Flavobacterium columnare, a Gram-negative fish pathogen belonging to the phylum Bacteroidota, causes columnaris disease in numerous commercially important freshwater fish species in the U.S. and globally. Columnaris disease negatively impacts all U.S. finfish aquaculture industries, leading to substantial financial ramifications estimated at $50 million annually. Due to the ectopic nature of the disease, it is highly contagious and deadly, especially in aquaculture, where fish population density is high allowing for greater disease transmission. Since there are unknown virulence factors contributing to columnaris disease pathogenesis, there are currently no effective prevention measures. While it is known that the secretion of extracellular proteins, including proteases, aggravates F. columnare infection, identification of the secreted virulence factors remains incomplete. A new protein secretion system, known as the type IX secretion system (T9SS), was shown to be required for F. columnare virulence. This T9SS secretes many soluble and cell-surface associated proteins, all of which are potential virulence factors. Thirty-five of the T9SS-secreted proteins were identified as predicted proteases, but their role in columnaris disease pathogenesis is largely unexplored.
Proteases are commonly recognized as crucial virulence factors among various pathogens, and they add to the complexity of F. columnare’s pathogenic mechanisms. The genome of F. columnare harbors numerous protease-encoding genes, potentially involved in nutrient acquisition and degrading fish tissues to promote virulence. In prior studies, our investigation into the F. columnare T9SS-deficient mutant strains (ΔgldN and ΔporV) unveiled a notable defect in proteolytic activity. In this study, we have undertaken a comprehensive exploration of individual protease functions by constructing a library of combinatorial protease mutants. We constructed a series of gene deletion mutants, each lacking either individual or multiple (up to ten) protease-encoding genes, and assessed them for proteolytic activity and any associated growth defects. Additionally, we used zebrafish and rainbow trout as model species to evaluate the pathogenic potential of these protease-deletion mutants through immersion challenges. While the majority of mutants retained their virulence, mirroring the wild-type strain, certain single or multi-deletion mutants displayed proteolysis defective phenotype and decreased infectivity. Specifically, a mutant devoid of ten proteases and another lacking the tail-specific protease (TspA) exhibited reduced virulence in rainbow trout fry trials compared to the wild-type strain, emphasizing the role of proteases in F. columnare virulence. Overall, our findings underscore the impact of secreted proteases on the pathogenesis of columnaris disease and their role as pivotal elements of F. columnare’s arsenal of virulence factors. These proteases could potentially serve as valuable targets for future vaccine development efforts.