Date of Award
Doctor of Philosophy
Sonia Bardy, Steven Forst, Osvaldo Jhonatan Sepulveda Villet, Charles Wimpee
Flavobacterium columnare, Virulence
The Gram-negative bacterium Flavobacterium columnare is the causative agent of columnaris disease and is responsible for significant economic losses in aquaculture. Little is known regarding the virulence factors produced by F. columnare, and control measures are limited. Like many members of the phylum Bacteroidetes, F. columnare uses the type IX secretion system (T9SS) to secrete enzymes, adhesins, and proteins involved in gliding motility. When a core component of this system was deleted in the wild type strain, the resulting mutant was avirulent in zebrafish, rainbow trout, and channel catfish infection studies. This suggests that the individual secreted proteins may function as virulence factors. Cell-free spent media was analyzed, and some of the most abundant secreted proteins were the chondroitinases, CslA and CslB that digest chondroitin, a component of fish connective tissues. The genes encoding CslA and CslB were deleted and the mutants were examined for virulence. Mutants lacking both genes failed to digest chondroitin but remained virulent in fish infection studies, indicating that chondroitinases are not essential for virulence under our conditions. Iron acquisition from the host during infection is important for pathogenicity and virulence of many bacterial pathogens. F. columnare genes predicted to function in iron uptake were identified by genome analyses. The genes encoding a heme-binding protein HmuY, siderophore synthesis and export proteins, the outer membrane iron receptors FhuA, FhuE, FecA, and IutA and components of an ATP binding cassette (ABC) transporter predicted to transport iron across the cytoplasmic membrane were deleted. The mutants were examined for defects in siderophore production, for growth defects in iron-limited conditions, and for virulence against zebrafish. Mutants lacking all siderophore activity were obtained. These mutants, and mutants with deletions in genes encoding outer membrane receptors, and components of the ABC transporter exhibited growth defects under conditions of limiting iron. Mutants deficient in single steps of the iron uptake pathways, such as heme binding by HmuY, siderophore synthesis, outer membrane transport, or inner membrane transport retained their virulence, whereas mutants lacking multiple steps exhibited decreased virulence. Zebrafish that survived challenge with the multiple step iron mutants exhibited partial immune protection against later infection by the wild type. The results suggest that iron acquisition systems are involved in virulence and that there may be some redundancies between components of these systems. Our work provides a better understanding of F. columnare pathogenesis mechanisms and may lead to development of an effective vaccine or other control measures to prevent or control columnaris disease.
Conrad, Rachel, "Roles of Secreted Proteins and Iron Utilization Proteins in Virulence of the Fish Pathogen Flavobacterium Columnare" (2021). Theses and Dissertations. 2768.