荚膜唾液酸缺失对猪链球菌2型与宿主相互作用影响的研究

发布时间：2018-03-21 23:16

本文选题：猪链球菌2型　切入点：荚膜　出处：《南京医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：猪链球菌(Streptococcus suis，S.suis)是一种重要的人兽共患病病原菌,根据其表面荚膜多糖(Capsular polysaccharide，CPS)的抗原性，可以分为1/2、1-31和33共33个血清型，其中猪链球菌2型(Streptococcus suis serotype2，S.suis2)是毒力最强，临床检出率最高的血清型。近年来多次出现S.suis2感染人事件，引起业界人士的高度关注。尤其是1998年和2005年，分别在我国江苏海安和四川资阳暴发了大规模的S.suis2感染人疫情，患者中出现高比例的、国内外罕见的链球菌中毒性休克综合症(Streptococcus toxic shock syndrome，STSS)症状，病情凶险，病死率极高。荚膜多糖是目前唯一被公认的S.suis2毒力因子，也是分离株高致病性所必需的组分。唾液酸作为荚膜多糖的单糖成分之一，已被证实是多种致脑膜炎链球菌的毒力相关因子，如在B群链球菌(Group B Streptococcus)中唾液酸被认为是一种重要的毒力因子，在细菌突破宿主血脑屏障过程中发挥重要作用。但唾液酸化的荚膜对S.suis2致病性的影响尚不清楚。本研究对已成功构建的中国强致病株05ZYH33的荚膜突变株Δcps2B、唾液酸突变株ΔneuB以及相应互补株cΔcps2B及cΔneuB进行比较研究，主要研究结果如下： 1.生物学性状研究：分析荚膜唾液酸合成相关基因在S.suis各血清型中的分布及生物信息学特征，比较野生株和突变株的生物学性状，解析荚膜唾液酸在05ZYH33致病过程中可能扮演的角色。研究发现，荚膜唾液酸编码基因cps2B、neuB在S.suis2及一些主要致病血清型分离株中常见，如1/2、1、14、19型等；与野生株相比，突变株菌体表面的显微结构发生显著的变化，ΔneuB菌体表面荚膜明显皱缩变薄，Δcps2B菌体表面荚膜缺失；Δcps2B和ΔneuB的唾液酸含量显著下降。 2.荚膜唾液酸对细菌毒力和宿主炎症反应的影响：比较实验菌株与宿主的相互作用，从动物水平分析不同菌株致病性的差异。小鼠毒力实验发现，荚膜多糖合成基因cps2B及唾液酸合成基因neuB缺失后，细菌毒力基本丧失，基因回复后毒力又恢复到野生株的水平；感染小鼠的外周血液中可见菌株的分布，与突变株相比，感染小鼠对野生株的清除能力较弱；脑组织病理切片分析，发现感染野生株小鼠脑组织可见显著的胶质细胞增生样结节，同时可见分散出血并伴随白细胞浸润；体外小鼠全血细胞体系刺激实验显示，突变株刺激后炎性因子MCP-1、IL-6的分泌水平显著高于野生株组，提示荚膜唾液酸可能在宿主对S.suis2的识别与应答中发挥抑制作用。 3.野生株和突变株ΔneuB与宿主细胞的相互作用：进一步从细胞水平分析野生株和突变株与宿主细胞的相互作用。一方面，比较野毒株与突变株对人上皮细胞（Hep-2）和内皮细胞（HBMEC）黏附和侵袭能力的差异；另一方面，比较野毒株与突变株分别与人单核细胞(Human THP-1monocytes，，THP-1)共孵育时的生存能力，并检测不同菌株刺激后THP-1细胞分泌炎症因子的水平。实验结果表明：荚膜唾液酸缺失后，病原菌对宿主上皮细胞和内皮细胞的黏附、入侵增强，而在宿主专职免疫细胞内的存活能力显著减弱。综上所述，本研究证实cps2B、neuB基因在荚膜唾液酸合成过程中的关键作用；证明荚膜唾液酸是S.suis2的重要毒力因子，初步阐明了荚膜及其唾液酸化在细菌对宿主组织细胞黏附与定植、入侵、播散等方面的作用，为深入阐释S.suis2荚膜唾液酸在S.suis2致病过程中的作用及其参与S.suis2逃避宿主固有免疫防御作用的分子机制奠定了基础。
[Abstract]:Streptococcus suis (Streptococcus suis S.suis) is an important zoonotic pathogen, according to the surface of the capsular polysaccharide (Capsular polysaccharide, CPS) antigenicity, can be divided into 1/2,1-31 33 and a total of 33 serotypes of Streptococcus suis serotype 2 (Streptococcus, including suis serotype2, S.suis2) is the most virulent, clinical detection rate of serum the highest. In recent years several S.suis2 infected people, caused great concern in the industry. Especially in 1998 and 2005, respectively, in China's Jiangsu Haian and Sichuan Ziyang large-scale outbreak of S.suis2 infection epidemic, the high proportion of patients in the domestic rare streptococcal toxic shock syndrome (Streptococcus toxic shock syndrome, STSS) symptoms, dangerous disease, high mortality rate.
Capsular polysaccharide is currently the only S.suis2 virulence factor is recognized, but also necessary for isolates of highly pathogenic components. Sialic acid as one of the monosaccharide compositions of capsular polysaccharide, has been confirmed as virulence factors of pathogenic streptococcus meningitis, as in group B Streptococcus (Group B Streptococcus) sialic acid is considered is an important virulence factor, bacteria play an important role in breaking the host blood brain barrier in the process. But the effect of sialylated capsule to the pathogenicity of S.suis2 is unclear. In this study, China strong pathogenic strain 05ZYH33 has been successfully constructed the capsular mutant Delta cps2B, Delta neuB sialic acid mutant and the corresponding complementary line C delta cps2B and C neuB were studied, the main results are as follows:
