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Characterization of Ehrlichia risticii binding, internalization, and proliferation in host cells by flow cytometry.
Abstract: The binding, internalization, and proliferation of Ehrlichia risticii in P388D1 cells and equine polymorphonuclear (PMN) leukocytes were studied by immunofluorescent staining and flow cytometric analysis. The binding of ehrlichiae to P388D1 cells at 4 degrees C was dose dependent, and the antigens of bound organisms were susceptible to pronase treatment. Additionally, the binding of ehrlichiae to P388D1 cells was diminished when either P388D1 cells or ehrlichiae were treated with 1% paraformaldehyde for 30 min or 0.25% trypsin for 15 min. These results indicate that the ehrlichial ligand and host cell receptor are likely surface proteins. Following incubation at 37 degrees C, bound E. risticii and/or its antigens were removed with pronase and indirect immunofluorescent staining in the presence of saponin was used to examine intracellular ehrlichiae. Our results indicate that E. risticii was internalized into P388D1 cells within 3 h and proliferated by 48 h of incubation. The microfilament-disrupting agent cytochalasin D and the transglutaminase inhibitor monodansylcadaverine were used to differentiate between phagocytosis (sensitive to cytochalasin) and receptor-mediated endocytosis (sensitive to monodansylcadaverine) of E. risticii by P388D1 cells. In concentrations that produced distinctive morphological changes and inhibited phagocytosis of polystyrene latex beads, cytochalasin D did not suppress the infectivity of E. risticii. Binding, internalization, or proliferation of E. risticii was not affected by cytochalasin D. However, monodansylcadaverine inhibited infection of E. risticii in a dose-dependent manner. The agent did not affect the attachment of ehrlichiae to host cells, but it did suppress internalization and proliferation. These results suggest that E. risticii is internalized by receptor-mediated endocytosis and that productive infection by E. risticii does not depend on phagocytosis by the P388D1 cells. Although E. risticii did not bind to the surface of equine PMN leukocytes at 4 degrees C, organisms were taken up by this cell at 37 degrees C. E. risticii, however, failed to survive in equine PMN leukocytes.
Publication Date: 1993-09-01 PubMed ID: 8359901PubMed Central: PMC281080DOI: 10.1128/iai.61.9.3803-3810.1993Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- U.S. Gov't
- P.H.S.
Summary
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This study explores how the bacteria Ehrlichia risticii, a cause of disease in horses, binds to, enters, and multiplies within certain host cells. The study found that the bacteria make use of a specific form of cell entry called receptor-mediated endocytosis, differing from the previously suspected mechanism of phagocytosis.
Research Objectives and Methods
- The researchers investigated the interaction between Ehrlichia risticii and P388D1 cells and equine polymorphonuclear (PMN) leukocytes. Specifically, exploring how the bacteria binds to, enters (internalizes), and multiplies within these host cells.
- Immunofluorescent staining and flow cytometric analysis were employed as techniques. These methods allowed for tracking and visualizing the bacteria’s interaction with the cells.
- Several interventions were used to clarify if the bacteria and host cell interaction depends on specific proteins, and the temperature effects were also considered.
Core Findings
- The study discovered that the binding of ehrlichiae to P388D1 cells is dose-dependent and is linked to specific surface proteins on both the bacteria and the host cells.
- Ehrlichia risticii was found to enter P388D1 cells within 3 hours and to start multiplying by 48 hours of incubation. This suggests the bacteria’s efficient use of host resources for its propagation.
- Utilizing cytochalasin D and monodansylcadaverine, agents known to either affect phagocytosis (cell ingesting particles – the process thought to facilitate E. risticii entry) or receptor-mediated endocytosis (another form of interaction where a cell engulfs external components), illustrated that E. risticii entry doesn’t rely on phagocytosis. Rather, it seems to depend on receptor-mediated endocytosis as monodansylcadaverine inhibited E. risticii infection.
- In the tests with equine PMN leukocytes, E. risticii failed to bind to the cells at 4°C but was internalized by the cell at 37°C. However, these cells did not support the survival of E. risticii, presenting an interesting case to further investigate the bacterial interaction with the equine immune cells.
Implications of the Study
- Understanding how E. risticii interacts with its host cells and the mechanisms it uses for this interaction is critical for advancing strategies to combat diseases caused by this bacterium.
- This research provides insights into the selective use of receptor-mediated endocytosis by E. risticii, which could direct future attempts to develop treatment strategies that block this interaction.
Cite This Article
APA
Messick JB, Rikihisa Y.
(1993).
Characterization of Ehrlichia risticii binding, internalization, and proliferation in host cells by flow cytometry.
Infect Immun, 61(9), 3803-3810.
https://doi.org/10.1128/iai.61.9.3803-3810.1993 Publication
Researcher Affiliations
- Department of Veterinary Pathobiology, College of Veterinary Medicine, Columbus, Ohio 43210-1092.
MeSH Terms
- Animals
- Bacterial Adhesion
- Cadaverine / analogs & derivatives
- Cadaverine / pharmacology
- Cell Division
- Cell Line
- Cytochalasin D / pharmacology
- Ehrlichia / pathogenicity
- Ehrlichia / physiology
- Endocytosis
- Flow Cytometry
- Horses
- Macrophages / microbiology
- Mice
- Mice, Inbred DBA
- Neutrophils / microbiology
- Paraldehyde / pharmacology
- Trypsin / pharmacology
Grant Funding
- R01 AI 30010 / NIAID NIH HHS
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Citations
This article has been cited 18 times.- Rogan MR, Patterson LL, Byerly CD, Luo T, Paessler S, Veljkovic V, Quade B, McBride JW. Ehrlichia chaffeensis TRP120 Is a Wnt Ligand Mimetic That Interacts with Wnt Receptors and Contains a Novel Repetitive Short Linear Motif That Activates Wnt Signaling. mSphere 2021 Apr 21;6(2).
- Cheng Z, Lin M, Rikihisa Y. Ehrlichia chaffeensis proliferation begins with NtrY/NtrX and PutA/GlnA upregulation and CtrA degradation induced by proline and glutamine uptake. mBio 2014 Nov 25;5(6):e02141.
- Fallon AM. Flow cytometric evaluation of the intracellular bacterium, Wolbachia pipientis, in mosquito cells. J Microbiol Methods 2014 Dec;107:119-25.
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