ECA-immunogenicity of Proteus mirabilis strains

Bacteria of the genus Proteus are opportunistic pathogens and cause mainly urinary tract infections. They also play a role in the pathogenesis of reactive arthritis (RA). Patients suffering from Yersinia-triggered RA often carry high titers of antibodies specific to enterobacterial common antigen (ECA). The immunogenicity of ECA has not received much attention thus far and studies have focused mainly on the ECA of Escherichia coli and Yersinia enterocolitica. In this paper the ECA-immunogenicity of Proteus mirabilis is elucidated using two wild-type strains (S1959 and O28) as well as their rough (R) derivative strains R110/1959, which expresses lipopolysaccharide (LPS) with a full core, and R4/O28, which expresses LPS with only an inner core. Rabbit polyclonal antisera were produced by immunization with boiled suspensions of the four P. mirabilis strains. The antisera were tested for the presence of antibodies specific to ECA by Western blotting using glycerophospholipid- linked ECA (ECA(PG) ) of Salmonella montevideo as antigen. Lipopolysaccharide (LPS) was isolated from the four strains by the hot phenol/water procedure in which ECA (PG) is co-extracted with LPS and by the phenol/chloroform/petroleum ether extraction that results in the isolation of LPS and/or LPS-linked ECA (ECA (LPS) ) free of ECA (PG) . The LPS preparations were tested for the presence of ECA by Western blotting using ECA-specific antibodies. The results demonstrated that all four P. mirabilis strains were ECA immunogenic. The rabbit antisera immunized by the four strains all contained ECA-specific antibodies. Analysis of the LPS preparations demonstrated that the P. mirabilis wild-type strains O28 and S1959 and the Ra mutant strain R110/1959 expressed ECA (LPS) , suggesting that it induced the anti-ECA antibody responses. Only the presence of ECA(PG) could be demonstrated in the Rc mutant strain R4/O28. These results therefore suggest that, similar to E. coli, LPS with a full core is also required as the acceptor of ECA for P. mirabilis strains to produce ECA (LPS) . Since ECA (>PG) is not immunogenic unless combined with some proteins, it is likely that ECA (PG) -protein complexes formed during the intravenous immunization with the Rc mutant strain R4/O28.