The bacterium Flavobacterium columnare is a world-wide pathogen causing columnaris disease and a pathogen in China causing bacterial gill-rot disease, a serious disease in several major cultured fish species including the grass carp Ctenopharyngodon idellus. The present study aimed to characterize the proteomic profiles of outer membrane proteins (OMPs) of F. columnare and gill mucus of grass carp. In addition, a comparative proteomic analysis was carried out to analyze extracellular proteins (ECPs) of virulent and avirulent strains of the bacterium.
Strains G4R3 and G18 of F. columnare were assessed to be virulent and avirulent, respectively, by immersion challenge. To identify the virulence factors of F. columnare, comparative proteomic analysis by two-dimensional polyacrylamide gel electrophoresis (2-DE) in combination with mass spectrometry and database search was conducted to analyze extracellular proteins of strains G4R3 and G18 at the late exponential growth phase. 34 differential expression spots were revealed by image analysis of 2-DE gels and further analyzed using in-gel tryptic digestion, peptide mass fingerprinting and tandem mass spectrometry. Seven spots representing three proteins, namely GldK, s-adenosylmethionine synthetase and putative membrane protein were identified as possible virulence-related factors in the virulent strain G4R3.
Outer membrane proteins of F. columnare were subjected to fractionation by SDS-PAGE, and then shotgun proteomic analysis. A total of 757 proteins were recognized, among which 110 proteins were identified with high-confidence. 30 proteins, which were previously characterized as products of genes encoding OMPs of F. columnare by screening the expression library of the bacterium, were also present in the 757 proteins.
The traditional two-dimensional ployacrylamide gel electrophoresis was employed, in combination with gel filtration chromatography as a third dimension, to separate gill mucus of healthy grass carp. It was revealed that 406 spots were present with isoelectric points between about 4 and 7, among which 99 spots were identified using mass spectrometric analysis, such as lectin-like proteins, transferrin, proteasome, histone-like proteins. These identified proteins may be involved in immune response of the fish.