The adhesion of Pseudomonas aeruginosa to high molecular weight human tear film species corresponds to glycoproteins reactive with Sambucus nigra lectin.
Pseudomonas aeruginosa is a pathogen gaining prevalence in contact lens-related corneal ulcers. Tear outflow protects the ocular surface, where high molecular weight tear glycoproteins bind bacteria for removal from the eye. The purpose of the present study was to identify glycoproteins in human tears involved in the adhesion of ocular P. aeruginosa isolates. Basal human tears were applied to a bacterial adhesion assay involving electrophoretic separation of tear components, transfer to nitrocellulose and incubation with biotin-labelled bacteria. Glycoproteins were further characterised using lectin profiling. The results showed large-dimension agarose gels were imperative for the detection of at least four glycoproteins with a migration >200 kDa, including species not previously identified. P. aeruginosa 6294 preferentially bound to a well-defined glycoprotein near the origin of the gel that, unlike other glycoproteins >200 kDa, reacted with Sambucus nigra lectin (sialic acid alpha2-6) but not WGA lectin (N-acetylglucosamine, sialic acid alpha2-3). Adhesion did not involve free biotin label or hydrophobic interactions. Also, the pre-incubation of separated tear glycoproteins with S. nigra lectin increased subsequent adhesion of 6294 to this tear glycoprotein. The less virulent Paer1 strain showed diffuse adhesion in the S. nigra-reactive region at the gel origin. In conclusion, an overlay adhesion assay was developed that identified slow-migrating sialylated glycoprotein species in human tears preferentially bound by P. aeruginosa ocular strains, and S. nigra lectin seemed to enhance the interaction. The study provides a basis for direct investigation of bacterial adhesion to glycoproteins with an apparent migration >200 kDa in tear fluid.
Aristoteli
Lina Panayiota
LP
Cooperative Research Centre for Eye Research and Technology, The University of New South Wales, Sydney NSW 2052, Australia. laristoteli@proteome.org.au
Willcox
Mark D P
MD
D000117:Acetylglucosamine / Q000378:metabolism; D001422:Bacterial Adhesion / Q000502:physiology; D019921:Biotinylation; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005136:Eye Proteins / Q000032:analysis / Q000378:metabolism*; D005260:Female; D006023:Glycoproteins / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D037102:Lectins / Q000032:analysis; D008297:Male; D008782:Methylurea Compounds / Q000494:pharmacology; D008970:Molecular Weight; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D037121:Plant Lectins / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000502:physiology*; D054788:Ribosome Inactivating Proteins; D013666:Tears / Q000737:chemistry* / Q000187:drug effects / Q000382:microbiology
D005136:Eye Proteins; D006023:Glycoproteins; D037102:Lectins; D008782:Methylurea Compounds; D037121:Plant Lectins; C050600:Sambucus nigra lectins; C004168:1,1,3,3-tetramethylurea; D054788:Ribosome Inactivating Proteins; D019158:N-Acetylneuraminic Acid; D000117:Acetylglucosamine