Antigenic and functional characterization of p57 produced by Renibacterium salmoninarum

Abstract

Renibacterium salmoninarum, the causative agent of bacterial kidney disease, produces large quantities of a 57-58 kDa protein (p57) during growth in broth culture and during infection of salmonid fish. Biological activities of secreted p57 include agglutination of salrnonid leucocytes and rabbit erythrocytes. We define the location of epitopes on p57 recognized by agglutination-blocking monoclonal antibodies (MAbs) 4Cl1, 4H8 and 4D3, and demonstrate that the majority of secreted p57 is a nlonomer that retains salrnonid leucocyte agglutinat~ng activity. The 3 MAbs bound a recombinant, amino-terminal fragment of p57 (211 aa) but not a carboxy-terminal fragment (315 aa) demonstrating that the neutralizing epitopes are located within the amino-terminal portion of p57. When combinations of the MAbs were used in an antigen capture ELISA. the epitopes recognized by the 3 MAbs were shown to be sterically separate. However, when the same MAb was used as both the coating and detection MAb, binding of the biotinylated detection MAb was not observed. These data indicate that the epitopes recognized by the 3 agglutination-blocking antibodies are functionally available only once per molecule and that native p57 exists as a monomer Similar ELISA results were obtained when kidney tissues from 3 naturally infected chinook salmon were assayed. Finally, a p57 monomer was purified using anion exchange and size exclusion chromatography that retained in vitro agglutinating activity. A model in which p57 is released from R. salmoninarum as a biologically active monomer during infection of salmonid fish is proposed.

Identification of a Third msa Gene in Renibacterium salmoninarum and the Associated Virulence Phenotype

“…This unexpected result may have been due to posttranscriptional regulation of total MSA levels, which would mask contributions from msa3. The MSA protein can be involved in host immune cell interactions, such as leukocyte adhesion (42,44), and a role for MSA in pathogenicity has been implicated (7,24,37). Close regulation of an important pathogenicity protein could be critical for appropriate function.…”

Mapping of Neutralizing Epitopes on Renibacterium salmoninarum p57 by Use of Transposon Mutagenesis and Synthetic Peptides

“…This region corresponds to the A1 repeat ( Fig. 3) of p57 (7,39). In summary, the binding profiles suggest that the 4H8 and 4D3 epitopes are distinct from one another, while the 4C11 epitope partially overlaps with both the 4H8 and 4D3 epitopes.…”

“…4) was unusual, as there was consistent binding to peptide 58 (amino acids 456 to 470) located outside the region (amino acids 78 to 210) mapped by transposon mutagenesis (Table 1). In addition, we previously failed to detect binding of MAb 4C11 to a recombinant, carboxy-terminal p57 protein (amino acids 242 to 557) which includes the peptide 58 sequence (39). Peptide 58 is not "sticky," as no other MAb bound to peptide 58 in the direct ELISA (Fig.…”

“…The peptide constructs include biotin-SGSG-peptide-amide, with SGSG as the linker designed by the manufacturer. The repeat regions in p57 consist of two A repeats (A1 and A2) and five B repeats (B1 to B5) (7,39). The invariant amino acids in the repeats are shown in boldface type.…”

“…Antibodies that bind reduced and SDS-denatured proteins often recognize linear epitopes. The murine MAbs 4H8, 4C11, and 4H3 recognize SDS-denatured and reduced p57 and also neutralize the agglutinating activity of the native protein (39,42). Thus, it was expected that the binding could be mapped to short linear sequences; however, transposon mutagenesis identified large regions that, when mutated, disrupt MAb binding.…”