The research goal is to understand the mechanisms by which IFN-gamma and various Ca2+ modulating agents induce ehrlichial killing. Hypothesis 1 is that IFN-gamma-activated monocytes inhibit the multiplication of Ehrlichia spp. by limiting the availability of intracellular iron. Hypothesis 2 is that ehrlichial ATP synthesis is Ca2+ dependent. Hypothesis 3 is that calmodulin antagonists and Ca2+ channel blockers, by inhibiting clathrin assembly, block maintenance and addition of increments of inclusion membrane and transferrin receptor recycling. Hypothesis 4 is that all these inhibitions make ehrlichiae incapable of resisting acidification and lysosomal fusion with inclusion vacuoles. To prove these hypotheses, the specific research aims are:
Aim 1. Evaluate the inhibitory mechanism of E. chaffeensis and E. sennetsu in human monocytes by IFN- by examining: 1) the effects of dosages of IFN-gamma at different time points, 2) the numbers and affinities of transferrin receptors with or without IFN-gamma treatment and reversibility of the inhibitory effect with iron-saturated transferrin, 3) inhibitory effect of chloroquine and NH4Cl on ehrlichial proliferation, and 4) iron-uptake mechanism by Ehrlichia spp.
Aim 2. Study the effect of Ca2+ on Percoll-density gradient purified ehrlichiae by measuring: 1) 14C-L-glutamine metabolism and ATP production by ehrlichiae in the presence of different concentrations of Ca2+, and 2) Ca2+ concentration in ehrlichiae by electron probe X-ray microanalysis.
Aim 3. Study the inhibitory mechanism of ehrlichiae through inhibition of clathrin assembly by: 1) fluorescence double labeling of infected monocytes with anti- clathrin IgG and anti-Ehrlichia spp. IgG after treatment with W-7, verapamil, or monodansylcadaverine, 2) high-resolution electron microscopy of platinum replicas of freeze-dried fractured cells, and 3) examining whether iron nitrilotriacetates can reverse the inhibition.
Aim 4. Examine whether acidification and lysosomal fusion are involved in ehrlichial killing induced by all these reagents by: 1) immunofluorescence and immunogold labeling with anti-vacuolar type H+-ATPase antibody, 2) measuring accumulation of weak bases in the vacuole containing ehrlichiae, and 3) examining lysosomal fusion by acid phosphatase cytochemistry, electron-dense tracer ThO2-preloading and - chasing method, and immunofluorescence and immunogold labeling with anti- lysosomal glycoprotein antibody.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI030010-05
Application #
2065367
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1990-08-01
Project End
1999-04-30
Budget Start
1995-05-01
Budget End
1996-04-30
Support Year
5
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Ohio State University
Department
Veterinary Sciences
Type
Schools of Veterinary Medicine
DUNS #
098987217
City
Columbus
State
OH
Country
United States
Zip Code
43210
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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