Gene Deletion Strategy To Examine the Involvement of the Two Chondroitin Lyases in Flavobacterium columnare Virulence

Abstract

cFlavobacterium columnare is an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. The F. johnsoniae ompA promoter (PompA) was fused to sacB to construct a counterselectable marker for F. columnare. F. columnare carrying PompA-sacB failed to grow on media containing 10% sucrose. A suicide vector carrying PompA-sacB was constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes, cslA and cslB, were deleted. The ⌬cslA and ⌬cslB mutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (⌬cslA ⌬cslB) was completely deficient in chondroitin lyase activity. Cells of F. columnare wild-type strain G 4 and of the chondroitin lyase-deficient ⌬cslA ⌬cslB mutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors of F. columnare but may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylum Bacteroidetes. C olumnaris disease can affect almost all species of freshwater fish, including cultured and wild fish, often resulting in heavy economic losses in aquaculture worldwide (1, 2). Its causative agent, Flavobacterium columnare, has been the focus of microbiological as well as aquaculture research for decades, with respect to diagnosis, epidemiology, pathology, preventive measures, and possible virulence factors (2-4). The genetic diversity of F. columnare has been recognized by the presence of at least three distinct genomovars (5-7). However, the pathogenesis of this important pathogen is still rather unclear. Although a few molecules, such as chondroitin AC lyase, have been reported as possible virulence factors (8-11), the lack of genetic manipulation systems suitable for F. columnare has been an obstacle in understanding the mechanisms involved in the pathogenesis of this bacterium. Staroscik et al. (12) developed techniques to introduce replicative plasmids into F. columnare by conjugation and also to construct mutants by insertional mutagenesis and transposon-mediated mutagenesis. However, both processes remain inefficient, and few subsequent reports utilizing these techniques have been published (12,13).A technique to construct in-frame deletions was recently developed for the genetically tractable bacterium Flavobacterium j...

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

“…Soluble proteins secreted by the T9SS included potential virulence factors, such as the chondroitin sulfate lyases CslA (AX766_RS05135) and CslB (AX766_RS01510), the predicted peptidases AX766_RS05330 and AX766_RS13405, and the predicted thiol-activated cytolysins AX766_RS03975 and AX766_RS13970. F. co- B 0 8 5 8 0 S R _ 6 6 7 X A 5 7 5 8 0 S R _ 6 6 7 X A 0 5 5 8 0 S R _ 6 6 7 X A lumnare C#2 CslA and CslB exhibited 96.6% and 98.1% amino acid identity with F. columnare strain G4 CslA and CslB, respectively (35). The identification of chondroitin sulfate lyases and peptidases in the cell-free culture fluids of wild-type cells that were absent or greatly reduced in cell-free culture fluids from the gldN mutant is consistent with the enzyme activity results shown in Fig.…”

“…However, definitive proof for the involvement or lack of involvement of these enzymes in virulence was lacking. Techniques for gene transfer (36) and gene deletion (35) were recently developed for F. columnare and allow experiments to test the roles of individual genes and proteins in virulence.…”

“…Recently, both secreted chondroitin sulfate lyases of F. columnare strain G4 were examined for their roles in virulence. Cells lacking both chondroitin sulfate lyases retained virulence when fish were challenged by intraperitoneal injection but exhibited decreased competitiveness during coinfection by immersion (35). Chondroitin sulfate is found in connective tissues, and the chondroitin sulfate lyases may allow the bacteria to penetrate fish tissues during infection.…”

“…Techniques to genetically manipulate F. columnare were recently developed (35,36), allowing an exploration of the roles of individual genes in columnaris disease. We used these tools to generate T9SS mutants.…”

Intensive aquaculture selects for increased virulence and interference competition in bacteria

“…Therefore, other possible genetic mechanisms causing the changes in virulence (transduction, chromosomal transformation, mutations) [64] remain to be solved, and will require whole-genome sequencing of several strains. Also, the recent achievements in genetic manipulation techniques [65,66] and genome sequencing [67] are likely to provide detailed insight into the mechanisms behind F. columnare pathogenicity.…”