ANTIGENIC DETERMINANTS OF THE OMPC PORIN FROM SALMONELLA-TYPHIMURIUM

The antigenic determinants of Salmonella typhimurium OmpC were investi gated by the analysis of cyanogen bromide (CNBr)-generated porin pepti des with antiporin monoclonal antibodies (MAbs). We identified six ban ds (f1 to f6) with estimated molecular masses of 35.5, 31.0, 25.0, 22. 5, 13.8, and 10.0 kDa, respectively, In addition, two small fragments (f7 and f8; 3.0 to 6.0 kDa) were detected only infrequently. The OmpC monomer or its CNBr-generated peptides were electrophoretically transf erred to a polyvinylidene difluoride membrane and then subjected to am ino acid composition analysis and N-terminal sequencing, A comparison of the amino acid composition data with known compositions of Escheric hia coli and Salmonella typhi OmpC showed some differences; however, t he amino acid sequences of 71 residues identified in S. typhimurium sh owed 88 and 98% identity,vith OmpC from E. coli and S. typhi, respecti vely. The screening of CNBr peptides with the 12 anti-(S. typhimurium) OmpC MAbs by Western blot (immunoblot), in conjunction with the predi ction of the OmpC folding pattern based on the known three-dimensional structure of E. coli OmpF, showed that four MAbs reacted with surface -exposed epitopes on loops L2, L8, and L4 to L7, four MAbs reacted wit h a region in the eyelet structure on loop L3, and four MAbs reacted w ith the buried epitopes on transmembrane beta strands, The MAbs reacti ng with surface-exposed loops showed no cross-reaction with E. coil Om pC, rr-hose sequence has diverged extensively from that of S. typhi an d (probably) S. typhimurium OmpC only in regions of the externally exp osed loops. In contrast, MAbs reacting with transmembrane beta strands , whose sequence is strongly conserved, showed strong cross-reaction w ith E. coli OmpC. These results show that comparison with the E. coil OmpF structure predicts the folding pattern of S. typhimurium OmpC rat her accurately and that evolutionary divergence in sequences is confin ed to the external loops, The possible roles of these surface-exposed and buried epitopes as potentially useful antigenic regions for diagno stic assays and vaccine development are discussed.