Abstract
Streptococcus suis causes meningitis, sepsis, and other serious infections in newborn and young pigs and in adult humans. The Gal alpha 1-4Gal-binding adhesin of S. suis was purified to homogeneity by ultrasonic treatment, fractional ammonium sulfate precipitation, and preparative polyacrylamide gel electrophoresis. Pigeon ovomucoid, a glycoprotein with Gal alpha 1-4Gal terminals, was used to detect the adhesin by blotting. The purified adhesin appeared as single band of an apparent size of 18 kDa and of a pI of 6.4; no disulfide bridges were present. The amount of adhesin as revealed by pigeon ovomucoid binding correlated with the hemagglutination activity of different S. suis strains. The purified adhesin bound to latex particles induced hemagglutination which was specifically inhibited with the same inhibitors as hemagglutination by the intact bacteria, thus demonstrating that the purified protein was the Gal alpha 1-4Gal-recognizing adhesin of S. suis. Two adhesin variants (PN and PO) with differing Gal alpha 1-4Gal binding specificity had the similar electrophoretic mobilities and the same N-terminal peptide sequences, indicating that they were closely related. This represents the first isolation of an adhesin with well-defined cell surface carbohydrate binding activity from Gram-positive bacteria associated with meningitis.
Highlights
The first event in the establishment of infectious diseases is the adhesion of bacteria to the surface of host cells [1]
Pigeon ovomucoid was shown to be highly active as an inhibitor of the hemagglutination induced by Gal␣1– 4Gal-binding S. suis [10]
Pigeon ovomucoid was tested as a ligand for the detection of the adhesin activity in S. suis cells in a dot binding assay
Summary
Materials—Pigeon ovomucoid was purified as described previously [12]. Hen ovomucoid, N-acetylgalactosamine, and N-acetylglucosamine were obtained from Sigma. Purified adhesin or sonicates were subjected to electrophoresis in 6% polyacrylamide gels and transferred to PVDF-P membranes as described above. The particles were suspended into 90 l of phosphate buffer A, and 0.4 g of either purified adhesin or bovine serum albumin was added in a volume of 10 l and the suspensions were incubated for 2 h at room temperature on a tilting table. 10 l of adhesin-covered latex particles were mixed with 10 l of the inhibitory compounds on a slide and incubated on ice. After 15 min, 20 l of sialidase-treated 4% human erythrocytes were added, and the mixture was incubated for 15 min on ice. Similar agglutination reactions were obtained in volumes of 5 to 25 l of latex with 5 to 25 l of sialidase-treated erythrocytes, respectively. Bovine serum albumincoated latex particles (0 – 0.1 mg/1.25 ϫ 108 particles) used as controls gave no hemagglutination reactions
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