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Pathogenesis of corneal infection: binding of Pseudomonas aeruginosa to specific phospholipids
N Panjwani1, Z Zhao, M B Raizman
1New England Eye Center, Tufts University School of Medicine, Boston, MA 02111, USA.
Abstract:
Clinical isolates of Pseudomonas aeruginosa were examined for binding interactions with phospholipids of corneal epithelium. Thin-layer chromatography (TLC) of lipids extracted from corneal epithelia followed by staining with an ammonium molybdate spray reagent revealed three phospholipid components, PL1, PL2, and PL3. The chromatographic mobility of PL1 was similar to that of the phospholipid standards phosphatidylinositol (PI) and phosphatidylserine (PS), which were not well resolved from one other; PL2 and PL3 comigrated with the standards phosphatidylcholine and phosphatidylethanolamine, respectively. By use of a TLC-bacterial overlay procedure, 35S-labeled P. aeruginosa organisms were shown to bind to PL1 but not to PL2 or PL3. P. aeruginosa binding to PL1 was concentration dependent. Alkaline methanolysis abolished the binding. PL1 was separated into two components, PL1-I and PL1-S, by chromatography on borate-treated TLC plates. Both PL1-I and PL1-S contained binding sites for P. aeruginosa. Mass spectral analysis identified PL1-I and PL1-S as PI and PS, respectively. Radiolabeled P. aeruginosa organisms were subsequently shown to bind to commercially available bovine PI and PS and synthetic dipalmitoyl-PS but not to other phospholipid standards, including bovine SM and PC or synthetic dioleoyl- and distearoyl-PC. A control Escherichia coli strain did not bind to either PS or PI. Tetramethylurea, a disrupter of hydrophobic associations, did not influence the binding of P. aeruginosa to PS or PI. P. aeruginosa bound to the monolayers of corneal epithelial cells. P. aeruginosa binding to the monolayer cultures as well as to rabbit corneas pretreated with exogenous PS and PI was significantly higher than that to those preincubated with PC or medium alone. The data suggest that phospholipids PS and PI present in mucus or on the cell surface may function as P. aeruginosa receptors and contribute to selective bacterium-host interactions responsible for initial colonization.
Insights
Pseudomonas aeruginosa binds to specific phospholipids, phosphatidylinositol (PI) and phosphatidylserine (PS), on corneal epithelial cells. These phospholipids act as receptors, facilitating bacterial colonization and infection.
Area of Science:
- Microbiology
- Ocular Surface Research
- Bacterial Pathogenesis
Background:
- Pseudomonas aeruginosa is a significant opportunistic pathogen causing severe eye infections.
- Understanding the initial steps of bacterial adhesion to the corneal surface is crucial for developing effective treatments.
- Corneal epithelial cells possess a complex lipid composition that may mediate bacterial interactions.
Purpose of the Study:
- To identify specific corneal epithelial phospholipids that bind Pseudomonas aeruginosa.
- To elucidate the role of these phospholipids in the initial adhesion and colonization of the cornea by P. aeruginosa.
Main Methods:
- Lipids were extracted from corneal epithelia and analyzed using thin-layer chromatography (TLC).
- A TLC-bacterial overlay assay with radiolabeled P. aeruginosa was employed to detect binding.
- Phospholipid components were identified using mass spectrometry and confirmed with commercially available standards.
- Binding assays were performed on corneal epithelial cell monolayers and ex vivo rabbit corneas.
Main Results:
- Three major phospholipid components (PL1, PL2, PL3) were identified in corneal epithelia.
- P. aeruginosa specifically bound to PL1, which was later identified as phosphatidylinositol (PI) and phosphatidylserine (PS).
- Binding was concentration-dependent and abolished by alkaline methanolysis.
- Exogenous PI and PS significantly enhanced P. aeruginosa binding to corneal cells and tissues.
Conclusions:
- Phosphatidylinositol (PI) and phosphatidylserine (PS) on the corneal surface act as key receptors for Pseudomonas aeruginosa.
- These phospholipid-bacterial interactions are critical for the initial colonization and pathogenesis of P. aeruginosa eye infections.
- Targeting these specific phospholipid-receptor interactions could offer novel therapeutic strategies against P. aeruginosa keratitis.