cholerae infection. As discussed in Chapter 1, oxygen regulation in the intestines is controlled through a combination of host-microbe interactions. How V. cholerae affects levels of gut oxygenation during infection and how it interacts with each of these factors that maintain physiological hypoxia in the gut has yet to be explored. Addressing alterations in host metabolism, changes in microbiota-produced short chain fatty acids, and perturbations to the resident aerotolerant microbial populations are key areas of research open for investigation as each plays a role in oxygen regulation in the gut. The first consideration in pursuing future research in this area requires a re-evaluation of the infant mouse model. The infant mouse model reflects human infection to the degree that the toxin-coregulated pilus is required for colonization of the infant mouse intestine, just as it is required for microcolony formation in the human gut (232, 276). However, cholera toxin, which directly elicits water efflux from human host tissues, does not cause similar diarrheal symptoms in the infant mouse. Additionally, the infant mouse 101 intestine is naïve both in terms of its innate immunity and composition of a commensal microbiota. To account for deficiencies of the infant mouse, at least in part, further research may benefit from both infant and adult rabbit infection models (277, 278). Both models elicit diarrheal-like symptoms characteristic of human disease, resulting in high fluid accumulation in the lumen of the intestines due to cholera toxin production. To establish oxygen-dependent V. cholerae growth in the rabbit models, we would infect the infant rabbit with our WT, Aero7, Δcbb3, Δbd-I, Δcbb3Δbd-I, and Δbd-IIΔbd-III strains. This experiment would further examine the role of the terminal respiratory oxidases in vivo and provide confidence for further work in these models. Additionally, examination of host factors that contribute to oxygen levels in the gut, through immunohistochemical staining of HIF regulated claudin-1 or GLUT-1 or, alternatively, pimonidazole dye detection (for examples) may help to investigate V. cholerae-induced oxygen changes in the gut following infection, although these would be limited to single time point measurements (33, 72, 155). The role of cholera toxin (CT) in supporting aerobic metabolism of V. cholerae is of interest as the toxin directly affects epithelial cell signaling (279) and drastically changes the luminal environment through diarrheal disease symptoms. In a comparison study of wild type and ΔctxA mutant in the infant rabbit, CT production led to induction of aerobic metabolic pathways (43). Production of CT was linked to increased expression of TCA metabolic genes in the ileum of rabbits (43), including expression of sucA, an indicator of aerobic metabolism (42, 280). In the same study, L-lactate metabolism was also induced in infecting V. cholerae cells (43). Shifts in host cell metabolism from oxygen- 102 dependent β-oxidation to anaerobic glycolysis leads to the secretion of lactate into the gut (11). By observing an increase in L-lactate metabolism, we hypothesize that V. cholerae CT is leading to a shift in host enterocyte cell metabolism. This shift results in excess oxygen no longer being used for β-oxidation to diffuse into the gut lumen, increasing luminal concentrations of oxygen. Lastly, CT has also been implicated in disrupting intestinal barrier integrity (281) which may contribute to oxygen availability in the gut to support V. cholerae growth and proliferation during infection. To probe the effects of CT on host cell-mediated gut oxygenation, purified CT could be administered to intestinal cell lines, such as polarized T84 epithelial cells (282), to determine if host cell metabolism changes as a result. This could be addressed through monitoring changes in the cell transcriptome with RNA-Seq. Additionally, we can apply fluidic devices that mimic the intestinal environment (283) to measure changes in oxygen availability (162) following administration of purified CT. This would directly test whether oxygen concentrations shift as a result of CT-mediated effects on host cells; although it may be challenging to delineate oxygen changes due to shifts in metabolism or weakening of barrier function in this experimental setup. Inflammation caused by infection is also of interest for future work. Although cholera is not traditionally considered an inflammatory disease, emerging lineages of V. cholerae isolates induce gross pathological alterations to infected tissues (224). Even in the absence of gross pathology changes, inflammatory markers are present in the infant mouse model, which may influence oxygen availability in the gut (221). Neutrophil 103 recruitment is essential for combating V. cholerae during early stages of infection in the adult mouse model (284) and this can lead to higher gut oxygenation. To investigate the role of inflammation for V. cholerae aerobic growth, adult mice can be pre-treated with TNBS (285) or DSS (286) to induce inflammation. The colonization capacity of WT V. cholerae in the inflamed gut can then be determined. Development of a 3-5 day old infant mouse inflammation model would also be beneficial for studying colonization of V. cholerae. To date, a 10-day old TNBS infant mouse model has been studied (287), but would need to be appropriately tested for V. cholerae colonization efficacy. As Haitian isolate strains were phenotypically linked to a hypervirulent strain (288), it may be worthwhile to identify and clone evolved genes whose products may contribute to increased inflammation into 7th pandemic El Tor and Classical V. cholerae to examine whether inflammation is maintained and if this benefits colonization. Genomic comparisons between the Haitian El Tor variant and the El Tor and Classical biotypes may provide insight into genetic factors that engender host inflammatory responses and support colonization in the inflamed intestine. Finally, as the intestinal microbiota also plays a significant role in oxygen regulation in the gut, examining interactions between V. cholerae and host microbiota would also be of interest. In the human intestinal tract, V. cholerae must compete with resident microbiota for niche establishment in order to cause disease. V. cholerae can directly kill resident microbes through its Type VI secretion system (45, 46) to reduce competitor populations, including other facultative anaerobes of the gut capable of utilizing oxygen for respiration. Additionally, changes in the microbiota as a result of diarrheal flushing of 104 resident populations is also anticipated to affect oxygen concentrations in the gut. Adult mice treated with streptomycin to alter the microbiota have been used to study V. cholerae infection, but this alteration may confound the interpretation of data from experiments aimed to understand how the microbiota contributes to oxygenation (270). V. cholerae infection of zebrafish alters host microbiota composition (289), and this model may be worth exploring as well. By removing short chain fatty acid-producing microorganisms, host cells can no longer obtain adequate amounts of butyrate to grow via oxygen-dependent β-oxidation, shifting host cells to anaerobic glycolysis, leading to an increase in oxygen diffusion to the gut lumen. The infant and adult rabbit models may be key to investigating this relationship. These models, particularly the adult model, would involve a more established microbiota where significant alterations to commensal taxa may be observed following V. cholerae infection. 105 APPENDICES 106 APPENDIX A Supplemental Material for Chapter 1 107 A.1 – Supplemental Methods A.1.1 – Vibrio cholerae Initial Growth on Sigma Type III Porcine Gastric Mucin Strains were grown on LB + 0.1mg/mL streptomycin plates overnight at 37ºC and a single colony isolate used to start a fresh LB + 0.1mg/mL streptomycin broth culture grown overnight 210rpm at 37ºC. Overnight cultures were washed in PBS and resuspended to an optical density 1.0 OD600. 