本文選題：豬鏈球菌2型　切入點：莢膜　出處：《南京醫(yī)科大學》2012年碩士論文　論文類型：學位論文

【摘要】：豬鏈球菌(Streptococcus suis，S.suis)是一種重要的人獸共患病病原菌,根據(jù)其表面莢膜多糖(Capsular polysaccharide，CPS)的抗原性，可以分為1/2、1-31和33共33個血清型，其中豬鏈球菌2型(Streptococcus suis serotype2，S.suis2)是毒力最強，臨床檢出率最高的血清型。近年來多次出現(xiàn)S.suis2感染人事件，引起業(yè)界人士的高度關(guān)注。尤其是1998年和2005年，分別在我國江蘇海安和四川資陽暴發(fā)了大規(guī)模的S.suis2感染人疫情，患者中出現(xiàn)高比例的、國內(nèi)外罕見的鏈球菌中毒性休克綜合癥(Streptococcus toxic shock syndrome，STSS)癥狀，病情兇險，病死率極高。 莢膜多糖是目前唯一被公認的S.suis2毒力因子，也是分離株高致病性所必需的組分。唾液酸作為莢膜多糖的單糖成分之一，已被證實是多種致腦膜炎鏈球菌的毒力相關(guān)因子，如在B群鏈球菌(Group B Streptococcus)中唾液酸被認為是一種重要的毒力因子，在細菌突破宿主血腦屏障過程中發(fā)揮重要作用。但唾液酸化的莢膜對S.suis2致病性的影響尚不清楚。本研究對已成功構(gòu)建的中國強致病株05ZYH33的莢膜突變株Δcps2B、唾液酸突變株ΔneuB以及相應(yīng)互補株cΔcps2B及cΔneuB進行比較研究，主要研究結(jié)果如下： 1.生物學性狀研究：分析莢膜唾液酸合成相關(guān)基因在S.suis各血清型中的分布及生物信息學特征，比較野生株和突變株的生物學性狀，解析莢膜唾液酸在05ZYH33致病過程中可能扮演的角色。研究發(fā)現(xiàn)，莢膜唾液酸編碼基因cps2B、neuB在S.suis2及一些主要致病血清型分離株中常見，如1/2、1、14、19型等；與野生株相比，突變株菌體表面的顯微結(jié)構(gòu)發(fā)生顯著的變化，ΔneuB菌體表面莢膜明顯皺縮變薄，Δcps2B菌體表面莢膜缺失；Δcps2B和ΔneuB的唾液酸含量顯著下降。 2.莢膜唾液酸對細菌毒力和宿主炎癥反應(yīng)的影響：比較實驗菌株與宿主的相互作用，從動物水平分析不同菌株致病性的差異。小鼠毒力實驗發(fā)現(xiàn)，莢膜多糖合成基因cps2B及唾液酸合成基因neuB缺失后，細菌毒力基本喪失，基因回復(fù)后毒力又恢復(fù)到野生株的水平；感染小鼠的外周血液中可見菌株的分布，與突變株相比，感染小鼠對野生株的清除能力較弱；腦組織病理切片分析，發(fā)現(xiàn)感染野生株小鼠腦組織可見顯著的膠質(zhì)細胞增生樣結(jié)節(jié)，同時可見分散出血并伴隨白細胞浸潤；體外小鼠全血細胞體系刺激實驗顯示，突變株刺激后炎性因子MCP-1、IL-6的分泌水平顯著高于野生株組，提示莢膜唾液酸可能在宿主對S.suis2的識別與應(yīng)答中發(fā)揮抑制作用。 3.野生株和突變株ΔneuB與宿主細胞的相互作用：進一步從細胞水平分析野生株和突變株與宿主細胞的相互作用。一方面，比較野毒株與突變株對人上皮細胞（Hep-2）和內(nèi)皮細胞（HBMEC）黏附和侵襲能力的差異；另一方面，比較野毒株與突變株分別與人單核細胞(Human THP-1monocytes，，THP-1)共孵育時的生存能力，并檢測不同菌株刺激后THP-1細胞分泌炎癥因子的水平。實驗結(jié)果表明：莢膜唾液酸缺失后，病原菌對宿主上皮細胞和內(nèi)皮細胞的黏附、入侵增強，而在宿主專職免疫細胞內(nèi)的存活能力顯著減弱。 綜上所述，本研究證實cps2B、neuB基因在莢膜唾液酸合成過程中的關(guān)鍵作用；證明莢膜唾液酸是S.suis2的重要毒力因子，初步闡明了莢膜及其唾液酸化在細菌對宿主組織細胞黏附與定植、入侵、播散等方面的作用，為深入闡釋S.suis2莢膜唾液酸在S.suis2致病過程中的作用及其參與S.suis2逃避宿主固有免疫防御作用的分子機制奠定了基礎(chǔ)。
[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.

【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R378

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