Complete genome sequence and immunoproteomic analyses of the bacterial fish pathogen Streptococcus parauberis

Abstract

Although Streptococcus parauberis is known as a bacterial pathogen associated with bovine udder mastitis, it has recently become one of the major causative agents of olive flounder (Paralichthys olivaceus) streptococcosis in northeast Asia, causing massive mortality resulting in severe economic losses. S. parauberis contains two serotypes, and it is likely that capsular polysaccharide antigens serve to differentiate the serotypes. In the present study, the complete genome sequence of S. parauberis (serotype I) was determined using the GS-FLX system to investigate its phylogeny, virulence factors, and antigenic proteins. S. parauberis possesses a single chromosome of 2,143,887 bp containing 1,868 predicted coding sequences (CDSs), with an average GC content of 35.6%. Whole-genome dot plot analysis and phylogenetic analysis of a 60-kDa chaperonin-encoding gene and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-encoding gene showed that the strain was evolutionarily closely related to Streptococcus uberis. S. parauberis antigenic proteins were analyzed using an immunoproteomic technique. Twenty-one antigenic protein spots were identified in S. parauberis, by reaction with an antiserum obtained from S. parauberis-challenged olive flounder. This work provides the foundation needed to understand more clearly the relationship between pathogen and host and develops new approaches toward prophylactic and therapeutic strategies to deal with streptococcosis in fish. The work also provides a better understanding of the physiology and evolution of a significant representative of the Streptococcaceae.

Fig. 1.

Circular representation of the S. parauberis KCTC11537BP genome. The outer circle indicates predicted coding regions on the forward DNA strand. The next circle (moving inward) indicates predicted coding regions on the reverse DNA strand. The next circle shows GC content (%) of the S. parauberis genome. The final circle shows GC skew, calculated as (G − C)/(G + C). Numbers on the outsides of circles indicate locations in the S. parauberis genome.

Fig. 2.

Dot blot analysis of the genome sequence of S. parauberis KCTC11537BP and six other species of Streptococcus (S. uberis NC_012004, S. pyogenes NC_003485, S. equi subsp. zooepidemicus NC_011134, S. mutans NC_004350, S. agalactiae NC_007432, and S. thermophilus NC_006449).

Fig. 3.

Phylogenetic relationship of the S. parauberis KCTC11537BP gap gene with those of 17 other streptococci (A), and that of the cpn gene with those of 13 other streptococci (B). Trees were calculated using the maximum-likelihood method, with 1,000 bootstrap replications.

Fig. 4.

The 2-DE profile of a whole-cell lysate of S. parauberis KCTC11537BP (A) and 2-DE immunoblotting using a flounder anti-S. parauberis KCTC11537BP serum (B). Arrows with numbers indicate protein spots analyzed by MALDI-TOF. MW, molecular weight (in thousands); pI, isoelectric point.