Abstract
The cell surface of Aeromonas salmonicida is covered by a regular surface array composed of a single species of protein, the A-protein (Phipps, B. M., Trust, T. J., Ishiguro, E. E., and Kay, W. W. (1983) Biochemistry 22, 2934-2939). The array, known as the A-layer, is the key virulence factor for this organism. Cells containing the A-layer specifically bound rabbit IgG and human IgM with high affinity (KD = 1.0 X 10(-6) M and 3.3 X 10(-6) M, respectively), but neither isogenic A-protein-deficient strains nor an Aeromonas hydrophila strain also possessing a regular surface array had binding activity. Selective removal of A-protein at pH 2.2 inactivated IgG binding. Structurally intact IgG was requisite for binding since both Fab and Fc fragments were inactive. Aeromonas A-protein did not share the same IgG binding sites as Staphylococcus aureus protein A. Purified A-protein bound IgG only weakly, but reassembled A-layer regained binding activity. Protein modification and perturbation of the A-layer indicated that no single amino acid residue was critical for binding, and that the binding site consisted of a native arrangement of at least four A-protein monomers in the layer.
Highlights
ImmunoglobulBininding by RegulSaurrfaAcreroafyA. salmonicida normal rabbit or human serum. Horseradish peroxidase-conjugated goat anti-rabbit IgG for Western blotting was supplied by Tag0 Inc
The cell surface of Aerornonw salrnonicida is cov- problem in commercial salmon culture
Whole Cell Immunoglobulin Binding". aureus SAC cells bind IgG in a cooperative manner [23], as shown in Fig. 1.A . salmonicida A450 cells bound IgG, but the binding was noncooperative (Fig. 1).The isogenic A-layer-negative strain A450-3 was virtually devoid of IgG binding activity (Fig. 1)
Summary
A. salmonicida normal rabbit or human serum. Horseradish peroxidase-conjugated goat anti-rabbit IgG for Western blotting was supplied by Tag0 Inc. The concentration of Ig was 1 X M.Binding wasallowed to occur for 40 min at room temperature with frequent vortexing and terminated by centrifugation for 30-40 s at 15,000X g.The cell pellet was washed once with PBS/TBA, andthe cell-bound label determined by y counting. The SAC cells bound 95% of the IgG, including that complexed with '2SI-A-protein,The SAC cells were sedimented by centrifugation for 30 s a t 15,000 x g, the pellet was washed once with PBS/TBA, and thecell-bound label was determined. Cells (100 mg wet weight) were suspended to 2.0 ml in the same buffer and the protein modification reagent added as a small volume of concentrate. Treated cells were centrifuged, washed twice, resuspended in PBS/TBA to 75 mg/ml (wet weight) (based on the assumption that no cell lysis had occurred), and "'I-IgG binding was assayed. The mixture was incubated a t room temperature for 30 min with shaking
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
