The Type IX Secretion System Is Required for Virulence of the Fish Pathogen Flavobacterium columnare

Abstract

Flavobacterium columnare, a member of the phylum Bacteroidetes, causes columnaris disease in wild and aquaculture-reared freshwater fish. The mechanisms responsible for columnaris disease are not known. Many members of the phylum Bacteroidetes use type IX secretion systems (T9SSs) to secrete enzymes, adhesins, and proteins involved in gliding motility. The F. columnare genome has all of the genes needed to encode a T9SS. gldN, which encodes a core component of the T9SS, was deleted in wild-type strains of F. columnare. The F. columnare ΔgldN mutants were deficient in the secretion of several extracellular proteins and lacked gliding motility. The ΔgldN mutants exhibited reduced virulence in zebrafish, channel catfish, and rainbow trout, and complementation restored virulence. PorV is required for the secretion of a subset of proteins targeted to the T9SS. An F. columnare ΔporV mutant retained gliding motility but exhibited reduced virulence. Cell-free spent media from exponentially growing cultures of wild-type and complemented strains caused rapid mortality, but spent media from ΔgldN and ΔporV mutants did not, suggesting that soluble toxins are secreted by the T9SS.IMPORTANCE Columnaris disease, caused by F. columnare, is a major problem for freshwater aquaculture. Little is known regarding the virulence factors produced by F. columnare, and control measures are limited. Analysis of targeted gene deletion mutants revealed the importance of the type IX protein secretion system (T9SS) and of secreted toxins in F. columnare virulence. T9SSs are common in members of the phylum Bacteroidetes and likely contribute to the virulence of other animal and human pathogens.KEYWORDS columnaris disease, gliding motility, type IX secretion system, virulence F lavobacterium columnare is a common fish pathogen that causes columnaris disease (1-3). F. columnare causes epidemics in wild and cultured fish and is a major problem in freshwater aquaculture worldwide, resulting in significant mortality. Many species of freshwater fish are susceptible to columnaris disease (4, 5). The virulence mechanisms of F. columnare are incompletely understood, and current control strategies are inadequate. F. columnare isolates are genetically diverse and have been assigned to multiple genomovars (6, 7). F. columnare genomovars I and II exhibit significant differences, including host ranges (8-10). Described outbreaks in salmonid aquaculture systems have almost invariably been associated with genomovar I strains

The Carboxy-Terminal Region of Flavobacterium johnsoniae SprB Facilitates Its Secretion by the Type IX Secretion System and Propulsion by the Gliding Motility Machinery

“…The importance of T9SSs in the nutrition, physiology, motility, and virulence of members of the phylum Bacteroidetes has been demonstrated (1,3,19,36,(38)(39)(40). Most of the proteins with easily predicted functions that are secreted by T9SSs have type A CTDs.…”

Gnotobiotic rainbow trout (Oncorhynchus mykiss) model reveals endogenous bacteria that protect againstFlavobacterium columnareinfection

“…The molecular basis of F. columnare pathogenicity is poorly understood, but was recently shown to rely on the secretion of largely uncharacterized virulence factors and toxins by the Flavobacterium type IX secretion system (T9SS) [50]. The high genetic variability of F. columnare and its broad host range constitute an important limitation for the identification of effective probiotics against this widespread pathogen.…”

Influence of native catfish mucus on Flavobacterium columnare growth and proteolytic activity

“…Li et al. () constructed mutants in two genes important for the T9SS ( gldN and porV ). Both mutants were defective for protease secretion in vitro and were avirulent in fish, demonstrating a functional role of proteases in the virulence of F. columnare .…”

“…The azocasein protease assay (Newton, Wood, & Hartley, ; Sarath, de la Motte, & Wagner, ) as described by Li et al. () was utilized to examine extracellular proteolytic activity after growth in mucus or MSB.…”