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Tyrosine phosphorylation is required for ehrlichial internalization and replication in P388D1 cells
1Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus 43210, USA.
Abstract:
Replication of Ehrlichia risticii was inhibited in P388D1 cells when a protein tyrosine kinase inhibitor (genistein or herbimycin A) was added after internalization of the organism at 3 h postinfection. Upon addition of genistein at day 1, 2, 3, or 4 postinfection, further proliferation of E. risticii was prevented. The inhibition was reversible, since regrowth of E. risticii occurred upon the removal of genistein. Genistein prevented spreading of E. risticii from P388D1 cells to THP-1 cells. Genistein did not prevent binding of [35S]methionine-labeled E. risticii to P388D1 cells but did prevent internalization of [35S]methionine-labeled E. risticii. 14CO2 production from L-[14C]glutamine in Percoll density gradient-purified E. risticii was not inhibited by genistein or herbimycin A, which suggests that these reagents did not directly inhibit ehrlichial energy metabolism. Double indirect immunofluorescence labeling with antiphosphotyrosine antibody and anti-E. risticii antibody revealed colocalization of tyrosine phosphoproteins with ehrlichial inclusions. There was, however, no colocalization of phosphotyrosine with phagosomes containing 0.5-microm-diameter fluorescent beads. Western immunoblot analysis revealed that 52- and 54-kDa proteins were tyrosine phosphorylated only in infected cells and that phosphorylation of these two proteins was reduced when infected cells were treated with genistein for 6 h. These results suggest that protein tyrosine phosphorylation is specific and essential for ehrlichial internalization, replication, and spreading in macrophages but not for binding.
Insights
Protein tyrosine phosphorylation is crucial for Ehrlichia risticii infection. Inhibiting this process prevents bacterial internalization, replication, and spread in host cells, but does not affect bacterial binding.
Area of Science:
- Microbiology
- Cell Biology
- Molecular Biology
Background:
- Ehrlichia risticii is an obligate intracellular bacterium that causes disease in animals.
- The mechanisms by which E. risticii infects host cells are not fully understood.
- Protein tyrosine phosphorylation plays a role in various cellular processes, including host-pathogen interactions.
Purpose of the Study:
- To investigate the role of protein tyrosine phosphorylation in Ehrlichia risticii infection.
- To determine if protein tyrosine kinase inhibitors affect E. risticii internalization, replication, or spread.
- To identify host cell proteins involved in E. risticii infection.
Main Methods:
- Treatment of infected P388D1 cells with protein tyrosine kinase inhibitors (genistein, herbimycin A).
- Assessment of E. risticii replication and spread using indirect immunofluorescence and cell culture.
- Analysis of protein tyrosine phosphorylation using Western immunoblotting and antiphosphotyrosine antibodies.
- Measurement of E. risticii metabolic activity.
Main Results:
- Protein tyrosine kinase inhibitors blocked E. risticii internalization and replication.
- Inhibition of protein tyrosine phosphorylation prevented E. risticii spread to new host cells.
- Tyrosine phosphorylation of specific host cell proteins (52 and 54 kDa) was observed during infection and reduced by inhibitors.
- Genistein and herbimycin A did not directly inhibit E. risticii energy metabolism.
- Phosphotyrosine colocalized with ehrlichial inclusions but not with phagosomes containing beads.
Conclusions:
- Protein tyrosine phosphorylation is essential for E. risticii internalization, replication, and spreading in macrophages.
- Host cell protein tyrosine phosphorylation is a specific and critical factor in E. risticii pathogenesis.
- Targeting host cell protein tyrosine kinases may represent a therapeutic strategy against ehrlichial infections.