Construction And Characterization Of Hylb And MP Deleted Mutant Srain In Piscine Streptococcus Agalactiae

Streptococcus agalactiae, also known as group B streptococcus (GBS), is a Gram-positive pathogen with a broad host range. S. agalactiae is the major pathogen causing neonatal pneumonia and meningitis in humans and mastitis in dairy cows. In the last decade, there has been an increase in reports of S. agalactiae infection in fish with septicemia and meningitis resulting in a disturbing case fatality rate of 100%. The investigation on pathogenesis of piscine Streptococcus agalactiae will be helpful for control of this bacterium infection.1 Cloning, expression and immunogenicity of hyaluronidase (HylB) of piscine Streptococcus agalactiaeHyaluronidase (HylB) is an important virulence factor of S. agalactiae. HylB gene was amplified from the genomic DNA of a tilapia S. agalactiae strain by PCR. The hylB gene contained a 2346 bp conserved functional domain. The purified hylB gene fragment was subcloned into pET28a(+) vector by BamH I and Xho I enzymes digestion. After identified by enzymes digestion and sequencing, the recombinant plasmid was transformed into BL21 (DM3), and induced to express by IPTG. SDS-PAGE analysis showed that a specific protein band with the molecular weight of 88.5 kD was obtained. To analyze the immunogenicity of the recombinant protein, rabbits were immunized by intracutaneous injection of the protein. ELISA and Western blot analysis revealed that the recombinant protein could be recognized by rabbit antiserum against HylB. The result showed that the recombinant protein possessed strong immunogenicity.2 Construction of the deleted mutants of hylb and mp gene in piscine S.agalactiaeTo knockout the gene hylB and mp from S. agalactiae GD201008-001, a thermosensitive suicide vector, pSET-4s, carrying the left arm and right arm, was constructed. The two arms were used as a template to generate a PCR fragment comprising the flanking sequences of the hylB genomic locus. The recombinant plasmid was electrotransformed into GD2010008-001 competent cells, and the strains were selected on Spc plates. The suspected double cross-over hylB and mp mutant strains were verified by PCR and qRT-PCR. The complementation vector pSET2 was constructed using a fragment containing the open reading frame coding for the HylB and Mp protein from the GD201008-001 genome. The complementation vector was electroporated into the mutant strain and screened by PCR.3 Characterization of the hylb and mp mutants in piscine S. agalactiaeTo investigate the function of S. agalactiae HylB and Mp protein, the study compared the biological characteristics of mutant, complemented strain and wild-type strain in vivo and vitro. The results showed that there were no significant differences in their growth characteristics. LD50 value of mp mutant in zebrafish was increased, and the colonization in bloods and brains of mice were decreased. The mp mutant displayed reduced survival in macrophages compared to the wild-type. The mp mutant culture supernatant induced a low level cytotoxicity to Bend.3 cells. It suggested that Mp was an important virulence factor of S. agalactiae, which might be involved in the survival of S. agalactiae in host macrophages. The investigation of hylB gene showed that only Hyl+ strains could grow utilizing hyaluronic acid (HA) as the sole carbon source, suggesting that Hyl permits the organism to utilize host HA as an energy source. LD50 determinations in zebrafish demonstrated that the hylb mutant was highly attenuated relative to the wild-type strain. Experimental infection of BALB/c mice revealed that bacterial loads in the blood, spleen and brain were significantly reduced in the hylB mutant compared to wild-type infected mice. The hylb mutant displayed reduced survival in macrophages compared to the wild-type, and stimulated a significantly higher release of pro-inflammatory cytokines, such as IL-1β,IL-6 and TNF-a, than the wild-type in macrophages as well as in mice. It showed that hyaluronidase has a strong influence on the intracellular survival of S. agalactiae and pro-inflammatory cytokine expression, suggesting that it plays a key role in S. agalactiae pathogenicity.