Date of Award

August 2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Mark J McBride

Committee Members

Ching-Hong Yang, Sonia L Bardy, Christopher C Quinn, Charles F Wimpee

Keywords

Adhesins, Aquaculture, Genetic manipulation, Gliding Motility, Secretion systems, Virulence factors

Abstract

Flavobacterium columnare is an opportunistic fish pathogen that causes columnaris disease in freshwater fish worldwide. Columnaris disease is of particular concern in aquaculture settings where high population density leads to stress and greater infection frequency. High mortality rates and inadequate methods of control exacerbate the problem, creating an urgent need to better understand the mechanisms underlying F. columnare virulence. F. columnare has a type IX secretion system (T9SS), as do many other members of the Bacteroidetes phylum. The F. columnare T9SS secretes cytolysins, proteases, adhesins, and other proteins. The T9SS is also involved in a unique type of movement called gliding motility. Mutants that lack T9SS function are completely avirulent. However, it is unclear which aspect of the T9SS is most critical in the infection process. Genetic manipulation of F. columnare is challenging, making identification of secreted proteins critical for virulence difficult. We identified a virulent wild-type F. columnare strain (MS-FC-4) that is more amenable to genetic manipulation. Deletion of core T9SS gene gldN disrupted protein secretion and gliding motility and eliminated virulence in zebrafish and rainbow trout. Deletion of T9SS gene porV disrupted secretion and virulence but not motility. Both mutants exhibited severe defects in secreted enzymatic activities, adhesion, biofilm formation, and attachment to zebrafish pectoral fins. Deletion of a gene encoding a predicted secreted cytolysin resulted in reduced virulence in zebrafish and rainbow trout, and a mutant lacking two predicted secreted peptidases exhibited reduced virulence in rainbow trout. The T9SS of F. columnare MS-FC-4 secretes thirty-eight peptidases, and peptidases are common virulence factors of other pathogens. The avirulent T9SS mutants, ΔgldN and ΔporV, show severe defects in secreted proteolytic activity. Mutants lacking single or multiple peptidase-encoding genes were assessed for proteolytic activity, growth defects, and virulence. Mutants lacking nine or ten peptidase-encoding genes exhibited reduced mortality in rainbow trout, indicating that secreted peptidases contribute to F. columnare virulence. Motility adhesin SprB is propelled along the cell surface by a rotary motor in a helical fashion, resulting in cell movement. SprF is predicted to function in secretion and localization of SprB. Motility mutants lacking sprB or sprF displayed similarly defective motility on glass and agar, but they exhibited differences in virulence. ΔsprB mutants exhibited virulence defects in rainbow trout and zebrafish, whereas ΔsprF mutants only displayed reduced virulence in juvenile rainbow trout. Truncation mutants of gldJ were constructed to untangle T9SS secretion and motility. ΔgldJ mutant cells were nonmotile and defective for secretion and virulence. Truncation mutants of gldJ were competent for secretion, but remained severely defective in motility. Truncation mutant gldJ563 displayed reduced virulence in juvenile rainbow trout, suggesting that motility may contribute to virulence. A better understanding of F. columnare virulence mechanisms will allow for better treatment and control of columnaris disease in aquaculture settings.

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