700µl of either LB, MCLMAN + 0.5% Mucin (Sigma Type III porcine gastric mucin), or MCLMAN minimal media was inoculated 1:1000 with the 1.0 OD600 culture. Triplicate 200µl aliquots were dispensed in a 96-well plate and optical densities recorded every 30min for 24h. A.1.2 – Complementation Plasmid Construction Complementation plasmid construct open reading frame inserts were generated by PCR using Phusion high-fidelity polymerase (Thermo Scientific). pMMB66EH vector backbones were generated by plasmid purification using Qiagen Mini Prep Kit and subsequent restriction digested using BamHI and HindIII at 37ºC for 1 hour followed by an additional 30 minutes at 37ºC with alkaline phosphatase from calf intestine (CIP) (New England Biolabs). Constructs were assembled using Gibson assembly (New England Biolabs) and were electroporated into electrocompetent S17 λpir E. coli and recovered on LB + 0.1mg/mL ampicillin agar plates. A.1.3 – Vibrio cholerae Complementation Strain Construction Complementation strains were made by mating S17 λpir E. coli pMMB66EH complementation strains with ΔaceE, ΔaceF, and ΔpflA mutant strains and recovered on LB + 0.1mg/mL ampicillin + 25 U/mL polymyxin B agar plates. Strains were verified 108 using pMMB66EH plasmid-specific primers to detect the full length insert and additional sequencing primers to verify construct sequence. A.1.4 – Complementation Growth Curves for ΔaceE and ΔaceF M9 + 0.5% glucose was prepared and 2mL prewarmed media inoculated 1:250 with 1.0 OD600 culture and grown 210rpm at 37ºC. For wild type control and ΔaceE complementation strains, 1mM isopropyl-β-D-thiogalactoside (IPTG) was added to induce ectopic expression from the complementation vector. For the ΔaceF complementation strain, 0.01mM IPTG was added to induce vector expression. At each timepoint, 100µl was removed for dilution series plating. A.1.5 – Complementation Growth Curves for ΔpflA M9 + 0.5% glucose 50mM fumarate media was prepared and 700µl was inoculated 1:250 with 1.0 OD600 culture. Triplicate 200µl aliquots were dispensed in a 96-well plate and grown statically at 37ºC in anaerobic conditions. Optical densities were recorded every 1h for 24h. For both WT control and pflA complementation strains, 1mM isopropyl-β-D-thiogalactoside (IPTG) was added to induce ectopic expression from the complementation vector. 109 A.2 – Supplemental Tables and Figures Table A.1. Transposon mutagenesis screen results. NR Well Location Locus Tag Gene Name Gene Description Gene Role 9-E1 VC2482 ilvH acetolactate synthase III, small subunit Amino acid biosynthesis 10-D1 VC0029 ilvE branched-chain amino acid amiotransferase Amino acid biosynthesis 9-E12 VCA0684 thiE thiamin-phosphate pyrophosphorylase Biosynthesis of cofactors, prosthetic groups, and carriers 17-F8 VC0061 thiC thiamin biosynthesis protein ThiC Biosynthesis of cofactors, prosthetic groups, and carriers 33-G10 VC1296 thiD phosphomethylpyrimidine kinase Biosynthesis of cofactors, prosthetic groups, and carriers 12-E7 VC2481 serA D-3-phosphoglycerate dehydrogenase Amino acid biosynthesis 28-B11 VC2345 serB phosphoserine phosphatase Amino acid biosynthesis 28-H7 VC2363 thrB homoserine kinase Amino acid biosynthesis 33-G6 VC2362 thrC threonine synthase Amino acid biosynthesis 10-F1 VC1169 trpA tryptophan synthase, alpha subunit Amino acid biosynthesis 33-H10 VC1170 trpB tryptophan synthase, beta subunit Amino acid biosynthesis 34-C5 VC1174 trpE anthranilate synthase component I Amino acid biosynthesis 5-H6 VC0705 pheA chorismate mutase/prephenate dehydratase Amino acid biosynthesis 25-C3 VC0056 aroE shikimate 5-dehydrogenase Amino acid biosynthesis 9-B1 VC0149 epsC general secretion pathway protein C Protein fate 28-E3 VC2725 epsL general secretion pathway protein L Protein fate 33-E3 VC1709 zinc protease, insulinase family Protein fate 1-F3 VC1181 cydD transport ATP-binding protein CydD Transport and binding proteins 12-G7 VC2270 ribE riboflavin synthase, alpha subunit Biosynthesis of cofactors, prosthetic groups, and carriers 23-C6 VC2268 ribE 6,7-dimethyl-8-ribityllumazine synthase Biosynthesis of cofactors, prosthetic groups, and carriers 28-H2 VC0094 ubiA 4-hydroxybenzoate octaprenyltransferase Biosynthesis of cofactors, prosthetic groups, and carriers 23-C8 VC2628 aroB 3-dehydroquinate synthase Amino acid biosynthesis 5-A12 VC2414 aceE pyruvate dehydrogenase, E1 component Energy metabolism pyruvate dehydrogenase, E2 component, 32-A1 VC2413 aceF dihydrolipoamide acetyltransferase Energy metabolism 27-A7 VC2544 fbp fructose-1,6-bisphosphatase Energy metabolism 4-H4 VC0604 acnB aconitate hydratase 2 Energy metabolism 26-A10 VC1487 conserved hypothetical protein Hypothetical proteins 3-C8 VC0849 conserved hypothetical protein Hypothetical proteins 110 Table A.2. Bacteria strain list. Strain Description Reference Vibrio cholerae V. cholerae C6706 El Tor biotype (Wild type) Wild type strain Waters Lab Collection V. cholerae ΔaceE (VC2414) Isogenic deletion strain This study V. cholerae ΔaceF (VC2413) Isogenic deletion strain This study V. cholerae ΔpflA (VC1869) Isogenic deletion strain This study V. cholerae ΔlacZ (VC2338) Isogenic deletion strain This study V. cholerae ΔtoxT (VC0838) Isogenic deletion strain Waters Lab Collection V. cholerae pMMB66EH (empty vector) Complementation verification strain This study V. cholerae ΔaceE pMMB66EH (empty vector) Complementation verification strain This study V. cholerae ΔaceE pMMB66EH-aceE Complementation verification strain This study V. cholerae ΔaceF pMMB66EH (empty vector) Complementation verification strain This study V. cholerae ΔaceF pMMB66EH-aceF Complementation verification strain This study V. cholerae ΔpflA pMMB66EH (empty vector) Complementation verification strain This study V. cholerae ΔpflA pMMB66EH-pflA Complementation verification strain This study Escherichia coli E. coli MCH100λpir pKAS32 (empty vector) Plasmid vector strain Lab Collection E. coli S17 pMMB66EH (empty vector) Plasmid vector strain Lab Collection E. coli S17 λpir 3-7 Cloning vector recipient Lab Collection 111 Table A.3. Primer list. Primer Name Primer Sequence (5' -> 3') Description Reference Mutant Construction Primers aceE Upstream Homology F GTGGAATTCCCGGGAGAGCTCAATATTTTTGCTGTTTAATCAACTCTTG pKAS32 construction primer This study aceE Upstream Homology R CATTACTTTTCCTACCTTCAAGGCGATCTATCCTTCTGTTGG pKAS32 construction primer This study aceE Downstream Homology F CCAACAGAAGGATAGATCGCCTTGAAGGTAGGAAAAGTAATG pKAS32 construction primer This study aceE Downstream Homology R CCGCGGACATGTACAGAGCTGCACGACTGGAGAAGCATG pKAS32 construction primer This study aceE aceE pKAS32 Seq Primer F1 GAACTGGAGAGACTGATAGTGGAAG pKAS32 sequencing primer This study VC2414 aceE pKAS32 Seq Primer F2 CAGAATTTTAAACTCTTACATCGC pKAS32 sequencing primer This study aceE pKAS32 Seq Primer R1 CAAAAGGTGCAGGTACTTCCAT pKAS32 sequencing primer This study aceE pKAS32 Seq Primer R2 CTGCACCTTCCGCTTCAA pKAS32 sequencing primer This study aceE Deletion Detection F CACCTCTAGCCCATCAAGTCC Isogenic deletion verification primer This study aceE Deletion Detection R GTAGTTCTGTACGTCTTCTTTCAGG Isogenic deletion verification primer This study aceF Upstream Homology F GTGGAATTCCCGGGAGAGCTTTCTTACTACAAAGAAGCGACTTC pKAS32 construction primer This study aceF Upstream Homology R TTTCGCCACCCGAGAATGCGCTTAAGCGTACAGCGGGTTGGT pKAS32 construction primer This study aceF Downstream Homology F ACCAACCCGCTGTACGCTTAAGCGCATTCTCGGGTGGCGAAA pKAS32 construction primer This study aceF Downstream Homology R CCGCGGACATGTACAGAGCTCTTTCGCTTCAACGGCGGTC pKAS32 construction primer This study aceF aceF pKAS32 Seq Primer F1 GTACAACGCTGAACGGTGAA pKAS32 sequencing primer This study VC2413 aceF pKAS32 Seq Primer F2 ATCGCAGCGACTGACTACAT pKAS32 sequencing primer This study aceF pKAS32 Seq Primer R1 CAGCGCATCGGTTGAATC pKAS32 sequencing primer This study aceF pKAS32 Seq Primer R2 GCTCATTTCGACCTCTTGTAGTC pKAS32 sequencing primer This study aceF Deletion Detection F TCCGTCAAATCGGTATCTACA Isogenic deletion verification primer This study aceF Deletion Detection R CGCATCGTAACGCTCAGCT Isogenic deletion verification primer This study pflA Upstream Homology F GTGGAATTCCCGGGAGAGCTTCACTGACTCGCAAAAAAG pKAS32 construction primer This study pflA Upstream Homology R AGAGGATGAAGAGCGCTTCTCAGTTATG pKAS32 construction primer This study pflA Downstream Homology F AGAAGCGCTCTTCATCCTCTCGACGTTATC pKAS32 construction primer This study pflA pflA Downstream Homology R TGCGCATGCTAGCTATAGTTAACTCGCGGTTCAGTTCAC pKAS32 construction primer This study VC1869 pflA pKAS32 Seq Primer F AATCTCAGACACCTTGTTTGAC pKAS32 sequencing primer This study pflA pKAS32 Seq Primer R GCCAGATATAAAGGGAGTTAAGC pKAS32 sequencing primer This study pflA Deletion Detection F GCTGTTGCTTCACTACTGAG Isogenic deletion verification primer This study pflA Deletion Detection R GGATTCTTGCATCGCATGATAC Isogenic deletion verification primer This study lacZ Upstream Homology F GTGGAATTCCCGGGAGAGCTGCCACCAAACACTAAGCTTC pKAS32 construction primer This study lacZ Upstream Homology R GCTCTCTGGCCCCTCAAGCCGAGGAGTAAAG pKAS32 construction primer This study lacZ Downstream Homology F GGCTTGAGGGGCCAGAGAGCCTTAAGGC pKAS32 construction