Portland State University PDXScholar Dissertations and Theses Dissertations and Theses Summer 9-26-2017 Uncovering Coxiella burnetii's Pathogenicity by Elucidating its Metabolism and Host Interactions Jess Annai Millar Portland State University Follow this and additional works at: https://pdxscholar.library.pdx.edu/open_access_etds Part of the Microbiology Commons Let us know how access to this document benefits ou.y Recommended Citation Millar, Jess Annai, "Uncovering Coxiella burnetii's Pathogenicity by Elucidating its Metabolism and Host Interactions" (2017). Dissertations and Theses. Paper 3937. https://doi.org/10.15760/etd.5821 This Thesis is brought to you for free and open access. It has been accepted for inclusion in Dissertations and Theses by an authorized administrator of PDXScholar. Please contact us if we can make this document more accessible: [email protected]. Uncovering Coxiella burnetii’s Pathogenicity by Elucidating its Metabolism and Host Interactions by Jess Annai Millar A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biology Thesis Committee: Rahul Raghavan Susan E. Masta Kenneth M. Stedman Portland State University 2017 ABSTRACT Coxiella burnetii, the etiologic agent of acute Q fever and chronic endocarditis, has a unique biphasic life cycle, which includes a metabolically active intracellular form that occupies a large lysosome-derived acidic vacuole. C. burnetii is the only bacterium known to thrive within such a hostile intracellular niche, and this ability is fundamental to its pathogenicity; however, very little is known about genes that facilitate Coxiella’s intracellular growth. This lack of knowledge of Coxiella’s basic biology and molecular pathogenesis is a critical barrier to developing more effective therapies. In this study, we aimed to understand both bacterial and host factors that have important roles during C. burnetii infections. Using an evolutionary genomics approach, we identified metabolic pathways that are critical to C. burnetii’s ability to grow intracellularly. Among those found, the most promising are fatty acid, biotin, and heme biosyntheses pathways. Coxiella has horizontally acquired extra copies of genes that enhance these processes; when these genes were disrupted, Coxiella’s growth was significantly inhibited. Also, by analyzing the host transcriptome, we identified human genes, including microRNA (miRNA) genes that are important during C. burnetii infections. Coxiella induces the expression of multiple anti-apoptotic miRNAs, which likely have a role in inhibiting apoptosis in order to sustain the intracellular replication of the pathogen. The biosynthetic pathways and miRNAs identified in this study are ideal targets for developing more effective therapeutic strategies against Q fever and its chronic and often fatal complications. i ACKNOWLEDGEMENTS I would like to thank my research advisor Rahul Raghavan of the Biology Department at Portland State University. He has provided continual support but also generous freedom that has allowed me to design aspects and conduct much of this project independently. I would also like to acknowledge Rahul and Tina Schroyer for their assistance in writing my thesis. Their feedback has helped me become a better writer. I want to thank Abraham Moses and Fenil Kacharia for their assistance with conducting all the physical experiments and other lab duties. I also want to thank Todd Smith for taking the time to supplement my learning in bioinformatics. I would also like to thank my thesis committee: Susan Masta and Kenneth Stedman. I appreciate their time and effort reviewing my paper and supporting my research and education. This work was supported by startup funds from PSU to Rahul Raghavan, as well as the American Heart Association Beginning Grant-in-Aid, Collins Medical Trust Research Grant, Medical Research Foundation of Oregon New Investigator Grant, and NIH National Institute of Allergy and Infectious Diseases grants (R03 AI123464-01A1, R15 AI126385-01A1). Fellowship and partial funding was provided by the Portland State Laurels Graduate Award, Elsa Jorgenson Award, President's Equal Access Scholarship, and Forbes-Lea Research Award at PSU. Outside funding was also provided by the Sigma Xi Grants-in-Aid of Research Copernicus Fund, as well as travel funds from American Society for Microbiology, Pacific Northwest Women in Science, Society for Applied Microbiology, and Society for the Study of Evolution. ii TABLE OF CONTENTS PAGE ABSTRACT ......................................................................................................................... i ACKNOWLEDGMENTS .................................................................................................. ii LIST OF TABLES ...............................................................................................................v LIST OF FIGURES ........................................................................................................... vi CHAPTERS 1. Introduction ..................................................................................................1 2. Horizontally acquired biosynthesis genes boost Coxiella burnetii’s physiology ....................................................................................................5 2.1 Abstract ............................................................................................5 2.2 Introduction ......................................................................................7 2.3 Materials and Methods ...................................................................10 2.4 Results and Discussion ..................................................................13 3. Coxiella burnetii and Leishmania mexicana residing within similar parasitophorous vacuoles elicit disparate host responses ..........................30 3.1 Abstract ..........................................................................................30 3.2 Introduction ....................................................................................32 3.3 Materials and Methods ...................................................................35 3.4 Results and Discussion ..................................................................39 4. Conclusion .................................................................................................51 iii PAGE REFERENCES ..................................................................................................................53 APPENDICES A. Additional Files ..........................................................................................72 B. Genome rearrangements can make and break small RNA genes ..............75 C. Emergence of new sRNAs in enteric bacteria is associated with low expression and rapid evolution ................................................................107 D. Accumulation and expression of multiple antibiotic resistance genes in Arcobacter cryaerophilus that thrives in sewage .....................................142 E. Whole-genome sequence of Coxiella burnetii Nine Mile RSA 439 (phase II, clone 4), a laboratory workhorse strain ...............................................175 iv LIST OF TABLES PAGE TABLES 2.1 Expression of biotin and heme biosyntheses genes in C. burnetii .............24 3.1 KEGG pathways enriched in Coxiella-infected and Leishmania-infected THP-1 cells ................................................................................................42 3.2 MicroRNAs perturbed by Coxiella and Leishmania infections .................49 v LIST OF FIGURES PAGE FIGURES 1.1 Biogenesis of Coxiella-containing vacuole (CCV) ......................................2 2.1 Horizontally acquired genes in C. burnetii ................................................15 2.2 Maximum Likelihood tree of CBU_0678, gained via HGT ......................18 2.3 Bayesian tree of CBU_0678, gained via HGT ...........................................19 2.4 A horizontally acquired fatty acid biosynthesis operon in C. burnetii ......21 2.5 Bayesian tree of CBU_0038, gained via HGT ...........................................22 2.6 Heme and biotin syntheses effect on C. burnetii’s growth ........................26 2.7 tRNAGlu2 was horizontally acquired by C. burnetii ..................................27 3.1 Identification of differentially expressed genes .........................................40 3.2 Protein-protein interaction analysis ...........................................................44 3.3 Validation by qPCR of expression levels estimated by RNA-seq .............45 3.4 Differential expression of mRNA isoforms ...............................................47 vi CHAPTER 1 Introduction Coxiella burnetii is the etiological agent of acute Q fever and a chronic disease commonly manifested as endocarditis (Maurin and Raoult 1999). C. burnetii has a unique biphasic lifestyle. The pathogen persists in the environment as a metabolically quiescent small cell variant (SCV), which transforms into a metabolically active large cell variant (LCV) within an acidic (pH ~4.5) lysosome-derived Coxiella-containing vacuole (CCV) (Figure 1.1; Voth and Heinzen 2007). Coxiella is the only bacterium known to thrive in such a compartment, and this ability is considered critical to its pathogenicity. CDC has classified Coxiella as a select agent due to its past use as a bioweapon, environmental stability, aerosol transmission, and extremely low infectious dose [ID50 of one to ten bacteria