Comparing the different morphotypes of a fish pathogen - implications for key virulence factors in Flavobacterium columnare

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.

Phenotypic characterization and genetic diversity of Flavobacterium columnare isolated from red tilapia, Oreochromis sp., in Thailand

“…Research has shown that the non‐rhizoid colonies are less virulent, and this may be due to differences in the organization of cells during growth, secreted proteins and production of outer membrane vesicles (Laanto et al . ).…”

Sickeningly Sweet: L‐rhamnose stimulates Flavobacterium columnare biofilm formation and virulence

“…F. columnare virulence has traditionally been evaluated through the extent of mortality produced by different isolates through the use of various bath immersion disease models (Laanto et al . ; Beck et al . ; Declercq et al .…”

Towards a model for Flavobacterium gliding

“…Flavobacteria are present in most aquatic and terrestrial environments. Filament-like structures were recently observed for Flavobacterium columnare [15]. Sequence alignment of F. johnsoniae and F. columnare SprB shows that while the two proteins have regions of similarity, they also have large regions that are not conserved.…”