primer This study lacZ lacZ Downstream Homology R TGCGCATGCTAGCTATAGTTTAGCACGTGAAGCCGGTG pKAS32 construction primer This study VC2338 lacZ pKAS32 Seq Primer F GATAACCAATCGCAAAACCAACTT pKAS32 sequencing primer This study lacZ pKAS32 Seq Primer R TCTCATCCGTCAAGGACATAGAAAC pKAS32 sequencing primer This study lacZ Deletion Detection F GAAATTGATCGGTCGATAGGCTG Isogenic deletion verification primer This study lacZ Deletion Detection R CCGAGTCCATAACTCTTATCCTTCTTA Isogenic deletion verification primer This study pKAS32 Multiple Cloning Site Seq Primer F GCCTCTAAGGTTTTAAGTTT pKAS32 specific sequencing primer Lab Collection pKAS32 pKAS32 Multiple Cloning Site Seq Primer R CTTTCAAGGTAGCGGTTACC pKAS32 specific sequencing primer Lab Collection toxT 1531 CAACTTCTGTAGTTAATGCAATTCCC toxT deletion verification primer Waters Lab Collection VC0838 1532 CCCTCCAGTAAATTTTCATAAATGTCG toxT deletion verification primer Waters Lab Collection Complementation Primer Sets pMMB66EH aceE ORF F CAGGAAACAGAATTCCCGGGATGTCTGACATGAAGCATGAC pMMB66EH aceE ORF construct primer This study pMMB66EH aceE ORF R CTCATCCGCCAAAACAGCCATTAAGCGTACAGCGGGTGG pMMB66EH aceE ORF construct primer This study aceE pMMB66EH aceE ORF Seq Primer 1 CTGCGTGCATCGAAGAAAGA pMMB66EH aceE ORF sequencing primer This study VC2414 pMMB66EH aceE ORF Seq Primer 2 CTGTTATGGGTAACGGTAAG pMMB66EH aceE ORF sequencing primer This study pMMB66EH aceE ORF Seq Primer 3 GTACCTGAAACTGGAAGAAG pMMB66EH aceE ORF sequencing primer This study pMMB66EH aceE ORF Seq Primer 4 GCGTGGATGGCGGGTGAC pMMB66EH aceE ORF sequencing primer This study pMMB66EH aceF ORF F CAGGAAACAGAATTCCCGGGTTGAAGGTAGGAAAAGTAATG pMMB66EH aceF ORF construct primer This study pMMB66EH aceF ORF R CTCATCCGCCAAAACAGCCATTACAGTACCAGACGACG pMMB66EH aceF ORF construct primer This study aceF pMMB66EH aceF ORF Seq Primer 1 AGCAGCGGCAGCACCAGC pMMB66EH aceF ORF sequencing primer This study VC2413 pMMB66EH aceF ORF Seq Primer 2 AGATCAAAGTGGCTACAGGCGA pMMB66EH aceF ORF sequencing primer This study pMMB66EH aceF ORF Seq Primer 3 GAGCAAAACGCGATGGAAGC pMMB66EH aceF ORF sequencing primer This study pflA pMMB66EH pflA ORF F CAGGAAACAGAATTCCCGGGATGTCTACCATTGGTCGAATTC pMMB66EH pflA ORF construct primer This study VC1869 pMMB66EH pflA ORF R CTCATCCGCCAAAACAGCCATCAATATTTTACGTTTGAGTGATAC pMMB66EH pflA ORF construct primer This study pMMB66EH Multiple Cloning Site Seq Primer F TGCATAATTCGTGTCGCTCA pMMB66EH specific sequencing primer This study pMMB66EH pMMB66EH Multiple Cloning Site Seq Primer R CTACGGCGTTTCACTTCTGA pMMB66EH specific sequencing primer This study RT-qPCR Primer Sets toxT toxT qPCR F ACTGATGATCTTGATGCTATGGAG qPCR Primer This study VC0838 toxT qPCR R CATCCGATTCGTTCTTAATTCACC qPCR Primer This study ctxA ctxA qPCR F TGGAATCCCACCTAAAGCAG qPCR Primer This study VC1475 ctxA qPCR R TTGTTAGGCACGATGATGGA qPCR Primer This study recA recA qPCR F GGGTAACCTCAAGCAATCCA qPCR Primer This study VC0543 recA qPCR R CCACTCTTCGCCTTCTTTG qPCR Primer This study tcpA tcpA qPCR F ACGCAAATGCTGCTACACAG qPCR Primer This study VC0828 tcpA qPCR R CCCCTACGCTTGTAACCAAA qPCR Primer This study 112 Table A.4. Purified porcine small intestinal mucin monosaccharide and sialic acid analysis determined by High-Performance Anion-Exchange Chromatography coupled with Pulsed Amperometric Detection (HPAEC-PAD). (GlycoAnalytics). Monosaccharide Amount (nmole/5µg of sample) Fucose 0.17 Galactosamine 0.14 Glucosamine 0.14 Galactose 0.17 Glucose 0.01 Mannose 0.02 Sialic Acid Amount (pmole/2µg of sample) Neu5Ac 8.54 Neu5Gc 6.90 113 Figure A.1. V. cholerae C6706 El Tor wild type growth in LB, minimal 0.5% mucin, and minimal media with no added carbon source. V. cholerae C6706 El Tor wild type growth in LB, minimal 0.5% mucin (Sigma Type III porcine gastric mucin) (MMu), and minimal media with no added carbon source (M). Data represent the average and SEM for three independent biological replicates. 114 Figure A.2. Complementation growth curves of ΔaceE and ΔaceF in M9 0.5% glucose media. ΔaceE and ΔaceF strains were complemented with IPTG-inducible vector pMB66EH. WT and ΔaceE strains were induced with 1mM IPTG whereas ΔaceF was induced with 0.01mM IPTG. (+) indicates the addition of IPTG inducer whereas (-) indicates the lack thereof. ‘pe’ denotes an empty vector control whereas ‘pE’ and ‘pF’ indicate the complementation plasmid contains the aceE and aceF open reading frames, respectively. Data represent the average and SEM for three independent biological replicates. 115 Figure A.3. Growth curves of WT, ΔaceE, and ΔaceF in LB media grown aerobically, anaerobically, or anaerobically supplemented with 50mM fumarate. Growth curves of WT (green circle) ΔaceE (dark grey square) and ΔaceF (grey triangle) in LB media grown (A) aerobically, (B) anaerobically, or (C) anaerobically supplemented with 50mM fumarate. Data represent the average and SEM for three independent biological replicates. 116 Figure A.4. Growth curves of WT, ΔaceE, ΔaceF in M9 0.2% Casamino acid media grown aerobically. Growth curves of WT (green circle), ΔaceE (dark grey square), ΔaceF (grey triangle) in M9 0.2% Casamino acid media grown aerobically. Data represent the average and SEM for three independent biological replicates. 117 Figure A.5. Complementation growth curve of ΔpflA in M9 0.5% glucose 50mM fumarate media grown anaerobically. ΔpflA strain was complemented with IPTG- inducible vector pMB66EH. WT and ΔpflA strains were induced with 1mM IPTG. (+) indicates the addition of IPTG inducer whereas (-) indicates the lack thereof. ‘pe’ denotes an empty vector control whereas ‘pA’ indicates the complementation plasmid contains the pflA open reading frame. Data represent the average and SEM for three independent biological replicates. 118 Figure A.6. Growth curves of WT and ΔpflA in LB media grown aerobically, anaerobically, or anaerobically supplemented with 50mM fumarate. Growth curves of WT (green circle) and ΔpflA (light grey square) in LB media grown (A) aerobically, (B) anaerobically, or (C) anaerobically supplemented with 50mM fumarate. Data represent the average and SEM for three independent biological replicates. 119 Figure A.7. Cholera toxin (CT) production for WT, ΔaceE, ΔaceF, and ΔtoxT strains. CT values relative to optical density (pg/mL/OD600) are reported under standard AKI toxin-inducing conditions (A-F) and anaerobic AKI conditions (G-H). (A- F) CT levels were measured at 4h, 5h, 6h, 7h, 8h, and 24h. (G-H) CT levels were measured at 8h and 24h. The optical densities for the biological replicates are displayed below the corresponding strain on the x-axis. Data represent the average and SEM for three biological replicates. Panel G is a duplicate graph of Figure 2.3B and is included here for convenient comparison with the 24h timepoint. 120 Figure A.8. Infant mouse colonization assays of WT and ΔpflA in the large intestine after 20h. (A) Mono-associated infections of 3-5 day old infant mice reported as CFU/g intestine. (B) Competition infections of 3-5 day old infant mice reported as a competitive index score calculated as a ratio of output versus input [(TargetOutput/ΔlacZOutput) / (TargetInput/ΔlacZInput)]. WT and ΔpflA strains were co- inoculated with a pflA+ ΔlacZ strain to determine the relative fitness of each test strain. Data for each experiment was obtained from 4-5 independent mouse colonization infections for WT and 7-8 mouse infections for ΔpflA. The bar represents geometric mean. Statistical analysis was performed using GraphPad PRISM where significance was tested on log transformed data by Student’s t-test; * indicates p<0.05. 121 Figure A.9. Growth curves of WT, ΔaceE, ΔaceF and ΔpflA in M9 0.3% acetate. Growth curves of WT (green circle), ΔaceE (dark grey square), ΔaceF (grey triangle) and ΔpflA (light grey inverted triangle) in M9 0.3% acetate. Data represent the average and SEM for three independent biological replicates. 122 Figure A.10. In vitro mono-culture and competition assays of WT, ΔaceE, ΔaceF, and ΔpflA in M9 0.5% glucose after 20h. (A-B) Mono-culture competitive index scores were calculated as a ratio of endpoint culture to initial culture of test strains versus a PDH+/PFL+ ΔlacZ strain grown in separate culture tubes; [(TargetEndpoint/ΔlacZEndpoint) / (TargetInitial/ΔlacZInitial)]. (C-D) Competitive index scores were calculated as a ratio of output versus input [(TargetOutput/ΔlacZOutput) / (TargetInput/ΔlacZInput)]. Strains were co-inoculated with a PDH+/PFL+ ΔlacZ strain to determine the relative fitness of each test strain. Data for each experiment was obtained from 3 biological replicates. 