Siderophores Produced by the Fish Pathogen Flavobacterium columnare Strain MS-FC-4 Are Not Essential for Its Virulence

Abstract

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.

Keywords: columnaris disease; fish pathogen; siderophore.

FIG 1

Map of region containing predicted F. columnare MS-FC-4 siderophore biosynthesis, export, uptake, and regulation genes. Black arrows indicate siderophore-related genes and the directions in which they are transcribed. Green arrows indicate genes flanking the siderophore gene regions. Numbers above the genes refer to the number of kilobase pairs (kbp) of sequence. Regions deleted in mutants are indicated by lines above each map. (A) The sidL locus spans 11 genes predicted to be involved in siderophore synthesis, export, uptake, or regulation. The gaps between genes containing predicted promoter sequences suggest organization as three operons. The sequence within each bracket is the region between the two coding regions, with Bacteroidota promoters shown in bold and highlighted yellow. (B) iucA/iucC-like gene, iucX, and nearby genes. The five genes in green shown upstream of C6N29_04165 appear unrelated to siderophores and instead form an apparent ParB-related, ThiF-related cassette (PRTRC) system, encoding proteins with TIGR03736, TIGR03737, TIGR03738, and TIGR03741 domains.

FIG 2

Siderophore activity for F. columnare wild type (WT), siderophore synthesis gene deletion mutants, and complemented mutants. Midexponential cultures were spotted onto chrome azurol S (CAS) plates and incubated at 28°C for 72 h. Siderophore presence is indicated by an orange halo around F. columnare growth. pRC48 is a complementation plasmid carrying iucA, and pRC56 is a complementation plasmid carrying iucX. This figure also includes chromosomal complementation strains in which genes labeled with “CC” were inserted at their original position in the chromosome. For the ΔiucX ΔiucA ΔiucC mutant, iucA was inserted back into the chromosome, and for the ΔsidL ΔiucX mutant, iucX was inserted back into the chromosome.

FIG 3

Growth of F. columnare strains in iron-rich tryptone yeast extract salt (TYES) and iron-limited (TYES medium without yeast extract [TS]) media. (A) Wild type (WT) and single iuc gene deletion mutants grown in TYES. (B) Wild type and single iuc gene deletion mutants grown in TS. (C) Multiple siderophore gene deletion mutants grown in TYES. A significant difference in peak cell density was seen between the WT and ΔsidL ΔiucX mutant (P < 0.05). (D) Multiple deletion mutants grown in TS. Significant differences in peak cell density were seen between the WT and ΔsidL mutants, between the WT and ΔiucX ΔiucA ΔiucC mutants, and between the WT and ΔsidL ΔiucX mutants (P < 0.05). Strains were grown at 28°C with shaking (200 rpm), and measurements were taken every 2 h for 36 h. The error bars represent standard error of the mean.

FIG 4

Growth of F. columnare wild-type, siderophore mutants, and complemented mutant strains in iron-limited (TS) medium. (A) Wild type (WT), ΔiucX ΔiucA ΔiucC mutant, ΔiucX ΔiucA ΔiucC mutant complemented with a plasmid pRC48, which carries iucA, and ΔiucX ΔiucA ΔiucC mutant chromosomally complemented by inserting iucA back into the chromosome. A significant difference in peak cell density was seen between the WT and the ΔiucX ΔiucA ΔiucC mutant (P < 0.05). (B) WT, ΔsidL ΔiucX mutant, ΔsidL ΔiucX mutant complemented with plasmid pRC56, which carries iucX, and ΔsidL ΔiucX chromosomally complemented by inserting iucX back into the chromosome. A significant difference in peak cell density was seen between the WT and ΔsidL ΔiucX mutant (P < 0.05). Strains were grown at 28°C with shaking (200 rpm), and measurements were taken every 2 h for 36 h. The error bars represent standard error of the mean.

FIG 5

Challenges of zebrafish with F. columnare wild-type and siderophore mutant strains at two doses. A total of 15 fish were challenged with each strain by immersion, and the percent survival was recorded. The avirulent ΔgldN mutant was used instead of a media control. (A) Adult zebrafish were exposed to wild-type MS-FC-4 (1.6 × 10⁶ CFU/mL), siderophore-deficient (2.3 × 10⁶ CFU/mL), and ΔgldN (6.1 × 10⁶ CFU/mL) strains. (B) Adult zebrafish were exposed to WT (8.0 × 10⁵ CFU/mL), siderophore-deficient (1.2 × 10⁶ CFU/mL), and ΔgldN (3.1 × 10⁶ CFU/mL) strains. The CFU values shown are the final challenge concentrations. The percent survival for fish challenged with the wild-type and ΔsidL ΔiucX mutant strains were not significantly different in either panel.

FIG 6

Challenge of rainbow trout with F. columnare wild-type and siderophore mutant strains. Rainbow trout fry were challenged with each strain by immersion, and the percent survival was recorded. The avirulent ΔgldN mutant was used instead of a media control. The strains examined were wild-type MS-FC-4 (3.4 × 10⁷ CFU/mL), siderophore-deficient mutant (2.2 × 10⁷ CFU/mL), and ΔgldN mutant (1.8 × 10⁷ CFU/mL). The CFU values shown are the final challenge concentrations. The percent survival for fish challenged with the wild-type and ΔsidL ΔiucX mutant strains were not significantly different.