Identification of a Third msa Gene in Renibacterium salmoninarum and the Associated Virulence Phenotype

Abstract

Renibacterium salmoninarum, a gram-positive diplococcobacillus, causes bacterial kidney disease, a condition that can result in extensive morbidity and mortality among stocks of fish. An immunodominant extracellular protein, called major soluble antigen (MSA), is encoded by two identical genes, msa1 and msa2. We found evidence for a third msa gene, msa3, which appears to be a duplication of msa1. Unlike msa1 and msa2, msa3 is not present in all isolates of R. salmoninarum. The presence of the msa3 locus does not affect total MSA production in culture conditions. In a challenge study, isolates possessing the msa3 locus reduced median survival in juvenile chinook salmon (Oncorhynchus tshawytscha) by an average of 34% at doses of <10 5 cells per fish compared to isolates lacking the msa3 locus. In contrast, no difference in survival was observed at the highest dose, 10 6 cells per fish. The phenotype associated with the msa3 locus and its nonuniform distribution may contribute to observed differences in virulence among R. salmoninarum isolates.Bacterial kidney disease (BKD) is a persistent, debilitating disease in salmonid fishes. Renibacterium salmoninarum, the etiological agent of BKD, accumulates primarily in the kidney, causing edema, granuloma development, and membranous glomerulonephritis (36) and resulting in morbidity and mortality (17). The prevalence of chinook salmon (Oncorhynchus tshawytscha) infected with R. salmoninarum can be as high as 90% in hatcheries (41) and 91% in outmigrating river populations (27). BKD is a persistent infectious disease problem in salmon culture (26), affecting wildlife conservation and restoration efforts. Twenty-six stocks of Pacific salmon and steelhead have been designated threatened or endangered by the United States federal government (23), and restoration activities such as captive rearing are hampered by recurrent outbreaks of BKD.Fish infected with R. salmoninarum mount an antibody response that is directed principally against a 57-kDa extracellular protein (5) called major soluble antigen (MSA). MSA has been implicated as a major pathogenicity determinant that is involved in host immunosuppression (6,15,19,40,45), leukocyte agglutination (43, 44), and virulence (7,24,37). MSA is encoded by two identical genes, msa1 and msa2 (25), and both genes are transcriptionally active (31). During a survey of R. salmoninarum isolates for mutations in the two msa genes, we discovered evidence of a third msa gene that was not present in all isolates. In this study we characterized the third msa gene and identified an associated virulence phenotype. MATERIALS AND METHODSBacterial strains and culture conditions. A total of 26 isolates of R. salmoninarum were used in this study. The geographic origin and source of each isolate are shown in Table 1. Liquid cultures were grown in modified KDM2 broth (1.0% Bacto Peptone [Difco], 0.05% yeast extract, 0.05% L-cysteine; adjusted to pH 6.5) (12) at 15°C. Plate cultures were grown on modified KDM2 agar (1% Bacto Peptone, 0.05% yeast extract...

Renibacterium salmoninarum p57 antigenic variation is restricted in geographic distribution and correlated with genomic markers

“…Interestingly, we identified 11 strains in the US and Canada that possess a putative third copy of msa, termed msa3. This finding confirms and extends a previous report that identified msa3 in 19 of 26 strains (Rhodes et al 2004). The high prevalence of strains containing msa3 and the previous correlation with increased virulence suggests that diagnostic assays identifying this variant may have relevance, particularly for regions outside of North America that are negative for this variant.…”

“…The coding regions of the msa1 and msa2 genes are identical, suggesting relatively recent gene duplication, and both genes are present in all characterized strains of R. salmoninarum (O'Farrell & Strom 1999, Wiens et al 2002. msa3 has not yet been fully sequenced, but the flanking regions resemble msa1 and this gene has only been identified in a subset of strains originating from the US (Rhodes et al 2004). Both msa1 and msa2 promoters are functional because integrated reporter plasmids were shown to be transcribed and translated during in vitro culture (Rhodes et al 2002).…”

“…A single gene encoding p57 was originally cloned and designated msa for major soluble antigen (Chien et al 1992), and subsequent investigation identified the presence of as many as 3 copies of msa in the bacterial genome depending on the R. salmoninarum isolate (Rhodes et al 2004). Experimental reduction in gene copy number correlated with reduced virulence, thus demonstrating a crucial contribution of this protein to virulence (Rhodes et al 2004, Coady et al 2006. The coding regions of the msa1 and msa2 genes are identical, suggesting relatively recent gene duplication, and both genes are present in all characterized strains of R. salmoninarum (O'Farrell & Strom 1999, Wiens et al 2002.…”

Genome Sequence of the Fish Pathogen Renibacterium salmoninarum Suggests Reductive Evolution away from an Environmental Arthrobacter Ancestor

“…Both copies of msa are linked to nearby IS994 sequences, and one msa copy is adjacent to the single degenerate ISRs3 sequence. Interestingly, a third copy is present in many clinical isolates, and these strains exhibit increased virulence compared to strains carrying two copies of msa (50). No homolog of the MSA gene has been found in any other sequenced bacterial genome.…”

Interpreting dual ELISA and qPCR data for bacterial kidney disease of salmonids

“…This primer-probe set is directed against a stable, single-copy intergenic region of the Renibacterium salmoninarum genome, and it can be used to quantify the number of bacterial cells present. Primer-probe sets directed against the major soluble antigen gene (msa) cannot be used for quantification, because R. salmoninarum has a variable number of copies of msa that are identical in sequence depending on the strain (Rhodes et al 2004). For DNA extractions using the ELISA pellet as the starting material, 20 to 25 mg of the pellet was directly removed.…”