123 APPENDIX B Supplemental Material for Chapter 2 124 B.1 – Supplemental Methods B.1.1 – Hydrogen Peroxide (H2O2) Sensitivity Assay Bacterial strains were grown ON in 4mL LB Str100 broth. After ~16h of growth, cultures were diluted to 1.0 OD600. In a 96-well plate all wells were filled with 100µl LB broth and in the second row 100µl of LB 20mM H2O2 (Fisher Chemical) was added and serially diluted to the last row where 100µl was removed leaving a volume of 100µl in all wells. In a 2mL tube, 1.7µl of fresh LB was inoculated 1:500 (2:1000 / 3.4µl) with the 1.0 OD600 cultures. Tubes were vortexed and inoculated media was distributed in duplicate columns for each strain and included blank LB control lanes. 96-well plates were grown at 37°C 210rpm on a plate shaker for 20h. After 20h, the OD600 was read and percent bacterial growth was determined in comparison to the first row which lacks added H2O2. B.1.2 – AKI Virulence Inducing Conditions Standard AKI Conditions Bacterial strains were struck on LB streptomycin (100µg/ml) plates and used to inoculate 4mL LB media. After ~16h bacterial strains were diluted to a 1.0 OD600. These cultures were then used to inoculate 40mL prewarmed AKI media 1:5000 (8µl) which were then inverted and set to grow static at 37°C for 4h. After the 4h timepoint, 19mL of culture was transferred to sterile 125mL Erlenmeyer flasks and grown at 37°C, 210rpm, for an additional hour to the experiment 5h timepoint. At both the 4h and 5h timepoint, samples for cholera toxin and TcpA quantification were taken. At the 4h and 5h timepoints, 20mL and 10mL of culture, respectively, were centrifuged at 4000rpm, 4°C, for 10min. For cholera toxin analysis, 1mL of supernatant was removed and stored at -80°C. For TcpA quantification, the bacterial pellet was resuspended in Resuspension 125 Buffer (50mM Tris-HCl pH 7.4, 50mM EDTA pH 8.0) and transferred to a 1.7mL Eppendorf tube where an equal volume of Lysis Buffer (1% SDS, 10mM Tris-HCl pH 7.4) was added. The bacterial solution was vortexed for 10s, boiled for 10min, and then stored at -80°C. Anaerobic AKI Conditions Bacterial strains were struck on LB streptomycin (100µg/ml) plates and used to inoculate 4mL LB deoxygenated media. After ~16h bacterial strains were concentrated and resuspended to a 1.0 OD600 anaerobically. These cultures were then used to inoculate 40mL prewarmed deoxygenated AKI media 1:5000 (8µl) which were then inverted and set to grow static at 37°C for 8h. After 8h, tubes were removed from the anaerobic chamber and 20mL volume centrifuged at 4000rpm, 4°C, for 10min. For cholera toxin analysis, 1mL of supernatant was removed and stored at -80°C. For TcpA quantification, the bacterial pellet was resuspended in Resuspension Buffer (50mM Tris- HCl pH 7.4, 50mM EDTA pH 8.0) and transferred to a 1.7mL Eppendorf tube where an equal volume of Lysis Buffer (1% SDS, 10mM Tris-HCl pH 7.4) was added. The bacterial solution was vortexed for 10s, boiled for 10min, and then stored at -80°C. B.1.3 – V. cholerae Terminal Oxidase Strain M9 Glucose Growth Curves Bacterial strains were struck on LB streptomycin (100µg/ml) and used to inoculate 4mL LB deoxygenated media. After ~16h bacterial strains were concentrated to a 1.0 OD600 anaerobically. These cultures were used to inoculate 700µl M9 0.2% Glucose 1:250 (2.8µl), vortexed, and aliquoted in triplicate 200µl volumes in a 96-well plate. Optical density was recorded every hour for the duration of the growth curve. Deoxygenated M9 126 0.2% Glucose was used for anaerobic growth curves and benchtop media used for aerobic growth curves. B.1.4 – Oxidase TMPD Test Strips Bacterial strains were tested for functional cytochrome c oxidase cbb3 using a rapid test DrySlide containing N1N1N’1N’-tetramethyl-p-phenylene-diamine dihydrochloride (Wurster’s blue; TMPD). Strains were grown on LB Str100 agar media 18-24h and cell collections spotted onto DrySlide using a wooden applicator. Color was allowed to develop, and images taken. B.1.5 – Whole Genome Sequencing Submission and Analysis DNA of select V. cholerae strains was submitted to the Microbial Genome Sequencing Center (MiGS) for whole genome sequencing following sample submission guidelines. Reads were trimmed for low quality base calls and aligned to NCBI reference genome ASM1308507v1, El Tor C6706 V. cholerae using Geneious software to generate genome assemblies. Genomes were aligned using Mauve and genomic polymorphisms recorded. B.1.6 – TcpA Western Protein Electrophoresis and Immunodetection Bacterial stains were grown under standard and anaerobic AKI conditions (199). Briefly, for standard AKI conditions, 40ml of AKI media was inoculated (1:5000) with a 1.0 OD600 bacterial cell suspension and incubated at 37°C static for 4hr and switched to shaking for one additional hour at which point sample cell pellets were harvested. For anaerobic AKI conditions, 40ml of deoxygenated AKI media was inoculated (1:5000) with a 1.0 OD600 bacterial cell suspension and incubated at 37°C static for 8hr in 127 anaerobic conditions at which point sample cell pellets were harvested. Bacterial cell pellets were first resuspended in resuspension buffer (50mM Tris-HCl pH 7.4, 50mM EDTA pH 8.0), lysed with addition of a lysis buffer (1% SDS, 10mM Tris-HCl pH 7.4), and boiled 10 minutes. After cell lysis, the total protein concentration of each sample was measured via Bradford assay (Sigma Aldrich). Samples were subsequently diluted to a final concentration of 0.5 µg total protein/µl. Samples were loaded on an SDS page gel which contained 12.5% acrylamide and run at 120 volts for 1.5 hours. Proteins were transferred to a nitrocellulose membrane using a semi dry electroblotter (Fisher Scientific) overnight at 35 mA. Membranes were blocked with 15 ml of blocking buffer (5% non-fat milk, 2% bovine serum albumin, 0.5% Tween-20, in Tris-buffered saline) for 1 hour at room temperature. α-TcpA antibodies were diluted 1:100,000 in 5% non-fat milk and incubated with the membranes for 1 hour at room temperature. Membranes were washed three times for five minutes with Tris-buffered saline. Goat anti-Rabbit IgG-HRP antibodies (Sigma Aldrich) were diluted 1:2,000 in 5% non-fat milk in Tris- buffered saline and incubated as before. Membranes were washed three times for five minutes with Tris-buffered saline, and then incubated with SuperSignal HRP Chemiluminescence substrate (Thermo Fisher) for five minutes at room temperature. Membranes were then imaged with an Amersham Imager 600. 128 B.2 – Supplemental Tables and Figures Table B.1. Whole Genome Sequencing SNP analysis annotation. Alignment Strain Chromosome Name Minimum Maximum Length Change Polymorphism Type Locus Protein Ana4 1A 1532419 1532419 1 G -> A SNP (transition) VC1492 Conserved Hypothetical Protein Insertion (tandem CCAGAA 306,404 306,403 0 (ACCAGA)11 -> (ACCAGA)12 bd -II 2B-1-1 2 repeat) VCA0283 Hypothetical Protein bd -III 3-2-1? Large col 1 626,770 626,774 5 -GTCGA Deletion VC2301 ChrR; anti-sigma factor for SigE; transcriptional regulator 129 Table B.2. CoMPAS sequencing reads. Sequencing Pool Target Locus Sequence Counts VC0838 VC1442 VC1844 VCA0872 VC1570 IP_1 16560 5896 13952 11529 7178 IP_2 47167 24594 44075 43971 32435 IP_3 60536 32368 51376 55351 42435 MP_1 62075 17503 3113 66325 32888 MP_2 56408 27681 5676 58797 30870 MP_3 63344 20741 4742 55125 38790 IP = Input Pool MP = Mouse Output Pool 130 Table B.3. Bacteria strain list. Strain Description Reference Vibrio cholerae V. cholerae C6706 El Tor biotype (Wild type) Wild type strain Waters Lab Collection V. cholerae ΔlacZ (VC2338) ΔVC2338 Isogenic deletion strain Lab Collection V. cholerae ΔtoxT (VC0838) ΔVC0838 Isogenic deletion strain Waters Lab Collection V. cholerae E7946 TND0195 ptac-TfoX ΔVC2417 ΔVC0766 ΔVC1807::Kan+ PMID: 28575400 V. cholerae E7946 TND0191 ΔVC2417 ΔVC0766 ΔVC1807::Spec+ PMID: 28575400 V. cholerae E7946 SAD030 Parent strain E7946 PMID: 28575400 V. cholerae E7946 SAD034 ΔVC1807::Kanr (50ug/mL) PMID: 28575400 V. cholerae E7946 SAD033 ΔVC1807::Specr (200ug/mL) PMID: 28575400 V. cholerae E7946 SAD530 ΔVC1807::Tmr (10ug/mL) PMID: 28575400 V. cholerae C6706 pMMB-tfoX 1 Waters WT + Ankur SM10 strain pMMB-tfoX plasmid This study V. cholerae C6706 Δ501bp recJ Δ501bp xseA pMMB-tfoX 1 Waters Δ501bp recJ Δ501bp xseA + Ankur SM10 strain pMMB-tfoX plasmid This study Terminal oxidase mutant strains pKAS32-derived isogenic deletion strains V. cholerae Δcbb 3 ΔVC1439-1442 This study V. cholerae Δbd -I ΔVC1844-43 This study V. cholerae Δbd -II ΔVCA0872-73 This study V. cholerae Δbd -III ΔVC1571-70 This study V. cholerae Δbd -I Δbd -II Δbd -III +cbb 3 Strain This study V. cholerae Δcbb 3 Δbd -II Δbd -III +bd -I Strain This