Purification and characterization of the cell surface virulent A protein from Aeromonas salmonicida

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Abstract
The predominant cell surface protein (A protein) of A. salmonicida was purified in high yield from outer membranes by using a combination of detergent and chaotropic extraction methods and exclusion and ion-exchange chromatography. The A protein was primarily monomeric, MW 50,000, but readily formed oligomers at high protein or low salt concentrations. Several isoelectric forms were distinguishable with purified protein as well as in situ on the cell surface. Neither phosphate nor carbohydrate was detectable. The A protein was hydrophobic in composition and the N-terminal sequence highly hydrophobic. From circular dichroism spectra the A protein exhibited 14% .alpha. helix and 19-28% .beta. structure and could also be readily crystallized. By fluorescent antibody staining the A protein was shown to cover the entire cell surface but was absent from A protein deficient mutants. This protein appears to have no apparent enzymatic activity but rather constitutes a macromolecular refractile protein barrier essential for virulence.