Hemolytic activity in Flavobacterium psychrophilum is a contact-dependent, two-step mechanism and differently expressed in smooth and rough phenotypes

The hemolytic activity of cells of smooth and rough phenotypic variants of the Gram-negative fish pathogen Flavobacterium psychrophilum was investigated in two different assays, a microplate and an agarose hemolysis assay, using rainbow trout erythrocytes. The smooth cells showed a high and the rough cells a negligible, concentration dependent, hemolytic activity in the microplate assay. Both smooth and rough cells showed a rather weak hemolytic activity, with two distinct hemolytic patterns, in the agarose assay. The hemolytic activity of the cells was not regulated by iron availability and cell-free extracellular products did not show any hemolytic activity. The smooth cells, in contrast to the rough cells, showed a high ability to agglutinate erythrocytes and both hemagglutination and hemolytic activity was impaired by treatment of the cells with sialic acid. The hemolytic activity was furthermore reduced after proteolytic and heat treatment of the cells. The results from the present study suggest that the hemolytic activity in F. psychrophilum is highly expressed in the smooth phenotype, and that it is a contact-dependent and two-step mechanism that is initiated by the binding of the bacterial cells to the erythrocytes through sialic acid-binding lectins and then executed by thermolabile proteinaceous hemolysins.