study V. cholerae Δcbb 3 Δbd -I Δbd -III +bd -II Strain This study V. cholerae Δcbb 3 Δbd -I Δbd -II +bd -III Strain This study V. cholerae Δcbb 3 Δbd -I +bd -II & + bd -III Strain This study V. cholerae Δbd -II Δbd -III +cbb 3 & bd -I Strain This study V. cholerae Δcbb 3 Δbd -I Δbd -II Variant (+bd -IIIV) +bd -IIIV Variant Strain This study MuGENT-derived knockout strains Mu V. cholerae Aero7 VC1442; MuVC1844-43; MuVCA0872-73; MuVC1571-70 This study Mu Mu V. cholerae cbb 3 VC1442 This study Mu Mu V. cholerae bd I VC1844-43 This study Mu Mu V. cholerae bd II VCA0872-73 This study Mu Mu V. cholerae bd III VC1571-70 This study Mu V. cholerae bd I Mu bd II Mu bd III Mu +cbb 3 Strain This study Mu Mu Mu Mu V. cholerae cbb 3 bd II bd III bd -I Strain This study Mu Mu Mu Mu V. cholerae cbb 3 bd I bd III +bd -II Strain This study Mu V. cholerae Mu cbb 3 Mu bd I Mu bd II +bd -III Strain This study Terminal reductase mutant strains MuGENT-derived knockout strains Mu V. cholerae Ana4 VC2656; MuVCA0678; MuVC1692; MuVC1950 This study Mu Mu V. cholerae Fum VC2656 (Fumarate reductase) This study Mu Mu V. cholerae Nit VCA0678 (Nitrate reductase) This study Mu Mu V. cholerae TMAO VC1692 (TMAO reductase) This study Mu Mu V. cholerae DMSO VC1950 (BSO reductase) This study Mu Mu Mu Mu V. cholerae Nit TMAO DMSO +Fum Strain This study Mu Mu Mu Mu V. cholerae Fum TMAO DMSO +Nit Strain This study Mu Mu Mu Mu V. cholerae Fum Nit DMSO +TMAO Strain This study V. cholerae Mu Fum Mu Nit Mu TMAO Mu +DMSO Strain This study Escherichia coli E. coli MCH100λpir pKAS32 (empty vector) Plasmid vector strain Lab Collection E. coli ET12567 ΔdapA Conjugation vector strain; diaminopimelic acid auxotroph PMID: 26166710 131 Table B.4. Primer list. Primer Name Primer Sequence (5' -> 3') Description Reference MuGENT Construction Primers AV MuGENT Mutant Seeking F CAAGCCGCTTAAACTGAATTAGC Common F oligo to detect all MuGENT inactivating mutations This study AV xseA MuGENT 2Kb Up F CATATTGCGGGGCACG ΔrecJ introduction from TND0195; WT repair from Waters C6706 F This study AV xseA MuGENT 2Kb Down ΔrecJ introduction from TND0195; WT repair from Waters C6706 R MuGENT R TTCTGGCCGCTCCATTG This study Construction AV recJ MuGENT 2Kb Up F TTAAAGTGAGAGGCGATTCTTTTAC ΔxseA introduction from TND0195; WT repair from Waters C6706 F This study Primers AV recJ MuGENT 2Kb Up R TTATTCAATTAGTTAGTATGCATGGGG ΔxseA introduction from TND0195; WT repair from Waters C6706 R This study AV DOG0223 xseA 501bp R to detect exoVII 501bp mutation in Vc – 449bp Fragment Confirming Primer R AGGATGCTGTTTATCAAGCTTGTG PMID: 28575400 AV VC2417 recJ 501bp R to detect recJ 501bp mutation in Vc – 550bp Fragment Confirming Primer R CAGTTGCAAACGCTCACAAAAC This study AV VC1442 cbb3 Gene GTGGTGTTACGACGAAAGTCGTGTTGGAAGTAAAAGTGTA F to create VC1442 knockout Inactivation Primer F caagccgcttaaactgaattagcAACAAAACTACAACTATACCGTGGTC This study AV VC1442 cbb3 Gene R to create VC1442 knockout Inactivation Primer R TGCCGTCTTGATAAGCCAAACG This study AV VC1844 Gene Inactivating GTTGCAACAACAAATTTTTTTTGTAGCAAAGTGTTATAGT F to create VC1844 knockout Primer F caagccgcttaaactgaattagcGACACAAAGGAGTTACCATGATCG This study AV VC1844 Gene Inactivating R to create VC1844 knockout Primer R AACGCCAGCGTTACCTAAAC This study AV VC1843 Gene Inactivating GTCTGTTCAGGATCAAGTCAACCGCCAAGTTGAAGCATAA F to create VC1843 knockout Primer F caagccgcttaaactgaattagcGGTGGGTACTGATTGGTGTG This study AV VC1843 Gene Inactivating R to create VC1843 knockout Primer R TATGGGCTGATGGAACCGAC This study AV VCA0872 Gene Inactivating CACTTCAAAAGCTGTGCTGAAATGCCTTCCAATTCTTGAT F to create VCA0872 knockout Primer F caagccgcttaaactgaattagcAATTATCGCGTTTGCAGTTCG This study AV VCA0872 Gene Inactivating R to create VCA0872 knockout Primer R CTGGATGTCGCATCCCAC This study AV VCA0873 Gene Inactivating TTATACGAGCATAAGTTGGCACAACAAGTACAATATTAGG F to create VCA0873 knockout Primer F caagccgcttaaactgaattagcGGCTGATCGGTGCATTGC This study AV VCA0873 Gene Inactivating R to create VCA0873 knockout Primer R TCCAGCTCGAGCCTTCTG This study Terminal oxidase AV VC1571 Gene Inactivating TAAAGATGGTGGTTAGTGTTAACCCCATCACTGGTTTGTT F to create VC1571 knockout primers Primer F caagccgcttaaactgaattagcTCCAATTCGCTGCCAATATCAG This study AV VC1571 Gene Inactivating R to create VC1571 knockout Primer R ACCATTATCGTCGTCACTCATG This study AV VC1570 Gene Inactivating ACCGCAAGTTTCACCAAGCTCTACAAAGGCCGCGTTATAA F to create VC1570 knockout Primer F caagccgcttaaactgaattagcTTATGTACGCGGTGTTGGATG This study AV VC1570 Gene Inactivating R to create VC1570 knockout Primer R GCACACGAATTTACGGTGATC This study AV VC1442 cbb3 Conf Prmr Mutant Verification Primer - 850bp Fragment New R TGC GAA CAG CAC GAG AGA C This study AV VC1844 bd I Conf Prmr Mutant Verification Primer - 1200bp Fragment New R ATA GTG GCC ATA CTG TTG GG This study AV VC1843 bd I Conf Prmr Mutant Verification Primer - 1000bp Fragment New R TAG CGT CAG CTT GCT TGA AG This study AV VCA0872 bd II Conf Prmr New R CGA GCA ACG GTG ACT ATG AG Mutant Verification Primer - 1500bp Fragment This study AV VCA0873 bd II Conf Prmr New R AAA TGG TAT GCG AGC GTG ATC Mutant Verification Primer - 1350bp Fragment This study AV VC1571 bd III Conf Prmr New R CAC CAA GAT CTG CAC AGG AAT C Mutant Verification Primer - 700bp Fragment This study AV VC1570 bd III Conf Prmr New R AGG AAA ACT AAA CCA ACG TTC TGC Mutant Verification Primer - 625bp Fragment This study AV VC1950 BSO Gene TCTTCCAGCACCATGCCAAAGATATGGTTAGCCACTGAGT F to create VC1950 knockout Inactivating Primer F caagccgcttaaactgaattagcTAAAAGGCACTGGTATGGCTGCCGGTG This study AV VC1950 BSO Gene R to create VC1950 knockout Inactivating Primer R AAAGCAGCAAGTCGGTTAAGAAC This study AV VC1692 TMAO Gene AAAACACTCTTCAGATTTCGCTGAAGGCCACCACTAAGCA F to create VC1692 knockout Inactivating Primer F caagccgcttaaactgaattagcAAGGTGTCGCAACCACCAG This study AV VC1692 TMAO Gene R to create VC1692 knockout Inactivating Primer R GCTGGCTTGAGGGATAACG This study AV VC2656 Fumarate Gene TTGAAAACTCTAGGTACTGAAACGAACTCAGTCCATAAAA F to create VC2656 knockout Inactivating Primer F caagccgcttaaactgaattagcTCGCAGTCATCGGCGC This study Terminal AV VC2656 Fumarate Gene R to create VC2656 knockout reductase Inactivating Primer R CTGGGTTCAACATCGCG This study primers AV VCA0678 Nitrate Gene CACTGGAAATACCTTTTCTGAACTCGAGGGTAATGTATAA F to create VCA0678 knockout Inactivating Primer F caagccgcttaaactgaattagcCGGCGGTTGCAGGGATC This study AV VCA0678 Nitrate Gene R to create VCA0678 knockout Inactivating Primer R GCTTAATACTTGGTCTGGGCTG This study AV VC1950 BSO Confirming Mutant Verification Primer - 500bp Fragment Primer R CGCCCGCCATACTCATATAG This study AV VCA0678 Nitrate Conf Prmr New R GAC ATC ACT TTG GTG TTT GGA TC Mutant Verification Primer - 1200bp Fragment This study AV VC1692 Conf Prmr 700bp AGA CTG GTG CGT TTC ACA G Mutant Verification Primer - 700 bp Fragment This study AV VC2656 Fumarate Mutant Verification Primer - 1000bp Fragment Confirming Primer R ACAGATAAATGGCAGACGTTCTTG This study 132 Table B.4 (cont’d) CoMPAS MASC-Primer Set AV 6-48 MuGENT AmpSeq F ACACTGACGACATGGTTCTACACAAGCCGCTTAAACTGAATTAGC MuGENT Mutant Seeking Amplicon Sequencing Primer F This study AV VC1442 AmpSeq 125R TACGGTAGCAGAGACTTGGTCTCTAATTGAGCGGCAATCAAAAC VC1442 Amplicon Sequencing Primer R 125bp This study AV VC1844 AmpSeq 125R TACGGTAGCAGAGACTTGGTCTTCCCTAGAGTGAGGGGAAC VC1844 Amplicon Sequencing Primer R 125bp This study Multiplex Allele- AV VCA0872 AmpSeq 125R TACGGTAGCAGAGACTTGGTCTAAATCGACTCCATGATAGCGAG VCA0872 Amplicon Sequencing Primer R 125bp This study Specific PCR AV VC1571 AmpSeq 125R TACGGTAGCAGAGACTTGGTCTGTGTTCGTAAATAACGCCATTTG VC1571 Amplicon Sequencing Primer R 125bp This study Sequencing Primers AV VC0838 toxT AmpSeq 125F ACACTGACGACATGGTTCTACAGTTCACTAAAATCTTACATTCTTGGTG VC0838 toxT Amplicon Sequencing Primer F 125bp This study AV VC0838 toxT AmpSeq 125R TACGGTAGCAGAGACTTGGTCTACCATTTACCACTTCAGAAAGG VC0838 toxT Amplicon Sequencing Primer R 125bp This study pKAS32 Mutant Construction Primers AV VC1844_43 1Kb Up F gtggaattcccgggagagctGTGAAGGCCCAGCTGCAC pKAS bd I Deletion Construct Upstream 1Kb Fragment This study AV VC1844_43 1Kb Up R tgagcgctccAGAACAACAATTACAATGTTAAAAAAACTATAACACTTTGC pKAS bd I Deletion Construct Upstream 1Kb Fragment This study AV VC1844_43 1Kb Dwn F ttgttgttctGGAGCGCTCATTATGTGG pKAS bd I Deletion Construct Downstream 1Kb Fragment This study AV VC1844_43 1Kb Dwn R tgcgcatgctagctatagttGTCACTGCAGTTTAACTCC pKAS bd I Deletion Construct Downstream 1Kb Fragment This study AV VC1844_43 Seq F 1Kb Up TGTTCTTGAGCCATTTCTCATT pKAS bd I Deletion Construct Sequencing Primer F This study AV VC1844_43 Seq R 1Kb Dwn CGTGCAATGTTTTCTTACCTAATGA pKAS bd I Deletion