Rapid Activation of Protein Tyrosine Kinase and Phospholipase C-γ2 and Increase in Cytosolic Free Calcium Are Required byEhrlichia chaffeensisfor Internalization and Growth in THP-1 Cells

Open Access
Publisher Website
Google Scholar
Abstract
Ehrlichia chaffeensis, a bacterium that cannot survive outside the eukaryotic cell, proliferates exclusively in human monocytes and macrophages. In this study, signaling events required for ehrlichial infection of human monocytic cell line THP-1 were characterized. Entry and proliferation ofE. chaffeensisin THP-1 cells were significantly blocked by various inhibitors that can regulate calcium signaling, including 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate and 2-aminoethoxydiphenyl borate (intracellular calcium mobilization inhibitors), verapamil and 1-{β-[3-(4-methoxyphenyl)propyl]-4-methoxyphenethyl}-1H-imidazole (SKF-96365) (calcium channel inhibitors), neomycin and 1-(6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione (U-73122) (phospholipase C [PLC] inhibitors), monodansylcadaverine (a transglutaminase [TGase] inhibitor), and genistein (a protein tyrosine kinase [PTK] inhibitor). Addition ofE. chaffeensisresulted in rapid increases in the level of inositol 1,4,5-trisphosphate (IP3) and the level of cytosolic free calcium ([Ca2+]i) in THP-1 cells, which were prevented by pretreatment of THP-1 cells with inhibitors of TGase, PTK, and PLC.E. chaffeensisinduced rapid tyrosine phosphorylation of PLC-γ2, and the presence of a PLC-γ2 antisense oligonucleotide in THP-1 cells significantly blocked ehrlichial infection. Furthermore, tyrosine-phosphorylated proteins and PLC-γ2 were colocalized with ehrlichial inclusions, as determined by double-immunofluorescence labeling. The heat-sensitive component of viableE. chaffeensiscells was essential for these signaling events.E. chaffeensis, therefore, can recruit interacting signal-transducing molecules and induce the following signaling events required for the establishment of infection in host cells: protein cross-linking by TGase, tyrosine phosphorylation, PLC-γ2 activation, IP3production, and an increase in [Ca2+]i.