【作者】 韩先干；

【导师】 陆承平；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2008， 博士

【摘要】 猪链球菌(Streptococcus suis, SS)是引起猪包括脑膜炎、败血症、心内膜炎、关节炎、肺炎等疾病的重要病原。根据其荚膜抗原的不同,猪链球菌分为35个血清型(1-34型,1/2型),其中猪链球菌2型(Streptococcus suis serotype 2, SS2)是一种重要的人畜共患病病原,可以通过直接接触传染人,是引起人和猪发病最常见的血清型。细菌通常都被认为是自由生活的单细胞生物,绝大部分细菌被认为是被动、独立地响应外界环境的变化,比如趋化性、趋磁性、趋光性等等；而细菌之间、细菌与其他动植物之间、细菌与环境之间被认为没有直接的联系。然而,最近的研究表明,密度感应(quorum sensing, QS)作为细菌中一种普遍存在的调控系统,在细菌间的信息交流中起着重要的作用。密度感应是细菌通过分泌信号分子(Autoinducer, AI)来监测细菌群体密度并协调细菌生物功能的信息交流机制,研究细菌与细菌之间的信息交流已成为微生物学研究的新热点。存在于革兰氏阴性和阳性菌中的luxS/AI-2型密度感应系统,可产生用于细菌种间交流的通用信号分子AI-2。细菌许多生理功能都受此系统的调节,包括质粒的转移、生物被膜(biofilm)的形成、毒力因子的调控和细菌发光等。细菌耐药性的出现迫切需要采用新的方式来进行SS2的防控,干扰或者阻断SS2的细菌密度感应系统,尤其是抑制luxS/AI-2型密度感应系统,可以成为防控SS2的新途径。本研究首次报道在SS2中存在luxS/AI-2型密度感应系统,通过以下6个方面对该系统进行了相关研究：1猪链球茵2型AI-2信号分子检测细菌的luxS基因参与信号分子AI-2合成,luxS/AI-2型密度感应系统参与调控细菌众多的生理功能。猪链球菌2型,是一种重要的人畜共患病病原,为探讨是否SS2也存在luxS/AI-2型密度感应系统,通过哈维弧菌(Vibrio harveyi) BB170对SS2的检测结果表明,SS2可以产生AI-2信号分子；对SS2基因组分析表明,SS2中存在的luxS基因,序列比对结果表明SS2的luxS基因与哈维弧菌的luxS基因有36%同源性和56%的相似性,与其他链球菌的luxS基因同源性都在80%以上,表明在不同的细茵中luxS基因具有高度的保守性；大肠杆菌DH5a互补实验证实在SS2中luxS参与SS2的AI-2信号分子合成。进一步的研究发现,AI-2的产生是生长依赖性的,在对数后期SS2产生的AI-2浓度最大。通过对luxS/AI-2介导的密度感应系统的研究,为进一步研究该系统在SS2中的作用奠定了基础。2重组蛋白LuxS与Pfs的表达与纯化在细菌体内信号分子AI-2的合成涉及Pfs和LuxS两个酶的催化作用。通过对SS2的luxS和pfs基因的测序、克隆和表达,获得了重组的LuxS和Pfs蛋白,用重组蛋白免疫新西兰白兔制备的免疫血清经Western blotting检测具有良好的免疫原性。对44株SS2分离株的luxS和pfs基因的检测结果表明,luxS和pfs在SS2中普遍存在,是SS2中的保守基因。通过对SS2重组蛋白LuxS与Pfs的表达和纯化,为AI-2信号分子的体外合成奠定了基础。3信号分子的体外合成及其影响因素AI-2参与调控细菌一系列重要的生物学功能,但由于缺少商业化的AI-2供应,限制了对AI-2在SS2中调控作用的研究。本实验利用表达纯化的SS2的LuxS和Pfs在体外催化SAH,其产物可以诱导报告菌株哈维弧菌BB170发光,表明SS2的LuxS和Pfs可以在体外催化SAH生成有活性的AI-2分子。进一步的研究表明,在SS2中AI-2的产生来源于S-腺苷甲硫氨酸(S-adenosylmethionine, SAM)代谢：SAM在甲基转移酶的作用下生成S-腺苷同型半胱氨酸(S-adenosylhomocysteine, SAH), SAH对细菌有毒害作用,细菌通过Pfs迅速降解SAH,产生S-核糖同型半胱氨酸(SRH),LuxS催化SRH形成有活性的AI-2分子。对影响AI-2体外合成因素的研究表明,AI-2的体外合成的最适温度是37℃,最适pH值是8.0；Cr3+,Al3+和Ba2+促进AI-2的体外合成；Fe2+和Ni2+抑制AI-2的体外合成；Hg2+,Cu2+和Mn2+强烈抑制AI-2的体外合成；而金属离子Li+,Mg2+和Zn2+对AI-2的体外合成基本没有影响。通过对AI-2体外合成及其影响因素的研究,为进一步研究AI-2对SS2的调控功能奠定了基础。4 AI-2的产生水平与pfs及luxS转录水平分析AI-2在茵体内的产生受多种因素的影响,例如营养条件、温度、离子强度、pH以及氧气浓度。为了研究影响SS2产生AI-2的因素,本实验利用荧光定量PCR技术研究了SS2不同时期AI-2的产生与luxS和pfs转录水平之间的关系,同时分析了葡萄糖、蔗糖以及NaCl对AI-2产生的影响。结果表明,在SS2中,AI-2的最大活性发生在对数生长后期,这和pfs的最大转录水平相一致,而luxS的最大转录水平却发生在稳定期。此外,NaCl和葡萄糖能促进AI-2分子的合成,而蔗糖对AI-2的生成没有显著影响。Real-time PCR分析表明,NaCl、蔗糖和葡萄糖都能显著增加luxS的转录水平,然而只有NaCl和葡萄糖能增加pfs的转录水平,蔗糖对pfs的转录水平没有显著的影响。这些结果表明,AI-2的产生水平与pfs转录水平一致,而与luxS转录水平并不一致。5噬菌体肽库筛选LuxS可能的抑制性多肽噬菌体展示技术是用来快速筛选抗体、酶、受体和细胞因子等靶蛋白配体的有效工具。本研究运用噬茵体展示技术,通过3轮的生物淘选和ELISA检测,筛选到20个阳性克隆,测序结果表明,在20个阳性克隆中有14个都含有相同的基序HSIR,表明LuxS含有与HSIR结合的结合位点,且该位点与HSIR的亲和力高。多肽抑制实验表明,多肽TNRHNPHHLHHV能部分抑制LuxS催化SRH生成AI-2,加入多肽TNRHNPHHLHHV后,LuxS催化SRH形成AI-2的浓度从200μmol降低到150μmol,抑制效率为25%。其余合成多肽对LuxS的活性基本没有影响。LuxS可能的抑制性多肽与高亲和力配体的发现,为开发潜在的抑制剂,抑制SS2的luxS/AI-2型密度感应系统以及SS2的防控提供了新的可能途径。6 AI-2对SS2粘附性的影响以及相关基因的调控AI-2作为细茵间的通用信号分子,参与细菌众多生理功能的调控,为了研究信号分子AI-2在SS2对HEp-2细胞粘附中的作用,在SS2对该细胞的粘附实验中加入不同浓度的AI-2。结果表明AI-2在浓度为4 umol时,SS2对该细胞的粘附性达到最大为144%,随着AI-2浓度的增加,SS2对该细胞的粘附性逐渐降低,当AI-2浓度为16umol时,SS2对该细胞的粘附性降低到58%。此外,荧光定量PCR结果表明AI-2促进pfs的转录,抑制luxS的转录；促进毒力因子cps、mrp、sly、gdh和fbps转录,抑制ef转录；促进编码具有免疫原性蛋白的基因MEP, hsp和AbpB的转录,抑制DAP的转录。进一步表明AI-2参与调控SS2众多的生理功能。通过对SS2 luxS/AI-2型密度感应系统研究,不仅为防控SS2感染,从细菌群体角度提供了新的思路,而且为进一步研究QS对SS2的调控功能及其机理奠定了基础。更多还原