Construct Sequencing Primer R This study AV VC1844_43 >1Kb Up F (2) TGGAAAAAGCGGGAGATTTAGC pKAS bd I Genome Deletion Verification Primer F This study AV VC1844_43 >1Kb Dwn R (2)AAAGCGGTCTTCAAAGGTATC pKAS bd I Genome Deletion Verification Primer R This study AV VCA0872_73 1Kb Up F gtggaattcccgggagagctTATGCTTACAATCTTGCAC pKAS bd II Deletion Construct Upstream 1Kb Fragment This study AV VCA0872_73 1Kb Up R gcgaatacctCCATCGTAAGATGGCTTTAC pKAS bd II Deletion Construct Upstream 1Kb Fragment This study AV VCA0872_73 1Kb Dwn F cttacgatggAGGTATTCGCATGTGGTATTTC pKAS bd II Deletion Construct Downstream 1Kb Fragment This study AV VCA0872_73 1Kb Dwn R tgcgcatgctagctatagttTCAGCATGAGCAAATAGC pKAS bd II Deletion Construct Downstream 1Kb Fragment This study AV VCA0872_73 Seq F 1Kb Up GCAGCATCCCGATCCAG pKAS bd II Deletion Construct Sequencing Primer F This study AV VCA0872_73 Seq R 1Kb Dwn GTCACTAAGTGCTCGTTTTACTA pKAS bd II Deletion Construct Sequencing Primer R This study AV VCA0872_73 >1Kb Up F TGGCAGCCAAACGCTAAG pKAS bd II Genome Deletion Verification Primer F This study AV VCA0872_73 >1Kb Dwn R GTGAGTGATGAGAATGATGTGAAG pKAS bd II Genome Deletion Verification Primer R This study Terminal oxidase AV VC1570_71 1Kb Up F gtggaattcccgggagagctGAGTTTGTGCTTGATAAAG pKAS bd III Deletion Construct Upstream 1Kb Fragment This study pKAS32 primers AV VC1570_71 1Kb Up R gtagtaaaggCTCTTTTCCTCAAAAAATGAAAC pKAS bd III Deletion Construct Upstream 1Kb Fragment This study AV VC1570_71 1Kb Dwn F aggaaaagagCCTTTACTACACTTGAATCCAAAC pKAS bd III Deletion Construct Downstream 1Kb Fragment This study AV VC1570_71 1Kb Dwn R tgcgcatgctagctatagttAATTGCCGGCTTAGGTGTG pKAS bd III Deletion Construct Downstream 1Kb Fragment This study AV VC1570_71 Seq F 1Kb Up TTATTATCACATTACTGCTGATTGC pKAS bd III Deletion Construct Sequencing Primer F This study AV VC1570_71 Seq R 1Kb Dwn GCGATCCGCAATCAAAAG pKAS bd III Deletion Construct Sequencing Primer R This study AV VC1570_71 >1Kb Up F GCCGCTTGTTTAATGTAGGC pKAS bd III Genome Deletion Verification Primer F This study AV VC1570_71 >1Kb Dwn R GCAACCCCCAATACCATTCG pKAS bd III Genome Deletion Verification Primer R This study AV cbb3 1Kb Up F gtggaattcccgggagagctGAGATCAATGCCGTGCCG pKAS cbb3 Deletion Construct Upstream 1Kb Fragment This study AV cbb3 1Kb Up R tattagaatgGCTTCCAATGTTTAATTAACTACACTTTTAC pKAS cbb3 Deletion Construct Upstream 1Kb Fragment This study AV cbb3 1Kb Dwn F cattggaagcCATTCTAATAACAGCCCC pKAS cbb3 Deletion Construct Downstream 1Kb Fragment This study AV cbb3 1Kb Dwn R tgcgcatgctagctatagttTTGAGCTAATACATGGCTTAG pKAS cbb3 Deletion Construct Downstream 1Kb Fragment This study AV cbb3 Seq F 1Kb Up GACTGGAATTCCGTCGAAC pKAS cbb3 Deletion Construct Sequencing Primer F This study AV cbb3 Seq R 1Kb Dwn CGTTGGAGAAGAGGGGAAG pKAS cbb3 Deletion Construct Sequencing Primer R This study AV cbb3 >1Kb Up F (2) GCATGCAAAAGCACAAGAC pKAS cbb3 Genome Deletion Verification Primer F This study AV cbb3 >1Kb Dwn R (2) CAAGTATGTTTTCATGGCATCG pKAS cbb3 Genome Deletion Verification Primer R This study pKAS32 Multiple Cloning Site SeqGCCTCTAAGGTTTTAAGTTT Primer F pKAS32 specific sequencing primer Lab Collection pKAS32 Multiple Cloning Site SeqCTTTCAAGGTAGCGGTTACC Primer R pKAS32 specific sequencing primer Lab Collection RT-qPCR Primer Sets recA recA qPCR F GGGTAACCTCAAGCAATCCA qPCR Primer PMID: 32873763 VC0543 recA qPCR R CCACTCTTCGCCTTCTTTG qPCR Primer PMID: 32873763 cbb3 cbb3 qPCR F ACAAGTGCCCTGTTTGCAAC qPCR Primer This study VC1442 cbb3 qPCR F AAGGTGATTGCAGCGGAAAC qPCR Primer This study bd I bd I qPCR F AGTTTGTTCACACCGTTGCG qPCR Primer This study VC1844 bd I qPCR F TGATGCCGCAATCGCAAATG qPCR Primer This study bd II bd II qPCR F TGGTCACGCTTGTCAAAACG qPCR Primer This study VCA0872 bd II qPCR F TGCATCCAACCGTTTGCAAC qPCR Primer This study bd III bd III qPCR F TTGGTGTGATGCTGTTTGGC qPCR Primer This study VC1571 bd III qPCR F AGCACCAGAATCCAAAACGC qPCR Primer This study 133 Figure B.1. MuGENT spectinomycin selective marker shows no fitness defect in vitro. Comparison of wild type and ΔlacZ C6706 V. cholerae strains with and without MuGENT spectinomycin selective marker in pseudogene VC1807. (A) Aerobic in vitro competition assay after 20h. (B) Aerobic growth curve assay. (C) Anaerobic in vitro competition assay after 20h. (D) Anaerobic growth curve assay. All assays were performed in LB media. Bars in in vitro competitions represent the arithmetic mean where error bars represent the standard error of the mean. Growth curves are an average of three biological replicates where error bars represent the standard error of the mean. 134 Figure B.2. cbb3 deficient strains and wild type grown anaerobically do not maintain a functional cbb3 oxidase complex. V. cholerae cultures were grown on LB agar plates and spotted onto a rapid test DrySlide containing N 1N1N’1N’- tetramethyl-p-phenylene-diamine dihydrochloride (Wurster’s blue; TMPD) that turns blue when reduced by cytochrome c oxidases. (A) V. cholerae cbb3 mutant strain spots and E. coli (cytochrome c deficient) control. (B) Wild type V. cholerae grown in aerobic, microaerobic, and anaerobic conditions. 135 Figure B.3. V. cholerae oxidases generally support the same pattern of growth in minimal M9 0.2% D-glucose media as seen in LB. (A-B) Single terminal oxidase deletion mutants grown in M9 0.2% D-glucose, aerobically and anaerobically, respectively. Inoculums for all growth experiments were prepared in anaerobic conditions. Growth curves are an average of three biological replicates where error bars represent the standard error of the mean. 136 Figure B.4. V. cholerae oxidases generally support the same pattern of growth in combinatorial MuGENT knockout strains. (A-B) Combinatorial terminal oxidase deletion mutant growth in LB. Inoculums were prepared anaerobically and subsequently grown in aerobic and anaerobic conditions, respectively. Triple deletion mutant strains have a ‘+’ with an oxidase name (e.g. +cbb3), indicating the sole remaining oxidase, with the other three oxidases disrupted by mutation. Growth curves are an average of three biological replicates where error bars represent the standard error of the mean. 137 Figure B.5. Terminal reductase mutants are variable for aerobic growth in the presence of cognate electron acceptor molecules. V. cholerae terminal reductase aerobic growth characteristics in LB in the presence and absence of alternative electron acceptors (A) 50mM fumarate, (B) 50mM trimethylamine-N-oxide (TMAO), (C) 50mM nitrate, and (D) 50mM dimethyl sulfoxide (DMSO). Inoculums were prepared aerobically. Closed symbols indicate LB growth media lacked an alternative electron acceptor whereas open symbols indicate LB growth media was supplemented with a given alternative electron acceptor. Growth curves are an average of three biological replicates where error bars represent the standard error of the mean. 138 Figure B.6. Functional terminal oxidases, but not alternative terminal reductases, are required for optimal colonization of the large intestine. Aero7 and Ana4 colonization of the large intestine in both monoassociated and competition infections. (A) Monoassociated infection of strain Aero7. (B) Competition infection of strain Aero7. (C) Monoassociated infection of strain Ana4. (D) Competition infection of strain Ana4. Bars represent the geometric mean. Horizontal dashed lines indicate the limit of detection (LOD) and red dots indicate recovered CFUs were below the LOD. Competitive index scores were calculated as [(MutantOutput/WTOutput) / (MutantInput/WTInput)]. Statistical analysis was performed using GraphPad PRISM. *, P < 0.05. A Mann-Whitney U-test was used in the determination of significance between WT and Aero7. A Student’s T-test was performed on log transformed data in the determination of signifance between WT and Ana4. 139 Figure B.7. Individual and combinatorial oxidase mutants colonize the large intestine more efficiently than the small intestine but reflect overall colonization patterns present in the small intestine. Monoassociated in vivo colonization assays in the large intestine for (A) single and (B) combinatorial oxidase deletion strains. Triple deletion mutant strains have a ‘+’ with an oxidase name (e.g. +cbb3), indicating the sole remaining oxidase, with the other three oxidases disrupted by mutation. Bars represent the geometric mean. Horizontal dashed lines indicate the limit of detection (LOD) and red dots indicate recovered CFUs were below the LOD. Statistical analysis was performed using GraphPad PRISM. *, P < 0.05. A Mann-Whitney U-test was used in the determination of significance between WT and Aero7 and WT and +bd-III whereas an Analysis of Variance with post-hoc Dunnett’s multiple comparisons test was conducted on log transformed CFU/g intestine for all other strain comparisons. 