First identification of Nocardia seriolae GapA adhesion function and its three B-cell epitopes with cell-binding...

Fish nocardiosis mainly caused by Nocardia seriolae (N. seriolae) is a serious threat to aquaculture. Bacterial adhesion to host cells mediated by adhesin is an initial step of pathogenesis. But it is not clear whether glyceraldehyde-3-phosphate dehydrogenase (GapA) is an adhesin of N. seriolae. Here, recombinant GapA protein (rGapA) was prokaryotic expressed, and its role in the bacterial adhesion to Ctenopharyngodon idella kidney cells was investigated by indirect immunofluorescence, protein-binding assay and adhesion inhibition assay. The results showed that an obvious green fluorescence was observed on the surface of the cells co-incubated with rGapA protein; the cytomembrane proteins of the cells pretreated with rGapA could react with anti-rGapA antibody; and the antibody significantly inhibited the adhesion ability of the bacteria. Subsequently, B-cell linear epitopes of GapA protein were identified by using a immunoinformatics approach combined with peptide ELISA and Western blot for the first time. It was found that four predicted epitopes (Ep_58-69 , Ep_139-150 , Ep_186-197 , Ep_318-329 ) could all react with anti-rGapA antibody and obviously inhibit the immunoreactivity between rGapA and anti-rGapA antibody, and they were confirmed as indeed B-cell linear epitopes of the protein. Furthermore, flow cytometry analysis found the percentage of positive cells co-incubated with FITC-labelled epitope peptides (Ep_139-150 , Ep_186-197 , Ep_318-329 ) was significantly higher than those in the FITC-labelled Ep_58-69 , unrelated control peptide and cell control. Collectively, GapA is an adhesin of N. seriolae, and epitope peptides (Ep_139-150 , Ep_186-197 , Ep_318-329 ) possess cell-binding activity, which are potential candidates for developing a multiple epitopes-based adhesin vaccine against fish nocardiosis.