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Secreted peptidases contribute to virulence of fish pathogen Flavobacterium columnare.

ABSTRACT: Flavobacterium columnare causes columnaris disease in freshwater fish in both natural and aquaculture settings. This disease is often lethal, especially when fish population density is high, and control options such as vaccines are limited. The type IX secretion system (T9SS) is required for F. columnare virulence, but secreted virulence factors have not been fully identified. Many T9SS-secreted proteins are predicted peptidases, and peptidases are common virulence factors of other pathogens. T9SS-deficient mutants, such as ΔgldN and ΔporV, exhibit strong defects in secreted proteolytic activity. The F. columnare genome has many peptidase-encoding genes that may be involved in nutrient acquisition and/or virulence. Mutants lacking individual peptidase-encoding genes, or lacking up to ten peptidase-encoding genes, were constructed and examined for extracellular proteolytic activity, for growth defects, and for virulence in zebrafish and rainbow trout. Most of the mutants retained virulence, but a mutant lacking 10 peptidases, and a mutant lacking the single peptidase TspA exhibited decreased virulence in rainbow trout fry, suggesting that peptidases contribute to F. columnare virulence.

SUBMITTER: Thunes NC

PROVIDER: S-EPMC9936825 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Secreted peptidases contribute to virulence of fish pathogen Flavobacterium columnare.

Thunes Nicole C NC Mohammed Haitham H HH Evenhuis Jason P JP Lipscomb Ryan S RS Pérez-Pascual David D Stevick Rebecca J RJ Birkett Clayton C Conrad Rachel A RA Ghigo Jean-Marc JM McBride Mark J MJ

Frontiers in cellular and infection microbiology 20230203

Flavobacterium columnare causes columnaris disease in freshwater fish in both natural and aquaculture settings. This disease is often lethal, especially when fish population density is high, and control options such as vaccines are limited. The type IX secretion system (T9SS) is required for F. columnare virulence, but secreted virulence factors have not been fully identified. Many T9SS-secreted proteins are predicted peptidases, and peptidases are common virulence factors of other ...[more]

PMID: 36816589

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Project description: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.

Project description:Flavobacterium columnare causes columnaris disease in wild and aquaculture-reared freshwater fish. F. columnare virulence mechanisms are not well understood, and current methods to control columnaris disease are inadequate. Iron acquisition from the host is important for the pathogenicity and virulence of many bacterial pathogens. F. columnare iron acquisition has not been studied in detail. We identified genes predicted to function in siderophore production for ferric iron uptake. Genes predicted to encode the proteins needed for siderophore synthesis, export, uptake, and regulation were deleted from F. columnare strain MS-FC-4. The mutants were examined for defects in siderophore production, for growth defects in iron-limited conditions, and for virulence against zebrafish and rainbow trout. Mutants lacking all siderophore activity were obtained. These mutants displayed growth defects when cultured under iron-limited conditions, but they retained virulence against zebrafish and rainbow trout similar to that exhibited by the wild type, indicating that the F. columnare MS-FC-4 siderophores are not required for virulence under the conditions tested. IMPORTANCE Columnaris disease, which is caused by Flavobacterium columnare, is a major problem for freshwater aquaculture. Little is known regarding F. columnare virulence factors, and control measures are limited. Iron acquisition mechanisms such as siderophores are important for virulence of other pathogens. We identified F. columnare siderophore biosynthesis, export, and uptake genes. Deletion of these genes eliminated siderophore production and resulted in growth defects under iron-limited conditions but did not alter virulence in rainbow trout or zebrafish. The results indicate that the F. columnare strain MS-FC-4 siderophores are not critical virulence factors under the conditions tested but may be important for survival under iron-limited conditions in natural aquatic environments or aquaculture systems.

Project description:Flavobacterium columnare causes columnaris disease in fish. Columnaris disease is incompletely understood, and adequate control measures are lacking. The type IX secretion system (T9SS) is required for F. columnare gliding motility and virulence. The T9SS and gliding motility machineries share some, but not all, components. GldN (required for gliding and for secretion) and PorV (involved in secretion but not required for gliding) are both needed for virulence, implicating T9SS-mediated secretion in virulence. The role of motility in virulence is uncertain. We constructed and analyzed sprB, sprF, and gldJ mutants that were defective for motility but that maintained T9SS function to understand the role of motility in virulence. Wild-type cells moved rapidly and formed spreading colonies. In contrast, sprB and sprF deletion mutants were partially defective in gliding and formed nonspreading colonies. Both mutants exhibited reduced virulence in rainbow trout fry. A gldJ deletion mutant was nonmotile, secretion deficient, and avirulent in rainbow trout fry. To separate the roles of GldJ in secretion and in motility, we generated gldJ truncation mutants that produce nearly full-length GldJ. Mutant gldJ563, which produces GldJ truncated at amino acid 563, was defective for gliding but was competent for secretion as measured by extracellular proteolytic activity. This mutant displayed reduced virulence in rainbow trout fry, suggesting that motility contributes to virulence. Fish that survived exposure to the sprB deletion mutant or the gldJ563 mutant exhibited partial resistance to later challenge with wild-type cells. The results aid our understanding of columnaris disease and may suggest control strategies.IMPORTANCEFlavobacterium columnare causes columnaris disease in many species of freshwater fish in the wild and in aquaculture systems. Fish mortalities resulting from columnaris disease are a major problem for aquaculture. F. columnare virulence is incompletely understood, and control measures are inadequate. Gliding motility and protein secretion have been suggested to contribute to columnaris disease, but evidence directly linking motility to disease was lacking. We isolated and analyzed mutants that were competent for secretion but defective for motility. Some of these mutants exhibited decreased virulence. Fish that had been exposed to these mutants were partially protected from later exposure to the wild type. The results contribute to our understanding of columnaris disease and may aid development of control strategies.

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Secreted peptidases contribute to virulence of fish pathogen Flavobacterium columnare.

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Secreted peptidases contribute to virulence of fish pathogen <i>Flavobacterium columnare</i>.

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