The Flavobacterium psychrophilum OmpA, an outer membrane glycoprotein, induces a humoral response in rainbow trout

Abstract

Aims: The purpose of this study was to characterize OmpA, a major glycoprotein isolated from the membrane fraction of Flavobacterium psychrophilum, and to evaluate its potential as antigenic unit in a possible vaccine. Methods and Results: The expression product of ompA is a 465‐amino‐acid protein precursor that contains a 21‐amino acid signal peptide and has overall homology (up to 60% identity) with similarly sized proteins of some bacteria belonging to the Flavobacteriaceae family. The carboxy‐terminal region contains the ‘OmpA/MotB’ domain/signature and five putative ‘Thrombospondin type 3 repeats’ domains have been identified in the central region. OmpA was clearly detected in the outer membrane fraction and its surface exposure was demonstrated. OmpA is one of the immunodominant antigens and binding of specific anti‐OmpA antibodies lead to cell lysis in the presence of complement. Fish immunized with OmpA emulsified with Freund’s adjuvant developed a high antibody titter. Conclusions: Collectively, the data obtained here indicate that OmpA may be involved in Fl. psychrophilum/host cell interactions and appears to be a potential immunogen for a vaccine. Significance and Impact of the Study: This study is one step in the direction of understanding pathogenesis of Fl. psychrophilum and development of future vaccine.

Analysis of the Flavobacterium psychrophilum outer-membrane subproteome and identification of new antigenic targets for vaccine by immunomics

“…Proteins separated by SDS-PAGE or by 2-D IEF/SDS-PAGE were electroblotted onto a nitrocellulose filter (Bio-Rad) using a Bio-Rad TransBlot electrophoretic transfer cell as specified by the manufacturer. The blots were blocked with 5 % BSA in PBS for 1 h at room temperature and then incubated for 1 h with anti-F. psychrophilum OmpA rabbit serum (Dumetz et al, 2007), with a pool of antisera (dilution 1 : 2000 in 0.05 % Tween 20 in PBS) collected from 20 healthy trout (generously given by Scott E. LaPatra, Clear Springs Foods, Inc., Buhl, Idaho, USA) or with a pool of antisera (dilution 1 : 1000 in 0.05 % Tween 20 in PBS) collected from 20 CWD-convalescent trout taken from an infected French farm. The washing steps were performed three times with PBS-Tween 20.…”

“…Many of these properties are related to four short protein loops that emanate from the protein to the outside of the cell (for a review see Smith et al, 2007). Although the N-terminal amino acid sequence of F. psychrophilum OmpA shows no similarity with any other OMPs of Gramnegative bacteria outside of the family Flavobacteriaceae, we have recently shown that OmpA contains five possible thrombospondin type 3 repeat : OmpA/MotB domains, suggesting that it might be involved in the interaction/ adherence of the pathogen with the host cells, as for other members of the OmpA family (Dumetz et al, 2007). In addition, OmpA is one of the immunodominant antigens, and antibodies raised against it are capable of killing flavobacterial cells in the presence of complement.…”

“…The purity of the OM preparation ( Fig. 1a; lane 2) was shown by immunoblot analysis using antibodies directed against an OM-specific marker (OmpA; Dumetz et al, 2007) and by an enzymic assay using NADH dehydrogenase activity as an inner-membrane marker (Fig. 1b).…”

“…They are OmpH/P18 (FP2098; Massias et al, 2004;Dumetz et al, 2006), OmpA (FP0156; Merle et al, 2003;Dumetz et al, 2007) and flavobacterial-specific protein antigen (FspA) (FP2019; Crump et al, 2005). All of them are probably involved in bacteria-host interactions since they are included in a set of antigenic flavobacterial proteins recognized predominantly by antisera from convalescent fish that were naturally infected.…”

“…Several surface components of F. psychrophilum have been implicated in flavobacterial pathogenesis and identified as possible vaccine and diagnostic candidate macromolecules; they include lipopolysaccharide O antigen (MacLean et al, 2001) and surface-exposed antigens [e.g. 20 kDa antigen and OmpA (Merle et al, 2003;Dumetz et al, 2007)], some of which may be good candidates for an F. psychrophilum subunit vaccine, such as the surface-localized Flavobacterium-specific protein (FspA; Crump et al, 2005). Indeed, a protective immune Abbreviations: ACN, acetonitrile; et al, 2000).…”