UC Irvine UC Irvine Electronic Theses and Dissertations Title Evolution of bacteriophages infecting Enterococcus from the human microbiome Permalink https://escholarship.org/uc/item/3dk3316p Author Wandro, Stephen Joseph Publication Date 2019 License https://creativecommons.org/licenses/by/4.0/ 4.0 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, IRVINE Evolution of bacteriophages infecting Enterococcus from the human microbiome DISSERTATION submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in Molecular Biology and Biochemistry by Stephen Joseph Wandro Dissertation Committee: Assistant Professor Katrine Whiteson, Chair Professor Jennifer Martiny Associate Professor Ilhem Messaoudi 2019 Chapter 1 © 2018 American Society for Microbiology Chapter 2 © 2017 American Society for Microbiology Chapter 3 © 2018 Frontiers Media All other materials © 2019 Stephen Wandro and Katrine Whiteson DEDICATION To my parents, Bob and Cathy Thank you for your love and support And to my partner, Linh Anh Let’s write the next chapter together ii TABLE OF CONTENTS Page LIST OF FIGURES iv LIST OF TABLES vi ACKNOWLEDGMENTS vii CURRICULUM VITAE viii ABSTRACT OF THE DISSERTATION x INTRODUCTION 1 CHAPTER 1: The Microbiome and Metabolome of Preterm 9 Infant Stool Are Personalized and Not Driven by Health Outcomes, Including Necrotizing Enterocolitis and Late-Onset Sepsis CHAPTER 2: Making It Last: Storage Time and Temperature Have 40 Differential Impacts on Metabolite Profiles of Airway Samples from Cystic Fibrosis Patients CHAPTER 3: Predictable molecular adaptation of coevolving 64 Enterococcus faecium and lytic phage EfV12-phi1 CHAPTER 4: Broad host range Brockvirinae phages infecting 93 Enterococcus and drive evolution of capsule synthesis genes CHAPTER 5: Phage cocktails can prevent the evolution of 120 phage-resistant Enterococcus SUMMARY AND FUTURE DIRECTIONS 138 REFERENCES 142 iii LIST OF FIGURES Page Figure 1.1 Study design schematic 26 Figure 1.2 Bacterial composition and bacterial load of preterm infant guts 27 Figure 1.3 Distance between bacterial communities over time 28 Figure 1.4 Alpha diversity 29 Figure 1.5 Metabolite profile of preterm infant fecal samples 30 Figure 1.6 Metabolite profile over time 31 Figure 1.7 Correlations between bacteria abundances and 32 metabolite intensities Figure S1.1 Bacterial composition of each set of twins 34 Figure S1.2 Average variation among infants of each metabolite 35 grouped by category Figure 2.1 16S rRNA gene sequence profiles of bacterial communities 52 Figure 2.2 Flow chart and study design schematic 53 Figure 2.3 Principal coordinates analysis (PCoA) of Bray-Curtis distances 54 calculated from the metabolite abundances in each sample Figure 2.4 Effect of storage at -20°C versus 4°C 55 Figure 2.5 The variables of importance from a supervised Random Forest 56 Figure 2.6 Intensities of several metabolites of potential clinical significance 57 Figure 2.7 Violin plot of the coefficients of variation 58 Figure S2.1 Heat map of a subset of metabolites detected by GC-MS after 60 storage at 4 °C iv Figure S2.2 Heat map of a subset of metabolites detected by GC-MS 61 after storage at -20 °C. Figure S2.3 Average percent change of each of the 239 detected metabolites 62 Figure S2.4 Total metabolite ion count (mTIC) of each sample over time 63 Figure 3.1 Experimental design of the three branches of the study 83 Figure 3.2 Growth dynamics of experimental coevolution 84 Figure 3.3 Host range analysis of phage and bacterial isolates 85 Figure 3.4 Frequency of common bacterial mutations over time 86 Figure 3.5 Phage EfV12-phi1 evolved tandem duplications in the 87 tail fiber gene to increase its infectivity Figure S3.1 Structural representation of RpoC with the positions 91 of the mutations highlighted in red Figure 4.1 Genomics of Enterococcus Brockvirinae phages 105 Figure 4.2 Host range of Brockvirinae Enterococcus phages 106 Figure 4.3 E. faecalis strains evolve mutations in Epa locus genes 107 to resist phage Figure S4.1 tRNAs in phage genomes 109 Figure 5.1 Phage cocktails prevent growth of resistant mutants 132 Figure 5.2 Cross-resistance patterns against Enterococcus phages 133 Figure 5.3 Vancomycin susceptibility of Enterococcus mutants 134 v LIST OF TABLES Page Table 1.1 Clinical and sampling information for all infants. 