纤维二糖利用基因簇对巴氏链球菌生长及毒力的影响  

The effect of cellobiose-utilization gene cluster on the growth and virulence of Streptococcus pasteurianus

作　　者：李鑫椿 王硕玥 吴宗福[1,2] LI Xinchun;WANG Shuoyue;WU Zongfu(College of Veterinary Medicine/Key Laboratory of Animal Bacteriology,Ministry of Agriculture and Rural Affairs/WOAH Reference Lab for Swine Streptococcosis,Nanjing Agricultural University,Nanjing 210095,China;Guangdong Provincial Key Laboratory of Research on the Technology of Pig-breeding and Pig-disease Prevention,Guangzhou 511400,China)

机构地区：[1]南京农业大学动物医学院/农业农村部动物细菌学重点实验室/世界动物卫生组织猪链球菌病参考实验室,江苏南京210095 [2]广东省养猪与猪病防控技术研究企业重点实验室,广东广州511400

出　　处：《南京农业大学学报》2024年第5期923-931,共9页Journal of Nanjing Agricultural University

基　　金：龙岩学院动物源性人兽共患病防控福建省高校工程研究中心开放基金课题(2021ZW001)。

摘　　要：[目的]本研究旨在明确纤维二糖利用基因簇对巴氏链球菌生长及毒力的影响。[方法]以肺炎链球菌基因组中纤维二糖利用基因簇为参考,同源性比对分析猪源巴氏链球菌WUSP067中纤维二糖利用基因簇,通过荧光定量PCR(RT-qPCR)、RT-PCR、缺失株构建、不同条件下的生长曲线测定、小鼠毒力试验等评价其对巴氏链球菌生长及毒力的影响。[结果]巴氏链球菌WUSP067基因组中存在2个潜在的纤维二糖利用相关基因簇(cellobiose locus, CL),将其分别命名为CL1和CL2,它们均包括编码磷酸转移酶系统和β-葡萄糖苷酶的基因。RT-qPCR结果显示,与添加10 g·L^(-1)葡萄糖的DMEM培养基相比,在添加10 g·L^(-1)纤维二糖的DMEM培养基中,CL1中的BglP1_(wp)和BglA_(wp)基因表达量无显著差异,而CL2中celA_(wp)和celB_(wp)基因表达极显著上调(P<0.000 1),分别上调49倍和33倍,提示其参与利用纤维二糖。选择CL2深入研究,RT-PCR结果显示,该基因簇由2个操纵子组成。构建编码β-葡萄糖苷酶的celA_(wp)基因缺失株,生长曲线分析表明,野生株和celA_(wp)基因缺失(ΔcelA_(wp))株在THB、DMEM+10 g·L^(-1)葡萄糖条件下生长情况无明显差异,但在DMEM+10 g·L^(-1)纤维二糖条件下,野生株能以纤维二糖为唯一碳源生长,而ΔcelA_(wp)表现出明显的生长缺陷,生长12 h时D_(600)仍低于0.5。以断奶仔鼠为感染模型,比较野生株和ΔcelA_(wp)株的毒力,每鼠攻毒剂量为3×10^(8) CFU,野生株攻毒组小鼠3日内全部死亡,而ΔcelA株攻毒组的小鼠死亡趋势较缓,14 d后存活率为20%(P<0.05)。[结论]猪源巴氏链球菌WUSP067能以纤维二糖为唯一碳源生长,CL2是纤维二糖利用相关基因簇,其中celA_(wp)基因不仅促进该菌利用纤维二糖,而且还影响该菌的毒力,在巴氏链球菌致病过程中发挥作用。[Objectives]This study aimed to clarify the effect of a cellobiose-utilization gene cluster on the growth and virulence of Streptococcus pasteurianus.[Methods]A similarity search was performed based on the cellobiose-utilization gene cluster of S.pneumoniae to analyze the homologous gene cluster in S.pasteurianus WUSP067.RT-qPCR,deletion mutant construction,RT-PCR,growth curve determination under different conditions,and mice infection experiments were performed to evaluate the effect of the cellobiose-utilization gene cluster on the growth and virulence of S.pasteurianus.[Results]Two potential cellobiose-utilization gene clusters(CL)were found in the WUSP067,named CL1 and CL2,respectively.Both of them included the phosphotransferase system andβ-glucosidase.RT-qPCR results showed no significant difference in the expression of BglP1 _(wp) and BglA _(wp) genes in CL1 in DMEM medium with 10 g·L^(-1) cellobiose.In contrast,the expression of celA _(wp) and celB _(wp) genes in CL2 were significantly up-regulated(P<0.0001)in DMEM medium with 10 g·L^(-1) cellobiose,with 49-fold and 33-fold,respectively,suggesting that they were involved in utilizing cellobiose.CL2 was chosed for further study,and RT-PCR results showed that it comprised two operons.A deletion mutant strain of the gene celA _(wp) encodingβ-glucosidase was generated.Growth curves analysis showed no significant difference in the growth of wild-type strain andΔcelA _(wp) in THB medium or DMEM medium supplemented with 10 g·L^(-1) glucose.However,in the presence of 10 g·L^(-1) cellobiose as the sole carbon source,compared with the wild-type strain,the growth ofΔcelA _(wp) was severely impaired.Its D 600 value of 12 h growth was lower than 0.5(P<0.0001).The virulence of the wild-type strain andΔcelA _(wp) was compared using a weaned mouse infection model.Mice infected with the wild-type strain with the dose of 3×10^(8) CFU per mouse died in three days,while the survival rate of mice infected with the same dose ofΔcelA _(wp) was 20%on the 14th day post

关 键 词：巴氏链球菌 纤维二糖 生长 毒力 

分 类 号：S852.611[农业科学—基础兽医学]