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MILLIGAN-THESISMASTERS-2015.Pdf Ehrlichia chaffeensis Type 1 Secreted Effector TRP47 is a Novel SUMOylated Nucleomodulin by Sarah L. Milligan, B.S. Thesis 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 Master of Science The University of Texas Medical Branch November 2015 ACKNOWLEDGEMENTS I wish to thank: Dr. Jere W. McBride, my supervising professor, for instilling in me the confidence to accomplish what I thought impossible. Dr. David H. Walker and Dr. Sanjeev K. Sahni for their flexibility and insight as members of my supervising committee. Tierra Farris for her immeasurable assistance in the development and execution of this project and for always having faith in my abilities as a student and a scientist. Xiaofeng Zhang for her generosity in sharing her kindness and professional expertise. Dr. Shubhajit Mitra for his patience and mentorship. Dr. Joan E. Nichols and Dr. Jose M. Barral for their support and guidance through a stressful transition. Claire Smalley for her steadfast friendship when I needed it most. All past and present members of the McBride lab for their friendship and contributions to my research. My family, especially my mother, father, and sister, for their unconditional love and understanding. My husband, Larry, for keeping me grounded and focused on my goals. This work has been funded by the National Institutes of Health. iii Ehrlichia chaffeensis Type 1 Secreted Effector TRP47 is a Novel SUMOylated Nucleomodulin Publication No._____________ Sarah L. Milligan The University of Texas Medical Branch, 2015 Supervisor: Jere W. McBride Ehrlichia chaffeensis secretes immunodominant and immunoprotective protein effectors into the host cell using a type I secretion system. The best-characterized of these effectors are the tandem repeat proteins (TRPs). In this study, E. chaffeensis TRP47 was identified as a target for host cell SUMOylation and as a novel nucleomodulin with DNA-binding activity and host nuclear localization. Using a microfluidic chip peptide array, a SUMOylation site in the TRP47 N-terminal domain was identified. A SUMO-2 modification was further confirmed using an in vitro SUMOylation assay with recombinant TRP47, and native polySUMOylated TRP47 was immunoprecipitated from E. chaffeensis-infected cell lysates. TRP47 was detected in host cell nuclei primarily at 24 and 48 hours post-infection by immunofluorescence microscopy. A domain in the N- terminal portion of the protein containing a variant MYND-binding motif was identified as the region responsible for TRP47 nuclear localization using ectopically expressed GFP-tagged TRP47 truncation constructs. Recombinant TRP47 also binds human genomic DNA in an electrophoretic mobility shift assay. Taken together, these data demonstrate that E. chaffeensis TRP47 is a nucleomodulin with DNA-binding activity and a non-canonical nuclear localization signal, and is post-translationally modified by SUMOylation, which is a novel modification among bacteria. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS ..............................................................................................III LIST OF TABLES .........................................................................................................VII LIST OF FIGURES .......................................................................................................VIII INTRODUCTION ..........................................................................................................1 Ehrlichiae and ehrlichiosis .......................................................................1 Ehrlichia chaffeensis life cycle and pathobiology ...................................2 Ehrlichia chaffeensis tandem repeat protein 47 .......................................3 The SUMO pathway and exploitation by pathogens ...............................4 Nuclear localization .................................................................................6 Bacterial nucleomodulins.........................................................................7 MATERIALS AND METHODS ......................................................................................8 Maintenance of cell lines and cultivation of E. chaffeensis .....................8 Antibodies ................................................................................................8 Immunofluorescence microscopy ............................................................9 Generation and analysis of fluorescent signal intensity profiles .............9 Microfluidic peptide chip array ...............................................................10 Immunoprecipitation of native polySUMOylated TRP47 .......................11 Expression constructs and site-directed mutagenesis ..............................12 Expression and purification of recombinant TRP47 ................................14 In vitro SUMOylation assay ....................................................................15 Ectopic expression of GFP-tagged TRP47 constructs .............................15 Electrophoretic mobility shift assay .........................................................15 RESULTS ....................................................................................................................17 Identification of the TRP47 SUMOylation site .......................................17 Detection of SUMOylated TRP47 during infection and in vitro .............18 SUMOylation and TRP47 protein-protein interactions ...........................19 Subcellular localization of TRP47 in host cells during infection ............22 Identification of the domain required for TRP47 nuclear localization ....26 v Confirmation of TRP47 DNA-binding activity and identification of functional domain ...........................................................................26 DISCUSSION ................................................................................................................29 REFERENCES ..............................................................................................................34 VITA ..........................................................................................................................40 vi LIST OF TABLES Table 1: Oligonucleotide primers used to create E. chaffeensis TRP47 expression constructs ..............................................................................................123 vii LIST OF FIGURES Figure 1: TRP47 protein domains and predicted SUMOylation sites ......................17 Figure 2: PolySUMOylation of TRP47 during infection and in vitro ......................18 Figure 3: Effect of TRP47 SUMOylation on colocalization with host proteins ..............................................................................................................Er ror! Bookmark not defined. Figure 4: Detection of native TRP47 by immunofluorescence microscopy. ............21 Figure 5: Cytoplasmic and nuclear distribution of TRP47 in host cells ...................23 Figure 6: Schematic of GFP-tagged TRP47 expression constructs ..........................24 Figure 7: Localization of ectopically expressed TRP47 truncation constructs. .......25 Figure 8: TRP47 DNA-binding activity ...................................................................27 viii INTRODUCTION Ehrlichiae and ehrlichiosis Ehrlichiae are obligately intracellular gram-negative bacteria belonging to the order Rickettsiales and are tick-bone zoonoses capable of causing serious and fatal disease in humans and a wide variety of animals (1). The genus Ehrlichia currently has six members: Ehrlichia chaffeensis, the causative agent of human monocytotropic ehrlichiosis (HME); Ehrlichia canis, which causes canine monocytic ehrlichiosis; Ehrlichia ewingii, which infects humans and dogs; Ehrlichia ruminantium, the etiological agent of heartwater in ruminants (2); Ehrlichia muris, which causes persistent infection in mice (3); and the newly characterized Ehrlichia muris-like agent (EMLA), which infects both humans and mice (4). Ehrlichia chaffeensis, the focus of this study, is transmitted by the lone star tick (Amblyomma americanum) and is maintained in nature through persistent infection of white-tailed deer (Odocoileus virginianus) (1). E. chaffeensis is only maintained transstadially in ticks, and thus relies on persistently infected white-tailed deer as a primary reservoir in nature. Humans are an incidental host and can become persistently infected, but this is extremely uncommon and occurs primarily in the immunocompromised (5). Acute E. chaffeensis infection, known as HME, is a serious and potentially fatal disease and one of the most prevalent life- threatening tick-borne illnesses in the United States. HME initially presents as an undifferentiated febrile illness with nonspecific flu-like symptoms (6). Administration of doxycycline or tetracycline improves patient outcome, but only if treatment is initiated very early during the course of illness (7). Undiagnosed HME, especially in elderly or immunocompromised patients, can progress to multi-organ failure and result in death. Unfortunately, prompt diagnosis is difficult and is often based primarily on clinical suspicion. It is likely that many HME infections are undiagnosed and go unreported, and seroepidemiologic and prospective studies suggest that infection rates in a variety of populations are much higher than what would be expected based on the prevalence rate reported by the CDC (2.5 cases per million persons in 2010) (8,
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