Evaluating the efficacy of an attenuated Streptococcus equi ssp. zooepidemicus vaccine produced by multi-gene deletion in pathogenicity island SeseCisland_4

Streptococcus equi ssp. zooepidemicus(SEZ) is a pathogen associated with a wild range of animal species. Frequent outbreaks have occurred in recent years in pigs, horses, goats and dogs which is liable to infect humans. There is a lack of efficient vaccines against this disease and the occurrence of antibiotic resistance may render drug therapies ineffective. In this study, gene deletion mutant(ΔSEZ) in pathogenicity islands SeseCisland_4 was constructed. The mutant ΔSEZ had a 52-fold decrease in 50% lethal dose(LD_(50)) and had less capacity to adhere epithelial cells. Importantly, immunization of mice with attenuated vaccine ΔSEZ at the dose of 10~2 colony-forming units(CFU) mL^(–1) elicited a significant humoral antibody response, with an antibody titer of 1:12 800. Therefore, 10~2 CFU mL^(–1) might be used as the appropriate immune dose for the attenuated vaccine ΔSEZ, which provided mice with efficient protection against virulent SEZ. In addition, the hyperimmune sera against 10~2 CFU m L–1 attenuated vaccine ΔSEZ could confer significant protection against virulent SEZ infection in the passive immunization experiment and exhibited efficient bactericidal activity in the whole blood assay. Meanwhile, no viable bacteria was detected in blood when mice were immunized with ΔSEZ at the dose of 10~2 CFU mL^(–1) via hypodermic injection. Thereafter, the mutant ΔSEZ at the dose of 10~2 CFU mL^(–1) could confer significant protection in mice and had less negative effects on host, which could be an effective attenuated vaccine candidate for the prevention of SEZ.

Immunoproteomic Assay of Antigenic Surface Proteins in Streptococcus equi ssp. zooepidemicus

Streptococcus equi ssp. zooepidemicus （S. zooepidemicus） is a zoonotic pathogen with worldwide distribution. Lacking suitable vaccine and virulent maker is still bottleneck to control this infection. An immunoproteomic approach has been used to screen the membrane-associated and cell wall-associated proteins of S. zooepidemicus isolate in China CY to discover vaccine candidate antigens and therapeutic agents. Finally, 11 membrane-associated proteins, and 13 cell wall-associated proteins were successfully identified. BLAST （www.sanger.ac.uk） results also indicated that nucleotide sequences of majority identified proteins shared high homology （60%） with S. zooepidemicus, except for AC1-3, AC5, AC8, and AC13. Moreover, genes for 7 of the identified proteins were detected from CY; compared with ST171, 3 of them （AM1, AM8 and AC11） were only found in virulent strains （CY）. All of the proteins identified in this study remain not to be reported in S. zooepidemicus. Some of the proteins serve a vital role in the immune system and reproduction of host species according to available data, while the functions of the rest were seldom researched.

Optimization of Fermentation Process for Hyaluronic Acid Production by Streptococcus zooepidemicus

Hyaluronic acid （HA） is a high molecular weight glycosaminoglycan consisting of alternating D-glucuronic acid and N-acetylglueasamine and plays ex- tremely important roles in many biological processes. In this study, we optimized fermentation process for the production of HA by Streptococcus zooepidemicus ATCC35246, including fermentation broth composition and various fermentation parameters. The experimental results showed that the optimal fermentation broth composition was： glucose 45 g/L, yeast extract 10 g/L, tryptone 12 g/L, KH2PO4 2 g/L, K2HPO4 . 3H20 2 g/L, MgSO4 · 7H2O 2 g/L, and （NH4 ）2SO4 0.4 g/L. The optimal parameters involved in fermentation was： liquid volume 20%, pH 6. 0, rotation speed 180 r/min, fermentation temperature 35 ℃, fermentation duration 18 h, CTAB concentration 25 mg/L. Under the optimized conditions, the yield of HA was 0. 305 g/L, which was dramatically improved by 43.87% compared to that of 0. 212 g/L before optimization.

