Human granulocytic anaplasmosis (HGA) is an emerging tick-borne disease caused by Anaplasma phagocytophilum, an obligate intracellular bacterium of neutrophils. A. phagocytophilum infection impairs neutrophil function by transcriptional reprogramming, where the reprogrammed neutrophil promotes inflammatory recruitment of new neutrophils, tissue injury, ineffective regulation of inflammation, and poor antimicrobial responses. We studied altered neutrophil function with A. phagocytophilum infection and focused on how the nuclear effector protein AnkA, when delivered into the host cell where it binds to promoters of genes regulated with infection, induces epigenetic chromatin remodeling and transcriptional reprogramming. The granulocyte transcriptome with A. phagocytophilum infection shows a number of differentially transcribed genes that promote infection [3-6]. Given the meager genomic resources of A. phagocytophilum, it is difficult to explain the extent of host transcriptional change and functional reprogramming by individual translocated effector proteins. This implies that the bacterium exerts influence over global gene transcription, including chromatin and histone remodeling, perhaps by targeting conserved mechanisms of transcriptional regulation such as in cellular differentiation and neoplasia. AnkA has properties that suggest function as a matrix attachment region-binding protein that could regulate access of chromosomal territories to transcriptional modifiers, a new paradigm in bacteria-host interactions. We hypothesize that AnkA binds to promoters of some transcriptionally regulated genes and modifies or recruits modifiers of epigenetic chromatin marks or transcription factors. In addition, we hypothesize that A. phagocytophilum reprograms the global neutrophil transcriptome by altering the epigenome through AnkA""""""""s action on nuclear matrix, chromatin, and transcriptional apparatus recruitment. We propose the following aims: 1. To identify AnkA binding sites in the CYBB promoter and to define AnkA domains or motifs involved in CYBB promoter binding and transcriptional activity. 2. To determine whether AnkA affects host gene transcription through direct action at the CYBB promoter or through recruitment of chromatin remodeling or transcription factors. 3. To determine whether AnkA functions as a matrix attachment region-binding protein that tethers DNA to nuclear matrix, regulates DNA loopscape, and permits docking of other chromatin modifiers in global transcriptional regulation. The effects that bacteria have over cellular transcription are increasingly recognized. Testing these hypotheses will provide evidence of a potentially powerful mechanism for prokaryotic control over eukaryotes. The long- term goals are to develop a mechanistic understanding of how bacteria with intimate host cell associations circumvent host functions. This information could allow rational preventions and therapies for HGA, but could also span biology and medicine, since such molecules could be engineered as epigenetic tools or therapies.

Public Health Relevance

Human granulocytic anaplasmosis (HGA) is a tick-borne disease caused by an intracellular bacterium, Anaplasma phagocytophilum that must live within neutrophils and similar cells. Interestingly, neutrophils are the major early host defense cells, and A. phagocytophilum alters their function to benefit survival of the bacteria. The altered function occurs mostly because the mRNA-producing machinery of the cell is altered by infection with this bacterium. We learned that one protein made by the bacterium, AnkA, is transported to the neutrophil's nucleus where mRNA is made, and its presence there changes how mRNA is made. In fact, the magnitude of changes in mRNA made by the cell are very difficult to explain based on AnkA altering mRNA made from individual genes. We propose to study the exact way that AnkA changes mRNA production from single genes, and to study whether it also affects the structure and function of chromosomes in a way that mRNA production is drastically altered. This information could provide evidence of an entirely new way for bacteria to control host cells, could define some aspects of normal cell function, and might provide new tools, even new drugs, for studying cells and their function in health and disease.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Host Interactions with Bacterial Pathogens Study Section (HIBP)
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Perdue, Samuel S
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Johns Hopkins University
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Reller, Megan E; Dumler, J Stephen (2018) Development and Clinical Validation of a Multiplex Real-Time Quantitative PCR Assay for Human Infection by Anaplasma phagocytophilum and Ehrlichia chaffeensis. Trop Med Infect Dis 3:
Dumler, J Stephen; Sinclair, Sara H; Shetty, Amol C (2018) Alternative Splicing of Differentiated Myeloid Cell Transcripts after Infection by Anaplasma phagocytophilum Impacts a Selective Group of Cellular Programs. Front Cell Infect Microbiol 8:14
Scorpio, Diana G; Choi, Kyoung-Seong; Dumler, J Stephen (2018) Anaplasma phagocytophilum-Related Defects in CD8, NKT, and NK Lymphocyte Cytotoxicity. Front Immunol 9:710
Paris, Daniel H; Dumler, J Stephen (2016) State of the art of diagnosis of rickettsial diseases: the use of blood specimens for diagnosis of scrub typhus, spotted fever group rickettsiosis, and murine typhus. Curr Opin Infect Dis 29:433-9
Dumler, J Stephen; Sinclair, Sara H; Pappas-Brown, Valeria et al. (2016) Genome-Wide Anaplasma phagocytophilum AnkA-DNA Interactions Are Enriched in Intergenic Regions and Gene Promoters and Correlate with Infection-Induced Differential Gene Expression. Front Cell Infect Microbiol 6:97
Li, Hao; Zheng, Yuan-Chun; Ma, Lan et al. (2015) Human infection with a novel tick-borne Anaplasma species in China: a surveillance study. Lancet Infect Dis 15:663-70
Walker, David H; Dumler, J Stephen (2015) The role of CD8 T lymphocytes in rickettsial infections. Semin Immunopathol 37:289-99
Chikeka, I; Dumler, J S (2015) Neglected bacterial zoonoses. Clin Microbiol Infect 21:404-15
Bakken, Johan S; Dumler, J Stephen (2015) Human granulocytic anaplasmosis. Infect Dis Clin North Am 29:341-55
Rennoll-Bankert, Kristen E; Garcia-Garcia, Jose C; Sinclair, Sara H et al. (2015) Chromatin-bound bacterial effector ankyrin A recruits histone deacetylase 1 and modifies host gene expression. Cell Microbiol 17:1640-52

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