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Anaplasma Phagocytophilum

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Anaplasma Phagocytophilum EFFECTOR BOTTLENECK: MICROBIAL REPROGRAMMING OF HUMAN NEUTROPHIL TRANSCRIPTION BY ANAPLASMA PHAGOCYTOPHILUM by Sara H Sinclair A dissertation submitted to the Johns Hopkins University in conformity with the requirements of the degree of Doctor of Philosophy. Baltimore, MD March 2015 Abstract Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium that infects human neutrophils. Once infected, the neutrophil shows marked functional aberrations, notably reduced antimicrobial activity, delayed apoptosis, and increased proinflammatory responses. The altered functions allow for bacterial propagation and spread to new host cells. Many of the observed phenotypic changes of the neutrophil are a result of transcriptional alterations. Furthermore, AnkA, a bacterial-derived effector localizes to the nucleus where it directly binds DNA, such as at the promoter of CYBB which encodes the NADPH oxidase component gp91phox. Binding of AnkA at CYBB results in downregulated transcription. Furthermore, histone deacetylase 1 (HDAC) activity is important for bacterial propagation and downregulation of multiple host defense genes. While the lab previously focused on characterizing the interaction between AnkA, CYBB, and HDAC1, we felt the data strongly suggested that A. phagocytophilum must have additional transcription-regulating effectors (nucleomodulins) to act on a global scale and to explain the magnitude of phenotypic changes observed within the neutrophil during the course of infection. A bioinformatic screen of the A. phagocytophilum genome for genes which could encode putative eukaryotic nuclear localization signals, and mass spectrometry of proteins in the infected cell nucleus yielded nearly 50 candidate nucleomodulins. Tracking of cellular localization with GFP-fusion peptides confirmed nuclear localization of seven genes, one of which (APH_0455) is also a type IV secretion substrate. Close investigation of microarray data gathered in 2005 comparing A. phagocytophilum-infected and uninfected neutrophils showed that large chromosomal ii territories were coordinately regulated. In addition, infected neutrophils were characterized by genome hypermethylation. Together these data further suggested that A. phagocytophilum infection influences transcriptional programs on a global scale, rather than relying solely on cis interactions and that AnkA likely is not the sole nucleomodulin at work. iii Acknowledgements I would like to thank my mentor Dr. J Stephen Dumler for always permitting me to work independently and being supportive of my ideas. His guidance and encouragement allowed me to grow into the scientist I am today. I needed to learn for myself when I had pursued something too long, and how to move on to the next questions. I would also like to thank the following current and former lab members for their continuous support both in and out of the lab: Kristen Bankert, Marguerite Lichay, Dennis Grab, Kyoung-Sung Choi, Diana Scorpio, Cindy Chen, Emily Clemens, Olga Nikolskaia, Lucy Hritzo, Carlos Borroto, and Jose Carlos Garcia-Garcia. I could not have asked for a better group of individuals to work with, and I have made life-long friends in the process. Additionally, I would like to thank my committee members Drs. Frank DeLeo, Stephen Baylin, and Srinivasan Yegnasubramanian for their insight and thoughtful criticism. Thank you to my family for encouraging me to leave Idaho and pursue graduate school. Without their love and support I would not have pursued research or a PhD. I would also like to thank my husband Dylan for moving across the country to support me and push me to work hard. Lastly, I would like to thank my friends who have been like family to me over the last five years. I know that I have made life-long friendships and connections and I would not be finishing now without their help. Between my friends and the lab, my PhD experience was both an enjoyable and fruitful experience. iv Table of Contents Chapter 1: The Effector Bottleneck: Chromatin Remodeling of Host Cell DNA via Epigenetic Mechanisms .......................................................................................................................... 1 References: ............................................................................................................................................. 5 Chapter 2: Anaplasma phagocytophilum: A model intracellular pathogen ............................. 7 Anaplasma phagocytophilum ............................................................................................................ 8 AnkA of Anaplasma phagocytophilum ....................................................................................... 10 References: .......................................................................................................................................... 13 Chapter 3: Anaplasma phagocytophilum induces genome-wide transcriptional changes in its host cell via chromatin remodeling ............................................................................................. 16 More than a cis interation ............................................................................................................... 17 Anaplasma phagocytophilum infection is associated with transcriptional changes over megabases in the human genome ................................................................................................. 21 References: .......................................................................................................................................... 27 Chapter 4: Global DNA methylation changes and differential gene expression in Anaplasma phagocytophilum-infected neutrophils. .................................................................... 29 Abstract ................................................................................................................................................ 30 Introduction ......................................................................................................................................... 31 Methods ................................................................................................................................................ 34 Results: ................................................................................................................................................. 41 Discussion ........................................................................................................................................... 59 Conclusion ........................................................................................................................................... 64 References ........................................................................................................................................... 65 Chapter 5: Bioinformatic and mass spectrometry identification of Anaplasma phagocytophilum proteins translocated into host cell nuclei .................................................... 70 Abstract ................................................................................................................................................ 71 Introduction ......................................................................................................................................... 72 v Methods ................................................................................................................................................ 74 Results .................................................................................................................................................. 85 Discussion ........................................................................................................................................... 98 Conclusion ......................................................................................................................................... 104 References: ........................................................................................................................................ 109 Chapter 6: Comparison and characterization of granulocyte cell models for Anaplasma phagocytophilum infection ............................................................................................................... 112 Abstract .............................................................................................................................................. 113 Introduction ....................................................................................................................................... 114 Materials and Methods .................................................................................................................. 117 Results ................................................................................................................................................ 123 Discussion ......................................................................................................................................... 136 References ......................................................................................................................................... 141 Chapter 7: Conclusions and Future Directions ........................................................................... 145 Curriculum Vitae 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