IN THE IMMATURE MOUSE, PSEUDOMONAS-AERUGINOSA PILI BIND A 57-KD (ALPHA-2-6) SIALYLATED CORNEAL EPITHELIAL-CELL SURFACE PROTEIN - A FIRST STEP IN INFECTION

Purpose. To test the hypothesis that in the unscarified immature eye, Pseudomonas aeruginosa pill bind glycoprotein receptors, one or more o f which are surface associated. Methods. Several methods-including rad ioiodination of bacterial pill and surface-associated corneal epitheli al proteins (CEPs), solid-phase binding assays, carbohydrate detection , and immunoblotting techniques in which periodate oxidation and prein cubation of blots with purified pill, neuraminidase, sialic acid, othe r sugars, and SNA and MAA lectins-were used to identify and characteri ze host proteins. Some of these proteins in the immature mouse corneal epithelium interacted with bacterial pill. Results. Seven proteins, w ith molecular weights from 14 to 66 kd were identified that strongly b ound PAK/PR1 pill. To determine if any protein(s) was cell surface loc alized, corneal epithelial surface membrane proteins were radioiodinat ed and examined using a pilus overlay assay and lectin analysis. Only one protein of 57 kd was cell surface labeled and bound pill in an ove rlay assay. This protein was alpha(2-6) sialylated, as shown by SNA bi nding. Furthermore, SNA lectin was able to block pilus binding to CEPs . I-125 labeling of pill and a solid-phase binding assay confirmed tha t pill bind to CEPs and, further, that binding could be competitively inhibited by excess unlabeled pill and that the receptors appeared sat urable. GlycoTrack reagents were used to show that the epithelial prot eins of the postnatal day 5 (P5) mouse cornea were glycosylated. Remov al of carbohydrates by preincubation of blots with periodate, or combi ning pill with sialic acid, eliminated pill binding. Pretreatment of b lots with either neuraminidase (N'ase) to decrease and/or remove siali c acid residues, or pretreatment with SNA lectin with specificity for alpha(2-6) linked sialic acid to galactose, also diminished pill bindi ng to CEPs. Other sugars or MAA lectin, specific for sialic acid alpha (2-3) linked to galactose, had no inhibitory effect. Conclusions. Thes e data show that a 57-kd surface membrane protein bound pill in the im mature 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 adhesin. The 57-kd protein is putati vely important in the initial interaction of pill with the unwounded o cular epithelium and may be the initial pathogenic event in this model .