Distribution of surface-exposed and non-accessible amino acid sequences among the two major structural domains of the S-layer protein of Aeromonas salmonicida.

ABSTRACT

The tetragonally arranged crystalline surface protein array (A-layer) of the fish pathogenic bacterium Aeromonas salmonicida is a virulence factor. Circular dichroism studies in the presence or absence of 0.1% sodium dodecyl sulfate showed that the secondary structure of A-protein, and its 39,439 molecular weight amino-terminal trypsin-resistant peptide, were altered. In both cases alpha-helix was increased significantly at the expense of beta-structure when SDS was added. Western and dot immunoblotting, immuno-microscopy and enzyme-linked immunosorbent assay with monospecific polyclonal antiserum and eight monoclonal antibodies specific for epitopes exposed on the surface of native A-layer showed that the 481 residue A-protein subunit and the surface of A-layer were conserved antigenically. Mimeotope analysis of nonapeptides representing the sequence of A-protein allowed identification of 146 residues in presumed linear epitopes accessible on the surface of A-layer. Inaccessible or non-epitopic residues accounted for 70% of the protein. The majority of inaccessible residues were in the N-terminal 301 residues of A-protein. Dispersed among these were 65 surface-accessible residues in five linear epitope clusters illustrating the complex folding of this major structural domain of A-protein. The C-terminal 180 residues carried fewer linear epitopes but contained the major region of A-layers surface-accessible sequence, including four linear epitopes in predominantly hydrophobic sequence. Four A-layer surface-binding monoclonal antibodies also bound to this minor structural domain, although the epitopes of only two were identified by mimeotope analysis. The epitopes of six A-layer surface-binding monoclonals could not be identified, suggesting that A-layer may also contain conformation dependent surface epitopes.