Expression of DuplicatemsaGenes in the Salmonid PathogenRenibacteriumsalmoninarum

Rhodes, Linda D.; Coady, Alison; Strom, Mark S.

doi:10.1128/aem.68.11.5480-5487.2002

Cited by 13 publications

(14 citation statements)

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“…The protein encoded by msa, MSA, has been implicated as a virulence factor in the pathogenesis of BKD, and previous work has shown that msa1 and msa2 are expressed in vitro (31). If a protein is expressed from msa3, the levels of MSA may be greater in isolates bearing msa3 than in isolates bearing only msa1 and msa2.…”

Section: Resultsmentioning

confidence: 99%

“…Because msa1 and msa2 are transcribed at a range of cell densities under in vitro conditions (31), it was expected that msa3 would contribute to MSA levels. Although sequence analysis revealed that the promoter region and ORF of msa3 are identical to those of msa1, the total level of MSA protein in cultured cells was not elevated in isolates possessing the msa3 locus.…”

Section: Discussionmentioning

confidence: 99%

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

Rhodes

Coady

Deinhard

2004

Appl Environ Microbiol

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Rhodes

Coady

Deinhard

2004

Appl Environ Microbiol

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“…A variant strain of R. salmoninarum displays both reduced MSA expression and lowered mortality upon infection (5,24,38), whereas R. salmoninarum isolates possessing an additional copy of the gene encoding MSA are more virulent than isolates lacking the additional copy (32). MSA is encoded by duplicate chromosomal loci (25), and both loci are expressed (33). These observations suggest that MSA is a significant virulence determinant, and we hypothesized that disruption of either chromosomal locus would result in a less virulent strain of R. salmoninarum.…”

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Both msa Genes in Renibacterium salmoninarum Are Needed for Full Virulence in Bacterial Kidney Disease

Coady

Murray

Elliott

et al. 2006

Appl Environ Microbiol

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Renibacterium salmoninarum, a gram-positive diplococcobacillus that causes bacterial kidney disease among salmon and trout, has two chromosomal loci encoding the major soluble antigen (msa) gene. Because the MSA protein is widely suspected to be an important virulence factor, we used insertion-duplication mutagenesis to generate disruptions of either the msa1 or msa2 gene. Surprisingly, expression of MSA protein in broth cultures appeared unaffected. However, the virulence of either mutant in juvenile chinook salmon (Oncorhynchus tshawytscha) by intraperitoneal challenge was severely attenuated, suggesting that disruption of the msa1 or msa2 gene affected in vivo expression.Bacterial kidney disease (BKD) has been reported in most areas of the world where salmonid species are found (4,11,13,20,27,28,35,37,42,46), and it is a principal source of morbidity and mortality among cultured salmon and trout. Acute BKD is characterized by ascites, swollen kidneys, exophthalmia, melanosis, and granulomatous lesions of internal organs such as the kidney, whereas asymptomatic carriers can complete an entire life cycle and successfully spawn (16). The infectious agent of BKD, Renibacterium salmoninarum, can be transmitted both horizontally among cohorts and vertically by intraovum inclusion (2,14). Current disease controls include broodstock segregation or culling, antibiotic treatment, and vaccination, but these methods have only moderate efficacy in preventing or treating the disease (1,30,32).R. salmoninarum is a gram-positive diplococcobacillus that grows very slowly and has fastidious nutritional requirements (8,12,39,40). R. salmoninarum produces abundant quantities of an extracellular, 57-kDa protein called p57 or major soluble antigen (MSA) (7,17,41). MSA is associated with the bacterial surface and is released into surrounding tissues, and it has been associated with leukocyte agglutination (7, 43, 44) and immunomodulation (3, 15, 41, 45). A variant strain of R. salmoninarum displays both reduced MSA expression and lowered mortality upon infection (5, 24, 38), whereas R. salmoninarum isolates possessing an additional copy of the gene encoding MSA are more virulent than isolates lacking the additional copy (32). MSA is encoded by duplicate chromosomal loci (25), and both loci are expressed (33). These observations suggest that MSA is a significant virulence determinant, and we hypothesized that disruption of either chromosomal locus would result in a less virulent strain of R. salmoninarum. The goals of the present study were to generate and characterize R. salmoninarum clones bearing disruptions of either msa1 or msa2, to compare the production of MSA protein in the mutants and the parental R. salmoninarum strain, and to evaluate the virulence of the mutant and parental R. salmoninarum strains for chinook salmon, Oncorhynchus tshawytscha. MATERIALS AND METHODSBacterial strains, media, and culture conditions. The parental R. salmoninarum strain used for transformation was the ATCC type strain 33209 (American Type Cult...

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“…This bacterium grows extremely slowly in vitro, and up to 6 weeks is required for primary isolation (26). While genetic tools for studying R. salmoninarum are available, the process used to introduce or inactivate specific genes is arduous and time-consuming (51). Because of these challenges to research with this organism, we completed and analyzed the R. salmoninarum genome sequence.…”

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

Wiens

Rockey

et al. 2008

J Bacteriol

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Renibacterium salmoninarum is the causative agent of bacterial kidney disease and a significant threat to healthy and sustainable production of salmonid fish worldwide. This pathogen is difficult to culture in vitro, genetic manipulation is challenging, and current therapies and preventative strategies are only marginally effective in preventing disease. The complete genome of R. salmoninarum ATCC 33209 was sequenced and shown to be a 3,155,250-bp circular chromosome that is predicted to contain 3,507 open-reading frames (ORFs). A total of 80 copies of three different insertion sequence elements are interspersed throughout the genome. Approximately 21% of the predicted ORFs have been inactivated via frameshifts, point mutations, insertion sequences, and putative deletions. The R. salmoninarum genome has extended regions of synteny to the Arthrobacter sp. strain FB24 and Arthrobacter aurescens TC1 genomes, but it is approximately 1.9 Mb smaller than both Arthrobacter genomes and has a lower G؉C content, suggesting that significant genome reduction has occurred since divergence from the last common ancestor. A limited set of putative virulence factors appear to have been acquired via horizontal transmission after divergence of the species; these factors include capsular polysaccharides, heme sequestration molecules, and the major secreted cell surface antigen p57 (also known as major soluble antigen). Examination of the genome revealed a number of ORFs homologous to antibiotic resistance genes, including genes encoding ␤-lactamases, efflux proteins, macrolide glycosyltransferases, and rRNA methyltransferases. The genome sequence provides new insights into R. salmoninarum evolution and may facilitate identification of chemotherapeutic targets and vaccine candidates that can be used for prevention and treatment of infections in cultured salmonids.

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Expression of DuplicatemsaGenes in the Salmonid PathogenRenibacteriumsalmoninarum

Cited by 13 publications

References 50 publications

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

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

Both msa Genes in Renibacterium salmoninarum Are Needed for Full Virulence in Bacterial Kidney Disease

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

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