Type IX secretion system effectors and virulence of the model Flavobacterium columnare strain MS-FC-4

Authors: THUNES, NICOLE - University Of Wisconsin; CONRAD, RACHEL - University Of Wisconsin; MOHAMMED, HAITHAM - University Of Wisconsin; ZHU, YONGTAO - University Of Wisconsin; BARBIER, PAUL - University Of Wisconsin; Evenhuis, Jason; PEREZ-PASCUAL, DAVID - Institut Pasteur - France; GHIGO, JEAN-MARC - Institut Pasteur - France; Lipscomb, Ryan; SCHNEIDER, JOHN - University Of Wisconsin; LI, NAN - University Of Wisconsin; ERBES, DEVON - University Of Wisconsin; Birkett, Clayton; Lafrentz, Benjamin; Welch, Timothy - Tim; MCBRIDE, MARK - University Of Wisconsin

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/22/2021
Publication Date: 11/24/2021
Citation: Thunes, N.C., Conrad, R.A., Mohammed, H.H., Zhu, Y., Barbier, P., Evenhuis, J., Perez-Pascual, D., Ghigo, J., Lipscomb, R.S., Schneider, J., Li, N., Erbes, D.H., Birkett, C.L., LaFrentz, B.R., Welch, T.J., McBride, M.J. 2021. Type IX secretion system effectors and virulence of the model Flavobacterium columnare strain MS-FC-4. Applied and Environmental Microbiology. 88(3). Article e01705-21. https://doi.org/10.1128/aem.01705-21.
DOI: https://doi.org/10.1128/aem.01705-21

Interpretive Summary: Flavobacterium columnare causes columnaris disease in freshwater fish and is a major problem for sustainable aquaculture. Genetic manipulation of F. columnare is inefficient, which has impeded identification of virulence factors. An F. columnare strain (MS-FC-4) that is easy to genetically manipulate was identified. This allowed construction of gene deletion mutants. Mutants deficient for protein secretion failed to cause disease in rainbow trout. A mutant lacking two secreted peptidases and a mutant lacking a secreted cytolysin each exhibited decreased virulence. The results establish F. columnare strain MS-FC-4 as a model to identify virulence factors, which may aid development of control measures and impact sustainable aquaculture.

Technical Abstract: Flavobacterium columnare causes columnaris disease in wild and aquaculture-reared freshwater fish and is a major problem for sustainable aquaculture. Little is known regarding the virulence factors involved in this disease and control measures are inadequate. The F. columnare type IX secretion system (T9SS) secretes many proteins and is required for virulence, but the secreted virulence factors are not known. Genetic manipulation of F. columnare is inefficient, which has impeded identification of secreted proteins that are critical for virulence. Here we identified a virulent wild-type F. columnare strain (MS-FC-4) that is highly amenable to genetic manipulation. The components of the T9SS and many proteins secreted by this system were identified. The ease of genetic manipulation allowed us to construct many mutants that disrupt the T9SS or that eliminate individual secreted proteins. Mutants lacking core components of the T9SS failed to cause disease in zebrafish and in rainbow trout. These mutants were deficient in secretion of many enzymes, in biofilm formation, and in attachment of the bacterial cells to fish fins and to other surfaces. Genes encoding ten secreted proteins were also deleted. A mutant lacking two peptidase-encoding genes exhibited decreased virulence in rainbow trout, and a mutant lacking a cytolysin-encoding gene had decreased virulence in rainbow trout and zebrafish. Secreted proteins are likely virulence factors and are targets for the development of control measures. The results establish F. columnare strain MS-FC-4 as a model to identify virulence factors. This may aid development of measures to control columnaris disease and impact fish health and sustainable aquaculture.