Construction And Characterization Of Nala And PepO Deleted Mutant Srain In Piscine Streptococcus Agalactiae

Streptococcus agalactiae,commonly known as group B streptococcus(GBS),which can cause mastitis in cows,neonatal meningitis and sepsis in humans,and meningitis in fish as previously reported,has a broad host range.Recently,S.agalactiae has become a major pathogen of many fish species,particularly harmful to warm-water fish,resulting great economic losses of the global aquaculture.Piscine S.agalactiae caused a 100 percent death rate on fish due to its strong pathogenicity and infectivity.Our previous study observed that expression of the nala and pepO genes that encodes N-acetyl muramidase and endopeptidase of S.agalactiae was upregulated thro ugh interaction with murine macrophages.In this study,we investigated the functions of nala and pepO genes.1 Construction of the deleted mutants of nala and pepO gene in piscine S.agalactiaeGD201008-001 isogenic mutants △nala,△pepO and complementation strains CAnala,C△pepO were constructed respectively by temperature-sensitive suicide vector pSET4s and Streptococcus-Escherichia coli shuttle vector pSET2.Then the isogenic mutants and complementation strains were verified by PCR,sequencing and qRT-PCR analysis of mRNA transcription level.2 Biological characterization of PepO in piscine S.agalactiaeThe pepO gene encodes the endopeptidase of S.agalactiae GD201008-001,a protein containing 361 amino acids.ELISA and Western-blot analysis showed that the recombinant protein recognized by rabbit antiserum against PepO possessed strong immunogenicity.The study analyzed the biological characteristics of pepO isogenic mutant,complemented strain and wild-type strain,including growth characteristics,virulence to zebrafish,capacity of anti-phagocytosis to RAW264.7 and intracellular survival,adhesion ability to HEp-2,the capacity of stimulating the secretion of cytokines in macrophages.There were no significant differences in all the above characteristics between the pepO mutant and the wild-type.The function of pepO need to be further investigated.3 Biological characterization of Nala in piscine S.agalactiaeThe study compared the biological characteristics of nala isogenic mutant and wild-type strain,including growth characteristics,bacterial form,autolytic activity,virulence to zebrafish,capacity of anti-phagocytosis to RAW264.7 and intracellular survival,adhesion ability to HEp-2,cytotoxicity on RAW264.7 and HEp-2.The results showed that the growth curve of the nala mutant showed an apparent decline in the logarithmic phase,but at the same time reached a plateau with the wild-type.Cell chains of the nala mutant became longer than that of the wild-type.Autolytic activity of the nala mutant decreased by 62.5%compared to wild-type.The nala mutant displayed a significant increase in the adherence to HEp-2 compared to the wild-type.The LD50 value of the nala mutant was increased up to four-ford in the zebrafish infection model.Based on the analysis above,we conclude that N-acetyl muramidase involves in bacterial growth,separation of daughter cells,bacterial autolysis and cell adhesion of S.agalactiae,suggesting that it might plays an important role in pathogenesis of S.agalactiae.