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Acanthamoeba binds to extracellular matrix proteins in vitro
V R Gordon1, E K Asem, M H Vodkin
1Department of Veternary Pathobiology, Purdue University, West Lafayette, Indiana.
Purpose:
To identify host-tissue amoeba interactions that may be important in the pathogenesis of Acanthamoeba keratitis, the ability of the opportunistic pathogen Acanthamoeba polyphaga to bind various components of the extracellular matrix (collagen type IV, laminin, or fibronectin) was examined in vitro.
Methods:
A polyphaga, isolated from a case of human amoebic keratitis, was used in the studies. In the experiments, 96-well plates were coated with 0-, 5-, 10-, 20-, or 50-micrograms/ml solutions of the basal lamina proteins laminin or collagen type IV, the extracellular matrix protein fibronectin, or casein (control). Amoeba were metabolically labeled with 35[S]-methionine, and 1x 10(4) labeled amoeba in phosphate buffered saline (PBS) were seeded per well and allowed to bind for 20 min. After washing with PBS, bound amoeba were solubilized with 1% sodium dodecyl sulphate (SDS) and scintillation counting was used to determine the number of bound amoeba.
Results:
Counts from casein and protein-free controls were not significantly different from each other (P > 0.05). There was a significant increase in the binding of 35[S]-labeled A. polyphaga to collagen IV, laminin, and fibronectin over controls (P < 0.0001) and the binding was concentration-dependent. The rank order of binding was collagen > or = laminin >> fibronectin. Alpha-methyl-mannopyranoside, but not fucose, inhibited binding of labeled A. polyphaga to collagen IV, laminin, and fibronectin in a concentration-dependent manner.
Conclusion:
In summary, the binding assays show that Acanthamoeba bind preferentially to collagen, laminin, and fibronectin, in that order, and that the adherence process is inhibited by mannose.
Insights
Acanthamoeba polyphaga binds to collagen, laminin, and fibronectin, key extracellular matrix components. Mannose inhibits this binding, offering insights into Acanthamoeba keratitis pathogenesis.
Area of Science:
- Microbiology
- Cell Biology
- Ophthalmology
Background:
- Acanthamoeba keratitis is a severe eye infection.
- Understanding host-pathogen interactions is crucial for treatment.
- Extracellular matrix (ECM) components play a role in tissue adherence.
Purpose of the Study:
- To investigate the in vitro binding of Acanthamoeba polyphaga to ECM proteins.
- To identify potential host-tissue interactions in Acanthamoeba keratitis pathogenesis.
Main Methods:
- Acanthamoeba polyphaga isolated from keratitis cases was used.
- Radiolabeled amoeba were incubated with ECM proteins (collagen IV, laminin, fibronectin) and casein (control) in 96-well plates.
- Scintillation counting measured amoeba binding to coated plates.
Main Results:
- Acanthamoeba polyphaga demonstrated significant, concentration-dependent binding to collagen IV, laminin, and fibronectin.
- Binding affinity followed the order: collagen ≥ laminin >> fibronectin.
- Mannose (alpha-methyl-mannopyranoside) inhibited amoeba binding, while fucose did not.
Conclusions:
- Acanthamoeba exhibits preferential adherence to specific ECM components.
- Mannose-sensitive binding suggests a role for mannose-binding lectins in Acanthamoeba adherence.
- These findings contribute to understanding Acanthamoeba keratitis pathogenesis and potential therapeutic targets.