The vital role of CovS in the establishment of Streptococcus equi subsp.zooepidemicus virulence

Streptococcus equi subsp.zooepidemicus(SEZ)is an important zoonotic agent.Here,a virulence-attenuated strain M35246 derived from natural variation of wild-type SEZ ATCC35246 was found.M35246 showed a deletion of 25contiguous genes as well as a loss-of-function mutation in covS.Subsequently,a 25-gene-deleted strain(ΔPI),a covS-mutant strain(Mcov S),and relevant complementary strains were constructed and investigated.M35246 and Mcov S were significantly less encapsulated and exhibited poorer anti-phagocytic capacity compared to wild-type SEZ.McovS was significantly more sensitive toβ-lactams,aminoglycosides,macrolides,and lincosamides than wild-type SEZ.M35246,McovS,andΔPI exhibited an increase in median lethal dose(LD_(50))in mice by 10~5,10~5,and 5 times when compared to wild-type SEZ,respectively.Neither M35246 nor McovS were isolated from mice 48 h after being challenged with approximately 2000 times the LD_(50)of wild-type SEZ.Transcriptome analysis showed that 668 significantly differentially expressed genes existed between McovS and wild-type SEZ.Numerous virulence factor-encoding genes and anabolicrelated genes in McovS that were involved in anti-phagocytosis,capsule formation,pathogenicity,and antibiotic resistance were downregulated significantly relative to the wild-type strain.This study revealed that the CovS plays a vital role in the establishment of SEZ virulence.

Identification and Characterization of Putative Virulent Genes in Streptococcus equi ssp. zooepidemicus

Suppression subtractive hybridization （SSH） was performed with virulent strain ATCC35246 and avirulent strain ST171 to identify novel genes associated with virulence in Streptococcus equi ssp. zooepidemicus （SEZ）. There were fourteen genomic regions that only presented in virulent strain ATCC35246. These regions encoded 14 proteins, some of them were homologous to proteins associated with cellular surface structure, molecular synthesis, energy metabolism, regulation, transport systems, and other unknown functions. Primers for 6 particular regions were designed from the already published SEZ sequence. Then, we used PCR to evaluate the distribution and conservation of these 6 DNA fragments in various SEZ strains collected from different sources, regions, groups, and times. The results showed that these 6 DNA fragments were widely distributed in SEZ strains, yet they were not existence in the avirulent strain ST171. Moreover, these fragments could not be detected in other Streptococcus groups.

Statistical modeling and optimization for enhanced hyaluronic acid production by batch culture of Sreptococcus zooepidemicus via the supplement of uracil

This work is aimed to achieve the optimal hyaluronic acid(HA)production by batch culture of Streptococcus zooepidemicus via the supplement of nucleotide bases using response surface methodology(RSM).First,the influence of nucleotide bases(adenine,guanine,cytosine,thymine,and uracil)on microbial HA production was investigated using fractional factorial design(FFD).By a 2^(5-2)FFD,uracil was found to be the most significant factor for cell growth and HA production,while the other nucleotide bases were shown to have no significant effects on cell growth and HA production.Also,the impact of uracil on cell growth and HA production was further investigated by RSM,where two variables were considered:uracil concentration and supplement time.The optimal uracil concentration and supplement time were found to be 0.051 g/L and 7 h,respectively,and the predicted maximal HA production reached 6.42 g/L.The maximal HA production increased from 5.0 g/L of the control without uracil supplement to 6.31 g/L at the optimal conditions in validation experiments.

马链球菌兽疫亚种hasB基因功能的研究

为了阐明马链球菌兽疫亚种(Streptococcus equi ssp.zooepidemicus,SEZ)荚膜的致病机理,使该菌引起的猪、马、牛等多种动物的链球菌病得到有效控制,试验采用Real-time PCR技术分析has B基因的体内表达特性,通过构建基因缺失突变菌株、调理吞噬试验等对has B基因的致病性进行系统评价。结果表明:相对于体外培养的细菌,SEZ感染小鼠脾脏分离细菌的mRNA表达均显著下调;基因缺失突变菌株Δhas B与亲本菌株的生长状况基本一致,但是致病力显著降低;基因缺失突变菌株Δhas B影响补体和巨噬细胞介导的免疫反应,使其抵抗宿主巨噬细胞的吞噬能力显著降低。

Purification and Structure Identification of Hyaluronic Acid

Polysaccharide produced by mutated strain of Streptococcus zooepidemicus was purified by the procedures including Savage method, quaternary ammonium compound precipitation, DEAE-cellulose(DE52) chromatography and Sephadex G-75 gel filtration. The structure of the purified polysaccharide has been characterized by means of chemical composition analysis,13C NMR spectrum, infrared spectrum and circular dichroism (CD). All the results showed that the purified polysaccharide was hyaluronic acid (HA). The single helix conformation of the purified HA was determined by Congo red experiment. The molecular weight of the HA was about 1.16×106D, which was measured by viscosity method.