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Copyright by Casey L. C. Schroeder 2016 Copyright by Casey L. C. Schroeder 2016 Rickettsia prowazekii and its ‘Junk DNA’: The Identification and Characterization of Small RNAs by Casey Lee Cody Schroeder, MS, MLS(ASCP), SM, SLS Dissertation Presented to the Faculty of the Graduate School of The University of Texas Medical Branch in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas Medical Branch October, 2016 Dedication I dedicate this dissertation to my daughter, who has the fortunate pleasure of possessing half of her mother’s genes. Sorry about the other half. Explore. Dream. Discover. For I learned far more in the woods than I did inside a building. Nature is calling. Answer it. Acknowledgments I would like to acknowledge those that were instrumental: -To the Flying Spaghetti Monster: For your noodly appendage has touched and guided my results. Without the logical conjecture based on overwhelming observable evidence, the conclusions in this dissertation would not have been possible. -The Universe: For had you not expanded 13.8 billion years ago and created the Earth 4.5 billion years ago, none of this research would have been physically possible. The continued spewing of chemically-rich star guts atomically binds this research to the universe and biologically to the Earth. For that, I am eternally grateful. -Dissertation Committee: My sympathies for forcing you to sit through, what probably felt like never-ending, biannual dissertation committee meetings with me. If it is any solace, they have ended………. hopefully. Specifically, Dr. Fofanov, you’re fired. Dr. Minnick, enjoy fly fishing with the grizzly bears. Dr. Valbuena, sweet job in California. Dr. Walker, live long and prosper. Dr. Sahni, I know, it’s about time. -Evolution: For you are the greatest show on Earth! -Space: For Douglas Adams said it best, you are big. Really big. Vastly, hugely, mind-bogglingly big. Thanks for that. iv -Homo neanderthalensis: Hell of a thing 40,000 years ago. On behalf of all Homo sapiens, sorry it did not work out. If it is any consolation, your demise allowed Homo sapiens to discover science and the scientific method. Without that, this dissertation would have never been accomplished. -Rickettsia prowazekii: It is truly unfortunate that I was required to sacrifice some of your living descendants. I am greatly saddened by their loss. Nevertheless, their contributions to this work and to rickettsiology in general will not be forgotten. -Don’t Panic! So long, and thanks for all the fish! -Oh, and SCIENCE RULES! v Rickettsia prowazekii and its ‘Junk DNA’: The Identification and Characterization of Small RNAs Publication No._____________ Casey L. C. Schroeder, MS, PhD, MLS(ASCP), SM, SLS The University of Texas Medical Branch, 2016 Supervisor: Sanjeev K. Sahni, PhD As the etiologic agents of Rocky Mountain spotted fever and epidemic typhus, Rickettsia are obligate intracellular Gram-negative pathogenic bacteria. Genomic sequencing determined that ~24% of the Rickettsia prowazekii genome was noncoding DNA due to genomic reduction. Regions of bacterial noncoding DNA were found to encode important post-transcriptional regulators of bacterial virulence and growth called bacterial small RNAs (sRNAs). These sRNAs are classified as either trans-acting, whose biogenesis is predominantly linked to intergenic regions, or cis-acting, encoded on the antisense strand of an open reading frame (ORF). Despite being present in a majority of other bacteria, the existence and function of sRNAs are unknown in Rickettsia. In order to explore the possibility of rickettsial sRNAs, a combination of bioinformatics and in vitro techniques was employed. The first objective identified over 1,700 sRNAs using the SIPHT analysis tool to analyze 16 different strains representing 13 rickettsial species. Strong σ70 promoters and Rho-independent terminators were detected in their respective positions for all candidate sRNAs predicted in R. prowazekii, R. typhi, R. conorii, and R. rickettsii. Next generation sequencing (NGS) performed at 3h and 24h on R. prowazekii- vi infected human microvascular endothelial cells (HMECs) validated the expression of 26 sRNA candidates. Six selected candidates were further confirmed using RT-PCR. The second objective analyzed the NGS data and found the expression of an additional 35 trans- and 23 cis-acting novel sRNAs in addition to well-conserved kingdom-wide sRNAs (6.5S, 4.5S, RNaseP_bact_a, α-tmRNA). Three novel sRNAs and the conserved sRNAs were confirmed using either Northern blot analysis or RT-PCR. Transcriptional start sites were determined using RLM-RACE and secondary structure predicted for five novel sRNAs and 6S RNA. The third objective examined comparison of sRNA expression between HMECs and AAE2 (arthropod host cells), and found that using qRT- PCR, four sRNAs that showed significantly higher levels of expression at 24h. Also, a closer examination of Rp_sR60 found an interaction with H375_0420, in which both sRNA and target were found to be significantly decreased at 3h and 24h in HMECs. During AAE2 infection, both of these targets were found to be consistently expressed at 0.5h, 3h, and 24h. Together, these results establish the presence and expression of sRNAs in R. prowazekii during host cell infection and suggest potential functional roles for these important post-transcriptional regulators in rickettsial biology and pathogenesis. vii TABLE OF CONTENTS List of Tables .............................................................................................................x List of Figures ............................................................................................................xi List of Abbreviations .................................................................................................xii Chapter 1 Introduction to Rickettsia and Bacterial Small RNAs ...............................1 Introduction .......................................................................................................1 Rickettsioses .....................................................................................................2 Rickettsial Transmission and Arthropod Vectors .............................................4 Genomics ..........................................................................................................5 Bacterial Small RNAs .......................................................................................9 Chapter 2 Methodology .............................................................................................17 Rickettsia Species and Strains ...........................................................................17 Prediction of sRNAs .........................................................................................17 Promoter prediction ..........................................................................................18 Target prediction ...............................................................................................18 Cell Culture and Rickettsial Infection ..............................................................20 RNA isolation ...................................................................................................21 RNA Sequencing ..............................................................................................22 Reverse Transcriptase PCR ..............................................................................23 Northern Blotting ..............................................................................................25 RNA Ligase-Mediated Rapid Amplification of cDNA Ends (RLM-RACE) ...25 Electrophoretic Mobility Shift Assay (EMSA) ................................................26 Chapter 3 Prediction of Small RNAs in the Genus Rickettsia ...................................33 Introduction .......................................................................................................33 Results ...............................................................................................................34 Discussion .........................................................................................................42 Chapter 4 Identification and Validation of Novel Candidate sRNAs within R. prowazekii strain Breinl .......................................................................................64 Introduction .......................................................................................................64 viii Results ...............................................................................................................65 Discussion .........................................................................................................72 Chapter 5 Define the expression profile of novel sRNAs between the virulent strains of R. prowazekii and their mechanistic role during host-pathogen and vector- pathogen interactions ...........................................................................................91 Introduction. ......................................................................................................91 Results ...............................................................................................................92 Discussion .........................................................................................................98 Chapter 6 Summary ...................................................................................................112 Appendix Additional Files .........................................................................................117
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