Structural relatedness of enteric bacterial porins assessed with monoclonal antibodies to Salmonella typhimurium OmpD and OmpC

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Structural relatedness of enteric bacterial porins assessed with monoclonal antibodies to Salmonella typhimurium OmpD and OmpC.(1)

The outer membrane (OM) of Salmonella typhimurium and other gram-negative bacteria contains a family of pore-forming proteins called porins. Escherichia coli K-12 produces two porins, OmpC and OmpF, whereas S. typhimurium LT-2 synthesizes three porin species, OmpC (36,000), OmpF (35,000), and OmpD (34,000).

Hybridomas were raised by immunizing BALB/c mice with S. typhimunum OmpD and OmpC trimers or monomers and subsequently fusing their immune lymphocytes with P3x63-Ag8.653 myeloma cells. Ten MAbs recognized native porin epitopes that are exposed on the cell surface as the Group 1. Although MAbs 3, 10, 15, 42, 81, and 82 bound to whole cells of rough strains, none recognized smooth bacteria. A single antibody (MAb 47) reacted with homologous trimer, either purified or in OM, but not in whole cells as the Group 2. MAbs 6 and 84 constituted a third distinct category of antibody that reacted with purified trimer but not with OM or intact whole cells as the Group 3. Twenty-six MAbs in group IV reacted strongly with homologous monomer in both ELISA and Western immunoblots as the Group 4. One MAb (48) gave a weak positive reaction with purified OmpC trimer and monomer as the Group 5. In Western blots of denatured OM, this antibody produced ladderlike bands in three different regions, suggesting recognizing both porin residues and LPS sugars. One antibody (13) gave a strong positive reaction with LPS, partially purified porins (both homologous and heterologous), OM, and intact whole cells as Group 6.

All the anti-OmpC MAbs and 12 of the 17 anti-OmpD MAbs reacted with all the

isolates tested which are 11 Salmonella serotypes (in seven different serogroups) and clinical isolates of Salmonella typhi and Salmonella paratyphi. These antibodies were tested similarly with seven nonenterobacterial species, including A. hydrophila, B. pertussis, Brucella suis, H. influenzae, L. pneumophila, N.gonorrhoeae, and P. aeruginosa, no clear cross-reactivity

was observed. Finally, the same antibodies were tested with 13 species of Enterobacteriaceae. The reactions of anti-OmpC MAbs could be interpreted on the basis of phylogenetic relationship among these bacteria. Groups with fairly close relationship with Salmonella species, such as E.coli, Shigella boydii, and Klebsiella pneumoniae, reacted with more than one-half of the MAbs.

Humoral immunity to infection by the Enterobactenaceae consists in part of antibodies to OM proteins and that purified OM proteins elicit protective immunity in animals. The physical shielding of surface epitopes by the LPS implies that the immunoprotection observed by immunization of mice with anti-porin antibodies in previous studies may have resulted from contaminating anti-LPS antibodies. OM protein epitopes may be transiently exposed at the cell surface, however, as a consequence of natural turnover of cell wall materials during certain periods (e.g., biogenesis of OM) in the life cycle of bacterial cells.

1. S. P. Singh, Y. Upshaw, T. Abdullah, S. R. Singh, P. E. Klebba, Structural relatedness of enteric bacterial porins assessed with monoclonal antibodies to Salmonella typhimurium OmpD and OmpC. J. Bacteriol. 174, 1965–1973 (1992).

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