1. biological traits: analysis of distribution and biological information of capsular sialic acid synthesis related genes in different serotypes of S.suis in characteristics, comparison of wild-type and mutant strains of biological characteristics, analysis of the capsular polysialic acid may play roles in the pathogenesis of 05ZYH33. The study found that the capsular sialic acid encoding gene cps2B, neuB in S.suis2 and some of the main pathogenic serotype isolates, such as 1/2,1,14,19; compared with wild-type, mutant cell surface microstructure changes significantly, neuB cell surface capsular were shrunken thinning, Delta cps2B cell surface capsular deletion; sialic acid content of cps2B and neuB decreased significantly.
2. effect of capsular sialic acid on bacterial virulence and host inflammatory response: interaction of strains and host comparative experiments, analysis of different pathogenic strains from the animal level. Found that the mice virulence experiments, capsular polysaccharide synthesis gene cps2B and sialic acid synthesis of neuB gene deletion, bacterial virulence gene loss, virulence and reply return to the wild strains of mice infected with strain level; distribution of visible in the peripheral blood, compared with the mutant mice infected on the wild strain scavenging ability is weak; analysis of brain tissue pathological slices, infection was found in wild-type mice brain tissue showed significant gliosis like nodules, scattered bleeding and the white blood cell infiltration system; whole blood cells of mice in vitro stimulation experiments showed that the mutant after stimulation of inflammatory factor MCP-1, the secretion level of IL-6 was significantly higher than that in wild-type group, suggesting that the capsule Sialic acid may play an inhibitory role in the identification and response of the host to S.suis2.
3. of wild-type and mutant neuB and host cell interactions: further analysis of wild and mutant strains and host cell interactions at the cellular level. On the one hand, compared with the wild strain and the mutant strain of human epithelial cells (Hep-2) and endothelial cells (HBMEC) differences in adhesion and invasion; on the other hand comparison, the wild strain and the mutant respectively with human monocytes (Human, THP-1monocytes, THP-1) Co incubated with the ability to survive, and the detection of inflammatory cytokines secretion of THP-1 cells after stimulation with different strains of level. The experimental results show that the capsular sialic acid deletion, adhesion of pathogenic bacteria to host epithelial cells and endothelial cell invasion enhancement of immune cells in the host and full-time viability was significantly reduced.
In summary, this study demonstrated that cps2B, the key role of neuB gene in the synthesis of sialic acid in the capsule; that capsular sialic acid is an important virulence factor of S.suis2, and illustrates the capsular sialic acid in bacteria to host tissue cell adhesion and colonization, invasion, spread effect and so on, which laid the foundation for the molecular mechanism of action a thorough interpretation of S.suis2 capsular sialic acid in the pathogenesis of S.suis2 and S.suis2 involved in the evasion of host innate immune defense.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

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