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Abstract
PURPOSE: To test the hypothesis that in the unscarified immature eye, Pseudomonas aeruginosa pili bind glycoprotein receptors, one or more of which are surface associated. METHODS: Several methods--including radioiodination of bacterial pili and surface-associated corneal epithelial proteins (CEPs), solid-phase binding assays, carbohydrate detection, and immunoblotting techniques in which periodate oxidation and preincubation of blots with purified pili, neuraminidase, sialic acid, other sugars, and SNA and MAA lectins--were used to identify and characterize host proteins. Some of these proteins in the immature mouse corneal epithelium interacted with bacterial pili. RESULTS: Seven proteins, with molecular weights from 14 to 66 kd were identified that strongly bound PAK/PR1 pili. To determine if any protein(s) was cell surface localized, corneal epithelial surface membrane proteins were radioiodinated and examined using a pilus overlay assay and lectin analysis. Only one protein of 57 kd was cell surface labeled and bound pili in an overlay assay. This protein was alpha (2-6) sialylated, as shown by SNA binding. Furthermore, SNA lectin was able to block pilus binding to CEPs. 125I labeling of pili and a solid-phase binding assay confirmed that pili bind to CEPs and, further, that binding could be competitively inhibited by excess unlabeled pili and that the receptors appeared saturable. GlycoTrack reagents were used to show that the epithelial proteins of the postnatal day 5 (P5) mouse cornea were glycosylated. Removal of carbohydrates by preincubation of blots with periodate, or combining pili with sialic acid, eliminated pili binding. Pretreatment of blots with either neuraminidase (N'ase) to decrease and/or remove sialic acid residues, or pretreatment with SNA lectin with specificity for alpha (2-6) linked sialic acid to galactose, also diminished pili binding to CEPs. Other sugars or MAA lectin, specific for sialic acid alpha (2-3) linked to galactose, had no inhibitory effect. CONCLUSIONS: These data show that a 57-kd surface membrane protein bound pili in the immature cornea and that for both this protein and the other nonsurface proteins, sialic acid alpha (2-6) linked to galactose was important in receptor recognition by the pilus adhesion. The 57-kd protein is putatively important in the initial interaction of pili with the unwounded ocular epithelium and may be the initial pathogenic event in this model.