Recently, tick-borne diseases caused by infection with two new human ehrlichiosis agents: Ehrlichia chaffeensis and human granulocytic ehrlichiosis (HGE) agent are increasingly recognized in the U.S. Ehrlichiae are small obligate intracellular gram-negative bacteria, which infect monocytes/macrophages or granulocytes. The overall goal of the proposed study is to unravel ehrlichiacidal mechanisms in host cells. Hypothesis 1 is that IFN-gamma inhibits monocytic ehrlichiae, but not the HGE agent by iron starvation through down regulation of TfR mRNA, and inhibition of Tf accumulation in ehrlichial inclusions. Hypothesis 2 is that ehrlichiae are inhibited by intracellular Ca2+-calmodulin modulating agents or protein kinase inhibitors, because establishment of ehrlichial replication-competent inclusion and/or its metabolism including iron acquisition, is Ca2+ and calmodulin and tyrosine phosphorylation dependent. Hypothesis 3 is that protein kinase A or adenylate cyclase inhibitors potentiate ehrlichiacidal effects, because these inhibitors abrogate the inactivation by ehrlichiae of the host cell's innate immunity including Jak-Stat pathway and reactive oxygen intermediate (ROI) generation. To test these three hypotheses, the Principal Investigator proposes the following aims:
Aim1. To examine the influence of IFN-gamma on transfer of 59FE from holoTf to Ehrlichia sp., on TfR mRNA expression and TfR accumulation in ehrlichial inclusions, and on the activity of cytoplasmic iron regulatory factors.
Aim 2. To evaluate the influence of Ca2+ ionophore, inhibitors of intracellular Ca2+ mobilization, Ca2+ channel blockers, calmodulin antagonists, phospholipase C inhibitor, and protein tyrosine kinase inhibitors on Ca2+ and inositol phosphates concentrations, localization of proteins involved in membrane docking, trafficking, and fusion, TfR and lysosome localization, and the proteins transglutaminated in ehrlichia-infected cells.
Aim 3. To examine mechanisms and roles of protein kinase activation A activation in ehrlichial infection by measuring cAMP levels, adenylate cyclase, phosphodiesterase, and protein kinase A activities under various conditions, and evaluating the influence of protein kinase A inhibitors on IFN-gamma ,A23187-, calmodulin antagonists-, or ROI-induced ehrlichial killing. This study will generate important knowledge on how ehrlichiae usurp the host cell's signaling and membrane sorting mechanisms for their survival. New data on the HGE agent and insights towards interference of the host cell's activation signal as a mechanism of intracellular survival will be gained. The knowledge will be exploited in identifying novel therapeutic and vaccine targets.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Bacteriology and Mycology Subcommittee 2 (BM)
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Baker, Phillip J
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Ohio State University
Veterinary Sciences
Schools of Veterinary Medicine
United States
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Mohan Kumar, Dipu; Yamaguchi, Mamoru; Miura, Koshiro et al. (2013) Ehrlichia chaffeensis uses its surface protein EtpE to bind GPI-anchored protein DNase X and trigger entry into mammalian cells. PLoS Pathog 9:e1003666
Xiong, Qingming; Rikihisa, Yasuko (2012) Subversion of NPC1 pathway of cholesterol transport by Anaplasma phagocytophilum. Cell Microbiol 14:560-76
Xiong, Qingming; Rikihisa, Yasuko (2011) The prenylation inhibitor manumycin A reduces the viability of Anaplasma phagocytophilum. J Med Microbiol 60:744-9
Rikihisa, Yasuko (2011) Mechanisms of obligatory intracellular infection with Anaplasma phagocytophilum. Clin Microbiol Rev 24:469-89
Gibson, Kathryn; Kumagai, Yumi; Rikihisa, Yasuko (2010) Proteomic analysis of Neorickettsia sennetsu surface-exposed proteins and porin activity of the major surface protein P51. J Bacteriol 192:5898-905
Rikihisa, Yasuko; Lin, Mingqun (2010) Anaplasma phagocytophilum and Ehrlichia chaffeensis type IV secretion and Ank proteins. Curr Opin Microbiol 13:59-66
Rikihisa, Yasuko (2010) Molecular events involved in cellular invasion by Ehrlichia chaffeensis and Anaplasma phagocytophilum. Vet Parasitol 167:155-66
Niu, Hua; Kozjak-Pavlovic, Vera; Rudel, Thomas et al. (2010) Anaplasma phagocytophilum Ats-1 is imported into host cell mitochondria and interferes with apoptosis induction. PLoS Pathog 6:e1000774
Rikihisa, Yasuko; Lin, Mingqun; Niu, Hua (2010) Type IV secretion in the obligatory intracellular bacterium Anaplasma phagocytophilum. Cell Microbiol 12:1213-21
Xiong, Qingming; Lin, Mingqun; Rikihisa, Yasuko (2009) Cholesterol-dependent anaplasma phagocytophilum exploits the low-density lipoprotein uptake pathway. PLoS Pathog 5:e1000329

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