ARS | Publication request: Comparative proteomic analysis of virulent and rifampicin attenuated Flavobacterium psychrophilum

Submitted to: Journal of Fish Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 11, 2011
Publication Date: June 12, 2012
Repository URL: http://handle.nal.usda.gov/10113/55470
Citation: Gliniewicz, K., Plant, K.P., Lapatra, S.E., Lafrentz, B.R., Cain, K., Snekvik, K.R., Call, D.R. 2012. Comparative proteomic analysis of virulent and rifampicin attenuated Flavobacterium psychrophilum. Journal of Fish Diseases. 35:529-539.

Interpretive Summary: Flavobacterium psychrophilum is an economically important bacterial pathogen of salmonid aquaculture worldwide. Previously, an attenuated strain of F. psychrophilum was developed by exposing the bacterium to the antibiotic rifampicin, and this strain showed promise as a live vaccine strain. The goal of this study was to identify differences between the vaccine strain and the parent strain in order to gain insight into the mechanism by the vaccine strain is unable to cause disease. Comparative proteomics identified fourteen differentially synthesized proteins between the two strains. The gene encoding one of these was cloned, expressed as a recombinant protein, and tested as a subunit vaccine. The protein failed to provide protection from experimental bacterial challenges. Analysis of the RNA polymerase gene sequences revealed a mutation in the vaccine strain. Bacterial RNA polymerases are involved in one step of protein synthesis, and this mutation may explain the altered protein synthesis that was observed in the vaccine strain. While the exact mechanism responsible for the attenuation of the vaccine strain is not completely understood, the proteins identified are a valuable resource to develop subunit vaccines and to determine their roles in the virulence of this important fish pathogen.

Technical Abstract: Flavobacterium psychrophilum is the etiologic agent of bacterial coldwater disease and rainbow trout fry syndrome. In this study we compared a wild-type strain (CSF 259.93) with a rifampicin resistant and virulence attenuated strain of F. psychrophilum (CSF 259.93B.17). The attenuated strain harboured a mutation in the rpoB gene consistent with resistance to rifampicin. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass-spectrometry demonstrated an altered proteome with eight proteins characteristic for the parent strain and six that were unique to the attenuated strain. Immuno-blotting with a diagnostic monoclonal antibody (FL-43) identified a putative antigen (FP1493) that was subsequently cloned, expressed as a recombinant protein, and confirmed as recognized by FL-43. 2D-PAGE, immuno-blotting with rainbow trout, Oncorhynchus mykiss (Walbaum), convalescent antisera and mass spectrometry of bacterial whole-cell lysates revealed several uniquely expressed immunoreactive proteins including FP1493. An FP1493 recombinant subunit vaccine was tested, but did not provide protection against challenge with the CSF259.93 strain. While the exact mechanism responsible for altered protein synthesis and attenuation of CSF 259.93.B17 is still unknown, the differentially expressed immuno-reactive proteins are a valuable resource to develop subunit vaccines and to identify proteins that are potentially involved in disease.