140 Figure B.8. TcpA production is functional in individual oxidase deletion mutants. (A) Western blot visualization of TcpA, a required virulence factor in V. cholerae pathogenesis, in both standard and anaerobic AKI conditions. (B) Densitometry analysis of TcpA production in standard AKI conditions. (C) Densitometry analysis of TcpP production in anaerobic AKI conditions. TcpA levels are displayed as relative to TcpA production in wild type cells. ImageJ was used to perform the densitometry analysis across three biological replicates. Horizontal bars represent the arithmetic mean where error bars represent the standard deviation of the mean. 141 Figure B.9. Terminal oxidase complexes are not required for cell survival under hydrogen peroxide stress. Minimum inhibitory concentration determination of individual oxidase deletion strains. Growth percentage was calculated as a function of optical density for test strains in various concentrations of H2O2 (0.15625mM, 0.3125mM, 0.625mM, 1.25mM, 2.5mM, 5mM, and 10mM) divided by the optical density for wild type V. cholerae grown in LB media without H2O2. All strains showed signs of growth reduction at 1.25mM H2O2 and were all entirely inhibited for growth at 2.5mM H2O2. Data points represent the arithmetic mean of three biological replicates with error bars representing the standard error of the mean. 142 Figure B.10. Variant +bd-III strain (+bd-IIIV) indicates that the bd-III oxidase, when expressed, is capable of supporting aerobic respiration in V. cholerae and colonization of the infant mouse. (A) Wild type and +bd-III strain (Δcbb3 Δbd-I Δbd- II) bd-III expression. Expression was determined for the primary subunit of the bd-III oxidase VC1571. Dots represent biological replicates of relative bd-III expression between +bd-III and wild type strains. Bars represent arithmetic mean with error bars representing the standard error of the mean. (B-C) Wild type and +bd-IIIV growth in LB. Inoculums were prepared aerobically and subsequently grown in aerobic and anaerobic conditions, respectively. Data points represent the mean of triplicate growth curves with error bars representing the standard error of the mean. (D) In vitro expression of terminal oxidases in +bd-IIIV strain. Expression was determined for the primary subunit of each oxidase complex (VC1442, VC1844, VCA0872, VC1571). Bars represent the arithemtic mean with error bars representing the standard error of the mean. (E) Monoassociated infection of +bd-IIIV in the small intestine. (F) Monoassociated infection of +bd-IIIV in the large intestine. Bars in monoassociated infections represent the geometric mean. 143 APPENDIX C Investigating the Mucin Response Network of Shiga-toxin Producing Escherichia coli (STEC) 144 C.1 – Introduction STEC causes disease in the large intestine of infected human hosts (290). Virulence factors encoded on the Locus of Enterocyte Effacement (LEE) of STEC stimulate the remodeling of actin filaments, forming a pedestal on host epithelial cells (291). STEC produces Shiga toxin (Stx), an AB5 class toxin that binds to the vascular epithelium. Stx kills host cells which leads to bloody diarrhea among other disease symptoms (292). Translocation of Stx to the kidney can cause hemolytic uremic syndrome, the most severe complication associated with STEC infection (293). Within the human intestine, the mucosal barrier functions as a barrier to infection and is composed of a loose and dense mucus layer in the large intestine (294). To be successful in causing disease, STEC has evolved mechanisms to bypass both layers of mucus and gain access to the host epithelium. Through a literature review, a collection of gene targets that comprise the STEC “mucin response network” has been generated (Table C.1). Genes part of this list are involved in mucin sugar metabolism, mucin cell signaling, and mucin degradation (mucinases). Previous work has shown sugar constituents of mucin to contribute to pathogenesis of STEC (295, 296). The LEE1 operon which encodes the LEE regulator ler has been shown to be controlled by sugar signaling (297). NagC and FusR act as transcriptional repressors of the LEE1 operon in the presence of N-acetylglucosamine (GlcNAc) and fucose, respectively, both constituents of host mucin (295, 296). This interaction leads to spatial control over the activation of virulence factor production in STEC within the intestinal tract to activate virulence factors nearer the host epithelium. The outer loose mucus layer contains 145 many free mucin constituents, such as GlcNAc and fucose, whereas the inner dense mucus layer contains fewer free sugars, leading to the de-repression of the LEE1 operon near the site of infection (295, 296). Table C.1. Shiga toxin-producing E. coli mucin response network. Shiga toxin-producing Escherichia coli O157:H7 str. EDL933 Mucin Response Gene Name Function/Pathway Reference Protein Mucinase stcE (pO157) Mucinase (298) sulfatase (Z2210) Mucinase (295) Transcriptional fusK (Z0462) Fucose (296) Regulators fusR (Z0463) Fucose (296) galR (Z4155) Galactose (299) galK (Z0927) Galactose (300) N- (301) acetylgalactosamine, agaR (Z4483) N-galactosamine nagC (Z0823) N-acetylglucosamine (295) mlc (Z2587) Mannose (302) nanR (Z4584) Neuraminic acid (303) Metabolic Pathways N- (301) acetylgalactosamine, agaS (Z4490) Galactosamine nagE (Z0826) N-acetylglucosamine (300) fucA (Z4117) Fucose (300) manA (Z2616) Mannose (300) manX (Z2860) Mannose (302) nanA (Z4583) Neuraminic acid (300) The purpose of this research was to primarily identify variations in the sequence profiles among STEC isolates. We hypothesized that changes in the mucin response network may be important for disease onset and disease outcome as mucin signaling has shown to be important for pathogen virulence (295, 296). Genotypic variation in STEC isolates may provide a deeper understanding of specific bacterial response pathways that are influential in causing disease and may be useful indicators for disease severity. 146 C.2 – Results A subset of published NCBI STEC genomes were analyzed for the presence of mucin response genes and for sequence variation among isolates. This collection included both O157 and non-O157 STEC isolates. Sequences for each mucin response gene listed in Table C.1 were aligned between isolates using E. coli K-12 as an outgroup comparison. Each gene sequence was aligned with MUSCLE in MEGA7 and phylogenetic maximum likelihood trees were constructed using 500 bootstrap replications. Maximum likelihood trees for each mucin response gene are depicted in Figures C.1-C.14. Initial analysis indicates STEC serogroups group together for all mucin response genes tested. In a number of genomic sequences, target genes exhibited sequence variation which may become targets of future investigation to determine changes to protein function and eventual disease outcome. O157:H7 str. TW14588 Sakai O157:H7 ASM886v1 86 O157:H7 str. EDL933 O157:H7 str. SS17 100 O55:H7 str. RM12579 O145:H28 str. RM12581 O145:H28 str. RM13516 88 O145:H28 str. RM12761 O103:H2 str. 12009 O103:H2 STEC ASM1074v1 O111:H- ASM1076v1 95 O104:H4 ASM29945v1 O26:H11 str. 11368 65 O104:H4 str. 2011C 3493 O104:H4 str. C22711 K12 ASM584v2 Figure C.1. Maximum likelihood tree of the STEC sulfatase Z2210 mucin response gene. The evolutionary history was inferred using Maximum Likelihood method based on the Tamura-Nei model. The highest likelihood tree is shown. The tree was constructed using a 500 bootstrap replication. 147 O157:H7 str. EDL933 O157:H7 str. SS17 O157:H7 str. TW14588 Sakai O157:H7 ASM886v1 O55:H7 str. RM12579 O145:H28 str. RM12581 O145:H28 str. RM13516 100 O145:H28 str. RM12761 Figure C.2. Maximum likelihood tree of STEC mucin response gene fusK. The evolutionary history was inferred using Maximum Likelihood method based on the Tamura-Nei model. The highest likelihood tree is shown. The tree was constructed using a 500 bootstrap replication. Sakai O157:H7 ASM886v1 O55:H7 str. RM12579 O157:H7 str. TW14588 O157:H7 str. SS17 O157:H7 str. EDL933 O145:H28 str. RM12581 O145:H28 str. RM13516 88 O145:H28 str. RM12761 Figure C.3. Maximum likelihood tree of STEC mucin response gene fusR. The evolutionary history was inferred using Maximum Likelihood method based on the Tamura-Nei model. The highest likelihood tree is shown. The tree was constructed using a 500 bootstrap replication. 