【Abstract】 Streptococcus suis is an important pathogen associated with a wide range of diseases in pigs, including meningitis, septicaemia, pneumonia, endocarditis, and arthritis. On the basis of the capsular polysaccharides,35 serotypes; have been identified (types 1-34 and 1/2). Serotype 2 is the most prevalent type in assoc ation with diseases in most countries.lt is also an important zoonotic agent for humans in contact with diseased pigs or their products, causing life threatening diseases.Quorum sensing is the cell population density-dependent regulation of gene expression by small signaling molecules,called autoinducers (AI).The only QS system shared by Gram-positive and Gram-negative bacteria involves the production of autoinducer-2 (AI-2). It has been proposed that AI-2 is a universal signaling molecule that functions in interspecies cell-to-cell communication. Recent reports have shown that,depending on the bacterium, AI-2 plays a role in the regulation of virulence-related genes,pathogenicity and biofilm formation.The worldwide spread of antibiotic resistance among emerging and re-emerging bacterial pathogens has underlined the need to develop new approaches in control the diseases of SS2.In this context, the disruption of bacterial quorum-sensing, especially luxSIA1-2 QS system in SS2, will be the aim of control SS2 infectivity and pathogenicity. 1 Dectation of AI-2 of Streptococcus suis serotype 2The only quorum sensing (QS) system shared by Gram-positive and Gram-negative bacteria involves the production of autoinducer-2 (AI-2). AI-2 has been reported to be a key player in the regulation of survival and virulence-related genes or processes.Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen. However,there have been no reports concerning the AI-2 in SS2. A homologue of luxS,the gene required for AI-2 synthesis in Vibrio harveyi (V.harveyi),was isolated from the SS2 genome. The amino acid identity to luxS in V. harveyi was 36% and>80% amino acid identity with various streptococcal luxS sequences. V.harveyi BB170 bioassay demonstrated luxS functionality in SS2 and its ability to complement the luxS-negative phenotype of Escherichia coli DH5a.Further studies showed that AI-2 activity peaked in the late exponential phase. Analysis of luxS/AI-2 QS will provid a solid basis for future research concerning the role of luxS/AI-2 QS in SS2.2 The expression,purification and preparation of multiantibody of LuxS and Pfs from Streptococcus suis Serotype 2LuxS and Pfs catalyze synthesis of the quorum-sensing signaling molecule autoinducer 2 (AI-2), which has been shown to control a variety of cellular processes.The expression and purification LuxS and Pfs from SS2 strain HA9801,two enzymes gave an apparent single protein band,the molecular mass of 21.74 and 28.44kDa on an SDS-PAGE, respectively.Two New Zealand White rabbits were immunized by subcutaneously injection with recombinant LuxS and Pfs proteins for preparation multiantibody.The results of western blotting showed that the LuxS and Pfs have immunogenicity.Moreover,44 strains SS2 have luxS and pfs,which demonstrating clearly that luxS and pfs are conserved in SS2.Such analyses will be the aim of future studies to elucidate the biosynthesis of AI-2 in SS2.3 Pathway of Biosynthesis Autoinducer-2 in vitro of Streptococcus suis Serotype 2Quorum sensing is the cell population density-dependent regulation of gene expression by small signaling molecules,called autoinducers. LuxS and Pfs catalyze the synthesis of the quorum-sensing signaling molecule autoinducer 2(AI-2),which has been shown to control a variety of cellular processes. Expressed and purified LuxS and Pfs were incubated with S-ribosylhomocysteine (SAH),the reaction products were able to induce the luminescence of Vibrio, harveyi BB170, demonstrating clearly that recombinant Pfs and LuxS synthesize AI-2 in vitro from SAH. The optimum pH and temperature for