33 Table S1.1 PERMANOVA table 36 Table S1.2 All identified metabolites measured by GC-MS 37 Table S2.1 PERMANOVA of Bray-Curtis distances between samples 59 for each patient Table 3.1 All mutations present in E. faecium TX1330 at the final timepoint 88 Table 3.2 All mutations present in phage EfV12-phi1 at the final timepoint 89 Table S3.1 Locus tags for genes mutating in Enterococcus faecium TX1330 92 Table 4.1 Brockvirinae Enterococcus phages from the Sequence 108 Read Archive Table S4.1 Brockvirinae phage information 110 Table S4.2 Enterococcus strain information 111 Table S4.3 Enterococcus mutations 113 Table S4.4 Phage mutations 116 Table 5.1 Enterococcus phage information 135 Table 5.2 Mutations in Enterococcus providing phage resistance 136 Table S5.1 Phage titers 137 vi ACKNOWLEDGMENTS None of this work would have been possible without the support and guidance of many people. To my parents: thank you for being a constant source of love and support. You set me up for success and set examples for the kind of person I want to be. Thank you to my advisor, Katrine Whiteson, who has been an amazing mentor and an endless source of fun ideas. Thank you for teaching me how to be a scientist and allowing me the freedom to pursue my interests. I will miss our lab dinners, Halloween murals, and Julefrokost celebrations. Thank you to my committee member Jennifer Martiny for helping me dive into the world of phages and experimental evolution. To my other committee member, Ilhem Messaoudi, thank you for your thoughtful advice and feedback. To all the past and present members of the Whiteson Lab, thank you for making these last five years so much fun. You have been a great group of people to work with and this work wouldn’t be possible without the support of the whole lab. Andrew, Whitney, Tara, and Joann, you are my valued colleagues. Thank you for all the help you have given me over the years and for all the good times at Whiteson Lab happy hours. Thanks to my undergraduate researchers, Bushra and Cyril, for being excellent helpers. To Clark, you are the hardest working individual I have ever met and your passion for science inspires me. Thank you for being my lab hands. Thank you Claudia for your advice and help in the lab. Thank you to the people of microbial group for providing me good discussions and good happy hours. Thank you to my wonderful partner Linh Anh. You are the best thing to come out of my graduate school experience. I am continually impressed by you as a scientist, a writer, a science communicator, and as a person. You give me support when I need it and are always there with me to celebrate all the big and little things. Financial support was provided by the Center for Virus Research at UC Irvine. Thank you to the Mortazavi lab for generously allowing us to use their sequencer. Chapter 1 was published with the permission of American Society for Microbiology. The text of this chapter is a reprint of the material as it appears in the mSphere. Chapter 2 was published with the permission of American Society for Microbiology. The text of this chapter is a reprint of the material as it appears in the journal mSystems. Chapter 3 was published with the permission of the Frontiers Media. The text of this chapter is a reprint of the material as it appears in the journal Frontiers in Microbiology. vii CURRICULUM VITAE Stephen J. Wandro Contact: [email protected] GitHub: swandro Education PhD ..................................................................................................... September 2014 – June 2019 University of California, Irvine Department: Molecular Biology and Biochemistry Thesis adviser: Katrine Whiteson GPA: 4.0 Master’s Degree ............................................................................... September 2014 – November 2017 University of California, Irvine Department: Molecular Biology and Biochemistry GPA: 4.0 Bachelor of Science ........................................................................................................................ June 2014 University of California, Los Angeles Major: Microbiology, Immunology, and Molecular Genetics GPA: 3.86 Skills • Proficient in programming in Python, R, Bash • Next generation DNA sequencing and analysis • Advanced molecular techniques • Scientific communication • Multivariate statistics • Genomics, metagenomics, metabolomics, transcriptomics analyses Lab Experience Whiteson Lab (UCI) ........................................................................................... Spring 2015 – June 2019 Earned a PhD investigating bacteria and phage communities in the human gut. My research involved using next-generation sequencing approaches and experimental evolution to study how bacteria and phage from the human gut co-evolve. Kohn Lab (UCLA) .............................................................................................. Fall 2011 – Summer 2014 As an undergraduate researcher, I contributed to a project for using lentiviral vectors for gene therapy to treat ADA linked Severe Combined Immunodeficiency. Training • Woods Hole Microbial Diversity