148 O26:H11 str. 11368 66 O103:H2 str. 12009 O111:H- ASM1076v1 O103:H2 ASM1074v1 O104:H4 str. C22711 95 O104:H4 ASM29945v1 O104:H4 str. 2009EL 2050 73 O104:H4 str. 2009EL 2071 O104:H4 str. 2011C 3493 O145:H28 str. RM13516 O145:H28 str. RM12581 100 O145:H28 str. RM12761 O157:H7 str. EDL933 O157:H7 str. SS17 O157:H7 str. TW14588 94 Sakai O157:H7 ASM886v1 O55:H7 str. RM12579 K12 ASM584v2 Figure C.4. Maximum likelihood tree of STEC mucin response gene galR. The evolutionary history was inferred using Maximum Likelihood method based on the Tamura-Nei model. The highest likelihood tree is shown. The tree was constructed using a 500 bootstrap replication. O157:H7 str. EDL933 O157:H7 str. SS17 99 O157:H7 str. TW14588 O55:H7 str. RM12579 46 Sakai O157:H7 ASM886v1 24 O111:H- ASM1076v1 100 O26:H11 str. 11368 O103:H2 STEC ASM1074v1 67 100 O103:H2 str. 12009 O145:H28 str. RM12581 O145:H28 str. RM13516 100 O145:H28 str. RM12761 O104:H4 str. 2009EL 2071 O104:H4 ASM29945v1 O104:H4 str. 2009EL 2050 99 O104:H4 str. 2011C 3493 O104:H4 str. C22711 K12 ASM584v2 Figure C.5. Maximum likelihood tree of STEC mucin response gene galK. The evolutionary history was inferred using Maximum Likelihood method based on the Tamura-Nei model. The highest likelihood tree is shown. The tree was constructed using a 500 bootstrap replication. 149 Sakai O157:H7 ASM886v1 O55:H7 str. RM12579 95 O157:H7 str. TW14588 O157:H7 str. SS17 99 O157:H7 str. EDL933 O145:H28 str. RM12581 47 O145:H28 str. RM12516 75 O145:H28 str. RM12761 O103:H2 STEC ASM1074v1 O103:H2 str. 12009 99 O111:H- ASM1076v1 72 O26:H11 str. 11368 O104:H4 str. C22711 O104:H4 ASM29945v1 O104:H4 str. 2009EL 2050 83 O104:H4 str. 2009EL 2071 O104:H4 str. 2011C 3493 K12 ASM584v2 Figure C.6. Maximum likelihood tree of STEC mucin response gene agaR. The evolutionary history was inferred using Maximum Likelihood method based on the Tamura-Nei model. The highest likelihood tree is shown. The tree was constructed using a 500 bootstrap replication. O157:H7 str. EDL933 O157:H7 str. SS17 100 O157:H7 str. TW14588 Sakai O157:H7 ASM886v1 97 O55:H7 str. RM12579 O104:H4 ASM29945v1 O104:H4 str. 2009EL 2050 O104:H4 str. 2009EL 2071 88 100 O104:H4 str. 2011C 3493 O104:H4 str. C22711 O145:H28 str. RM12581 O145:H28 str. RM13516 98 O145:H28 str. RM12761 O103:H2 ASM1074v1 O103:H2 str. 12009 100 O111:H- ASM1076v1 69 O26:H11 str. 11368 K12 ASM584v2 Figure C.7. Maximum likelihood tree of STEC mucin response gene nagC. The evolutionary history was inferred using Maximum Likelihood method based on the Tamura-Nei model. The highest likelihood tree is shown. The tree was constructed using a 500 bootstrap replication. 150 O104:H4 str. 2009 EL 2071 O104:H4 str. 2011C 3493 O104:H4 str. 2009EL 2050 65 O104:H4 ASM29945v1 O103:H2 STEC ASM1074v1 85 63 O103:H2 str. 12009 O104:H4 str. C22711 O111:H- ASM1076v1 O26:H11 str. 11368 O145:H28 str. RM13516 100 O145:H28 str. RM12761 O145:H28 str. RM12581 O157:H7 str. EDL933 99 O157:H7 str. SS17 O157:H7 str. TW14588 99 Sakai O157:H7 ASM886v1 O55:H7 str. RM12579 K12 ASM584v2 Figure C.8. Maximum likelihood tree of STEC mucin response gene mlc. The evolutionary history was inferred using Maximum Likelihood method based on the Tamura-Nei model. The highest likelihood tree is shown. The tree was constructed using a 500 bootstrap replication. O157:H7 str. EDL933 O157:H7 str. SS17 100 O157:H7 str. TW14588 Sakai O157:H7 ASM886v1 97 O55:H7 str. RM12579 O104:H4 ASM29945v1 O104:H4 str. 2009EL 2050 O104:H4 str. 2009EL 2071 87 100 O104:H4 str. 2011C 3493 O104:H4 str. C22711 O145:H28 str. RM12581 O145:H28 str. RM13516 100 O145:H28 str. RM12761 O103:H2 STEC ASM1074v1 O103:H2 str. 12009 99 O111:H- ASM1076v1 71 O26:H11 str. 11368 K12 ASM584v2 Figure C.9. Maximum likelihood tree of STEC mucin response gene nanR. The evolutionary history was inferred using Maximum Likelihood method based on the Tamura-Nei model. The highest likelihood tree is shown. The tree was constructed using a 500 bootstrap replication. 151 O104:H4 str. 2011C 3493 O104:H4 str. C22711 55 O104:H4 str. 2009EL 2071 O104:H4 str. 2009EL 2050 O104:H4 ASM29945v1 72 O103:H2 STEC ASM1074v1 O111:H- ASM1076v1 O26:H11 str. 11368 O103:H2 str. 12009 O157:H7 str. SS17 O145:H28 str. RM13516 65 O145:H28 str. RM12761 O145:H28 str. RM12581 97 O157:H7 str. EDL933 O157:H7 str. TW14588 Sakai O157:H7 ASM886v1 O55:H7 str. RM12579 K12 ASM584v2 Figure C.10. Maximum likelihood tree of STEC mucin response gene fucA. The evolutionary history was inferred using Maximum Likelihood method based on the Tamura-Nei model. The highest likelihood tree is shown. The tree was constructed using a 500 bootstrap replication. O104:H4 str. 2011C 3493 O104:H4 str. C22711 86 O104:H4 str. 2009EL 2071 O104:H4 str. 2009EL 2050 100 O104:H4 ASM29945v1 69 O103:H2 ASM1074v1 O103:H2 str. 12009 72 O111:H- ASM1076v1 65 O26:H11 str. 11368 O145:H28 str. RM13516 100 O145:H28 str. RM12761 O145:H28 str. RM12581 O157:H7 str. EDL933 94 O157:H7 str. SS17 O157:H7 str. TW14588 100 Sakai O157:H7 ASM886v1 O55:H7 str. RM12579 K12 ASM584v2 Figure C.11. Maximum likelihood tree of STEC mucin response gene nagE. The evolutionary history was inferred using Maximum Likelihood method based on the Tamura-Nei model. The highest likelihood tree is shown. The tree was constructed using a 500 bootstrap replication. 152 O104:H4 str. 2009 EL 2017a O104:H4 str. 2011C 3493 O104:H4 str. 2009EL 2050 86 O104:H4 ASM29945v1 O103:H2 ASM1074v1 100 66 O103:H2 str. 12009 O104:H4 str. C22711 O111:H- ASM1076v1 83 O26:H11 str. 11368 O55:H7 str. RM12579 O157:H7 str. EDL933 100 O157:H7 str. SS17 67 O157:H7 str. TW14588 Sakai O157:H7 ASM886v1 O145:H28 str. RM12581 O145:H28 str. RM13516 100 O145:H28 str. RM12761 K12 ASM584v2 Figure C.12. Maximum likelihood tree of STEC mucin response gene manA. The evolutionary history was inferred using Maximum Likelihood method based on the Tamura-Nei model. The highest likelihood tree is shown. The tree was constructed using a 500 bootstrap replication. O104:H4 str. 2009 EL 2071 O104:H4 str. 2011C 3493 O104:H4 str. 2009EL 2050 O103:H2 str. 12009 65 O104:H4 ASM29945v1 O104:H4 str. C22711 O111:H- ASM1076v1 64 O26:H11 str. 11368 O103:H2 STEC ASM1074v1 O145:H28 str. RM13516 99 O145:H28 str. RM12761 O145:H28 str. RM12581 99 O55:H7 str. RM12579 O157:H7 str. EDL933 97 O157:H7 str. SS17 66 O157:H7 str. TW14588 Sakai O157:H7 ASM886v1 K12 ASM584v2 Figure C.13. Maximum likelihood tree of STEC mucin response gene manX. The evolutionary history was inferred using Maximum Likelihood method based on the Tamura-Nei model. The highest likelihood tree is shown. The tree was constructed using a 500 bootstrap replication. 153 O157:H7 str. EDL933 O157:H7 str. SS17 72 O157:H7 str. TW14588 Sakai O157:H7 ASM886v1 100 O55:H7 str. RM12579 O145:H28 str. RM12581 O145:H28 str. RM13516 85 89 O145:H28 str. RM12761 O104:H4 ASM29945v1 O104:H4 str. 2009EL 2050 O104:H4 str. 2009EL 2071 100 O104:H4 str. 2011C 3493 O104:H4 str. C22711 O111:H- ASM1076v1 100 O26:H11 str. 11368 O103:H2 ASM1074v1 O103:H2 str. 12009 K12 ASM584v2 Figure C.14. Maximum likelihood tree of STEC mucin response gene nanA. The evolutionary history was inferred using Maximum Likelihood method based on the Tamura-Nei model. The highest likelihood tree is shown. The tree was constructed using a 500 bootstrap replication. C.3 – Future Directions Through partnership with the Manning lab at Michigan State University, which has collected an extensive library of STEC genomes in partnership with the Michigan Department of Health and Humans Services (MDHHS), we would look to perform a similar genome analysis among isolates. To strengthen our analysis, target gene sequences would be aligned with MUSCLE in MEGA7 and phylogenetic trees constructed by maximum likelihood with 1000 bootstrap replications and neighbor joining as a secondary tree building method. From this analysis, genes of interest can be determined and assessed through genetic manipulation of STEC to compare mucin response capacity. 154 As isolates were obtained in partnership with the MDHHS, epidemiological data concerning patients and clinical disease presentation are available to link to individual STEC genomes. To do this, univariate analysis of the mucin response genes in STEC will investigate potential associations with clinical outcomes listed in Table C.2 (304). A Chi-squared analysis with be performed to determine the Odds Ratio with a 95% confidence interval and associated p-value, which will be calculated and reported for each analysis; with a value P < 0.05 considered as significant (305). Drawing these associations allows for a better understanding of mucin response pathways that contribute to disease outcome (306). 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