biosynthesis AI-2 in vitro were 8.0 and 37℃, respectively. The biosynthesis AI-2 in vitro was stimulated by Cr3+,A13+ and Ba2+ and was inhibited by Fe2+, Ni2+, respectively. It was strongly inhibited by Hg2+, Cu2+and Mn2+,while enzyme activity was not affected by Li+, Mg2+and Zn2+.In this study,we identified the pathway of AI-2 synthesis in SS2,and analyzed the impact factor of biosynthesis AI-2 in vitro, which provided a solid basis for future research concerning the importance of AI-2 in SS2.4 Analysis of profile of luxS and pfs transcription in Streptococcus suis serotype 2In this study,we investigated AI-2 production and analyzed the relationship between the transcription level of luxS and pfs and AI-2 production in SS2.The results showed that the maximum level of AI-2 occurred during the late exponential phase, which is consistent with the maximum transcription profile of pfs; however, the maximum transcription profile of luxS occurred in the stationary phase. Further studies showed that sodium chloride or glucose increased the production of AI-2, the results of real time PCR assays showed that NaCl, glucose and sucrose increased the amount of luxS-mRNA, while only NaCl and glucose increased the amount of pfs-mRNA; sucrose had no significant on the amount of pfs-mRNA.Furthermore, although glucose and sucrose increased luxS-mRNA by the same amount, sucrose did not raise the level of AI-2 or transcription of pfs-mRNA.The results presented here demonstrate that the level of transcription of pfs is tightly correlated to the level of AI-2 production, while the level of transcription of luxS does not correlate with the level of AI-2 production.5 Biological activity and identification of a peptide inhibitor of LuxS from Streptococcus suis serotype 2Phage display is a powerful technology that allows selection of short peptide ligands with high binding affinities to proteins of interest from a large pool of random peptide permutations. AI-2 is produced in bacteria that express the gene luxS.In the present study,expressed and purified LuxS from Streptococcus suis serotype 2 was used to catalyze the substrate SRH in a reaction that leads to the production of AI-2.The biological activity of the in vitro synthesized AI-2 was demonstrated in a Vibrio harveyi strain BB170 bioassay.Phage-encoded peptides that specifically interact with the LuxS enzyme were selected following three rounds of phage display.One such peptide inhibitor (TNRHNPHHLHHV) of LuxS was shown to partially inhibit the enzyme’s activity.Furthermore,14 peptides containing the consensus sequence HSIR showed high affinity with LuxS.The selected and characterized specific inhibitor as well as the high-affinity ligands may facilitate the identification of new vaccination targets,thereby opening up new approaches in the development of therapeutic drugs.6 Effects of AI-2 on adherence and gene transcription of SS2AI-2 has been shown to control a variety of cellular processes.In this study, to investigate whether AI-2 affect the capability of SS2 to adhere HEp-2 epithelial cells,we tested the adherence of SS2 by adding different concentration AI-2. The results showed that the maximal adherence of SS2 is 144% at the concentration of 4 umol AI-2. While,it lost 42% of the adherence at the concentration of 16 umol AI-2.Further study by Real-time PCR showed that AI-2 increased the transcription of cps, mrp,sly, gdh,fbps,orf2,MEP,hsp and AbpB, while decreased the transcription of luxS, ef and DAP. These findings will be of benefit to future studies of the role of AI-2 in SS2.更多还原