Proteomic analysis of the fish pathogen Flavobacterium columnare

Abstract

Background: Flavobacterium columnare causes columnaris disease in cultured and wild fish populations worldwide. Columnaris is the second most prevalent bacterial disease of commercial channel catfish industry in the United States. Despite its economic importance, little is known about the expressed proteins and virulence mechanisms of F. columnare. Here, we report the first high throughput proteomic analysis of F. columnare using 2-D LC ESI MS/MS and 2-DE MALDI TOF/TOF MS.

Results: Proteins identified in this study and predicted from the draft F. columnare genome were clustered into functional groups using clusters of orthologous groups (COGs), and their subcellular locations were predicted. Possible functional relations among the identified proteins were determined using pathway analysis. The total number of unique F. columnare proteins identified using both 2-D LC and 2-DE approaches was 621, of which 10.95% (68) were identified by both methods, while 77.29% (480) and 11.76% (73) were unique in 2-D LC and 2-DE, respectively. COG groupings and subcellular localizations were similar between our data set and proteins predicted from the whole genome. Twenty eight pathways were significantly represented in our dataset (P < 0.05).

Conclusion: Results from this study provide experimental evidence for many proteins that were predicted from the F. columnare genome annotation, and they should accelerate functional and comparative studies aimed at understanding virulence mechanisms of this important pathogen.

Figure 1

2-D map of proteins extracted from Flavobacterium columnare. Circled spots were excised and analyzed by MS. Numbers indicate proteins with known IDs while NDs indicate unidentified proteins.

Figure 2

Comparison of COG categories. Proteins identified in this study (621 proteins in both 2-D LC and 2-DE analysis) and predicted from the Flavobacterium columnare draft genome (2,882 ORFs) were organized into COG functional groups. Percentages were calculated by dividing the number of proteins in the particular COG category by the number of unique proteins in each analysis. COG categories are as follows: J, translation, ribosomal structure and biogenesis; K, transcription; L, replication, recombination, and repair; D, cell cycle control, cell division, chromosome partitioning; M, cell wall/membrane/envelop biogenesis; N, cell motility; O, post-translational modification and protein turnover, chaperones; T, signal transduction mechanisms; U, intracellular trafficking, secretion, and vesicular transport; V, defense mechanisms; C, energy production and conversion; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism; R, general function prediction only; S, function unknown. "No hit" group indicates non-significant short or low-complexity sequences. Query proteins not belong to any of the currently-defined COGs (23.51% in 2-D LC and 2-DE analysis and 36.33% in predicted ORFs) were not included in the figure for clarity.

Figure 3

Subcellular locations of Flavobacterium columnare proteins determined by PSORTb prediction. Percentages were calculated by dividing the number of proteins in the particular subcellular location by the number of unique proteins in each analysis (548 proteins in 2D-LC, 141 proteins in 2-DE, and 2,882 ORFs predicted from the draft genome). 2-D LC and 2-DE indicates proteins identified in this study, while ORFs indicates proteins predicted from the whole genome. Unknown category includes proteins with multiple subcellular locations or unknown location.

Figure 4

Amino acid metabolism related pathways in Flavobacterium columnare. Amino acid metabolism related pathways include glutamate; histidine; selenoamino acid; alanine and aspartate; glycine, serine, and threonine metabolism; lysine biosynthesis; lysine degradation; and valine, leucine, and isoleucine degradation pathways. Entities shown in oval represent genes involved in this pathway. Filled ovals represent proteins detected in this study. Unconnected genes were removed from the figure to reduce complexity.