Abstract
BackgroundFlavobacterium columnare (Bacteroidetes) is the causative agent of columnaris disease in farmed freshwater fish around the world. The bacterium forms three colony morphotypes (Rhizoid, Rough and Soft), but the differences of the morphotypes are poorly known. We studied the virulence of the morphotypes produced by F. columnare strain B067 in rainbow trout (Onconrhynchus mykiss) and used high-resolution scanning electron microscopy to identify the fine structures of the cells grown in liquid and on agar. We also analysed the proteins secreted extracellularly and in membrane vesicles to identify possible virulence factors.ResultsOnly the Rhizoid morphotype was virulent in rainbow trout. Under electron microscopy, the cells of Rhizoid and Soft morphotypes were observed to display an organised structure within the colony, whereas in the Rough type this internal organisation was absent. Planktonic cells of the Rhizoid and Rough morphotypes produced large membrane vesicles that were not seen on the cells of the Soft morphotype. The vesicles were purified and analysed. Two proteins with predicted functions were identified, OmpA and SprF. Furthermore, the Rhizoid morphotype secreted a notable amount of a small, unidentified 13 kDa protein absent in the Rough and Soft morphotypes, indicating an association with bacterial virulence.ConclusionsOur results suggest three factors that are associated with the virulence of F. columnare: the coordinated organisation of cells, a secreted protein and outer membrane vesicles. The internal organisation of the cells within a colony may be associated with bacterial gliding motility, which has been suggested to be connected with virulence in F. columnare. The function of the secreted 13 kDa protein by the cells of the virulent morphotype cells remains unknown. The membrane vesicles might be connected with the adhesion of cells to the surfaces and could also carry potential virulence factors. Indeed, OmpA is a virulence factor in several bacterial pathogens, often linked with adhesion and invasion, and SprF is a protein connected with gliding motility and the protein secretion of flavobacteria.
Highlights
Flavobacterium columnare (Bacteroidetes) is the causative agent of columnaris disease in farmed freshwater fish around the world
Bacteria in the biofilm are covered by an extracellular polymeric substance (EPS) layer that protects the cells from hostile environmental factors [7]
outer membrane vesicles (OMVs) are abundant in the extracellular material of many gram-negative bacteria, including Helicobacter pylori, Myxococcus xanthus and Pseudomonas aeruginosa [8,9,10], and they are often associated with virulence
Summary
Flavobacterium columnare (Bacteroidetes) is the causative agent of columnaris disease in farmed freshwater fish around the world. Bacteria in the biofilm are covered by an extracellular polymeric substance (EPS) layer that protects the cells from hostile environmental factors [7]. OMVs are abundant in the extracellular material of many gram-negative bacteria, including Helicobacter pylori, Myxococcus xanthus and Pseudomonas aeruginosa [8,9,10], and they are often associated with virulence. The planktonic OMVs of P. aeruginosa contained virulence factors such as LasA protease precursor, elastase LasB and alkaline protease whereas these were missing from the biofilm OMVs, indicating that planktonic cells may be important mediators of disease [13]. The role of OMVs has been studied extensively in many other pathogenic bacteria, and there is no doubt of their significant role in the virulence of bacterial pathogens [14]
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