【作者】 操敏；

【导师】 唐家琪；

【作者基本信息】 第三军医大学 ， 微生物学， 2012， 博士

【摘要】 2型猪链球菌（Streptococcus suis serotype2, SS2）是一种重要的人畜共患病病原体,不仅可感染猪导致脑膜炎、肺炎、关节炎、败血症等,甚至可以致急性死亡，每年给全球养猪业造成重大经济损失；偶尔也可通过伤口、呼吸道等途径感染人，患者临床表现为脑膜炎、败血症等疾病，但多为散发，预后较好。值得关注的是我国在1998和2005年分别暴发两起SS2同时大规模感染猪和人的公共卫生事件，而且有大约1/3的病人出现中毒性休克综合征（TSS）的临床表现，这类病人病情发展快，病程凶险，病死率高达62.7%⁸1.3%。我们课题组对于这一情况进行了全面研究，国内外首次报道SS2感染引起新的临床病型——链球菌中毒性休克综合征（streptococcal toxic shock syndrome,STSS），认为SS2的感染宿主范围和致病性都发生重大变化，流行菌株可能发生毒力变异，文章发表后引起国内外学术界的广泛关注。随后，我们对两次流行的代表菌株98HAH12和05ZYH33进行了全基因组测序和组功能注释，结果发现引发两次大流行的SS2强毒株基因组中均带有一段89kb的DNA片段，该片段在结构上具备了毒力岛的特点，接着我们的研究又发现该片段编码的二元信号转导系统（TCSTS）SalK-SalR是一个致病性正调控分子开关，该片段编码的Ⅳ型分泌系统（Type IV secretion system，T4SS）既可参与中毒性休克的发病机制，又能参与89K片段的切离、复制和转移，从功能上证明89K是流行菌株通过基因水平转移获得的一个毒力岛（PAI89K），使原菌株变成了强毒株。近年来，在东南亚和我国这种SS2强毒株的高致病、高病死和高流行态势引起世界上越来越多的研究团队对SS2致病机制进行研究，但多数报道致力于探讨确定或潜在毒力因子的致病作用，包括荚膜多糖、溶血素、EF、MRP、Sortase A和Dipeptidylpeptidase IV等。本课题组从全基因组出发先后阐明了毒力调控相关元件二元信号转导系统SalK-SalR、CiaRH，孤儿调控因子CovR、Rgg等的转录调控功能和致病作用。近年研究证明，细菌之间存在信息交流，许多细菌都能合成并释放一种称为自诱导物质（autoinducer，AI）的信号分子，这一物质的释放依赖于细菌密度，因此称为密度感应（quorun sensing，QS）。目前已发现多种密度感应系统，其中LuxS/AI-2QS是革兰阳性和阴性细菌共同的信号系统，其信号分子是呋喃酰硼酸二酯（furanosylboratediester）类分子，即AI-2信号分子，它能被不同种属细菌识别，其合成依赖存在于许多菌属序列中的保守的luxS基因编码的LuxS蛋白酶。AI-2合成酶LuxS在超过80种细菌中都有同源物，高度保守，产生的AI-2结构相似，能被不同细菌识别，被认为是细菌种内和种间交流的通用语言。此外，LuxS还是活性甲基循环的重要代谢酶，在代谢平衡中起重要作用。经过长期的研究，LuxS/AI-2系统已被证实在多种细菌的生物膜形成、毒力、代谢、运动、抗生素敏感性等方面起作用，在有的细菌中还具有对基因组进行全局调控的功能。SS2中有关密度感应系统的存在和功能尚不清楚。本研究在SS2中国强毒株05ZYH33中鉴定了一个LuxS同源类似物，据此探讨其是否具有基因组网络调控，感应和应答环境变化，调节细菌的生长和毒力活动以适应不同生境的功能。1.对05ZYH33中luxS基因的生物信息学分析、克隆和原核表达经生物信息学分析发现，位于05ZYH33基因组负链上的luxS基因编码一个166aa的蛋白，其分子中含有3个保守的锌2+结合位点His58, His61和Cys127，还有一个AI-2合成所必需的氨基酸（Gly82），其氨基酸序列与其它链球菌属的LuxS蛋白有较高的一致性（＞80%），结构模拟显示该蛋白采用LuxS蛋白相似的折叠方式，如4个β-折叠加上3个α-螺旋，上述特点提示其为LuxS蛋白家族的功能蛋白。基因注释分析显示该基因与其2个相邻基因在相反链上，提示它为独立的操纵子。此外，我们利用pET-32a原核表达系统对包含luxS OFR及其上游可能的启动子、下游区域的730bp基因进行了体外表达，获得了LuxS融合表达蛋白。2. LuxS基因敲除株与功能互补株的构建05ZYH33株luxS敲除突变株是用敲除质粒上的壮观霉素抗性基因（Spc^R）等位置换luxS基因获得。我们首先分别将luxS基因上下游约1000bp的两个DNA片段克隆到pUC18质粒的多克隆位点上，然后将壮观霉素抗性基因（SpcR）引入上下游基因中间,得到敲除载体pUC::luxS。将pUC::luxS质粒电转化05ZYH33感受态细菌，筛选具有壮观霉素抗性的SS2菌落，采用组合PCR、Southern杂交和RT-PCR等方法验证luxS编码基因已被Spc^R基因所替换，从而获得具有壮观霉素抗性的luxS基因敲除突变株ΔluxS。互补质粒的构建通过将包含luxS OFR、其上游可能的启动子及下游区域基因片段，克隆至大肠杆菌－猪链球菌穿梭质粒pVA838中获得，然后将互补重组质粒电转化突变株ΔluxS的感受态细胞，通过双抗性筛选，PCR和双酶切鉴定获得luxS功能互补株C-ΔluxS。3. luxS缺失对05ZYH33表型和毒力的影响在相同培养条件下观察野生株05ZYH33与ΔluxS突变株表型差异的结果如下：革兰染色后光镜下可见突变株细菌聚集成簇,链变短，与野生株有明显差异；透射电镜下可见突变株荚膜比野生株变薄；通过测OD600值绘制生长曲线发现，△luxS生长速度比野生株变慢，对数期滞后，添加DPD不能恢复其生长能力；AI-2发光活性实验证实, luxS表达产物能产生AI-2活性, luxS缺失使得突变株的AI-2活性显著降低;氧压试验表明，△luxS对较高浓度H₂O₂耐受能力增强，添加DPD无变化，不能回复该表型变化；粘附实验证实，与野生株相比△luxS对Hep-2和HUVEC细胞的黏附能力下降，添加DPD不能回复其粘附能力；测定唾液酸含量发现，△luxS唾液酸含量比野生株降低；仔猪感染实验证明，△luxS对动物的致死率明显下降，对两组感染动物的病理学观察也显示突变株所致动物组织的病理损伤明显减轻；用等比例的野生株／突变株混合菌感染仔猪，对易感组织中的细菌进行培养和计数，结果发现luxS缺失突变株在各组织中的定植能力明显弱于野生株。因此，luxS缺失导致SS2对仔猪的致病性减弱。在功能互补株C-△luxS中，上述表型均恢复至近似于野生株水平。4.基因芯片分析突变株△luxS转录谱变化及AI-2调控基因应用Agilent基因芯片，从转录水平比较野生株和突变株的基因表达差异，同时用定量PCR验证芯片检测结果，以结果相差2倍以上为差异表达标准。与野生株相比，luxS突变株共有312个基因发生了差异表达，占05ZYH33总基因数14.5%。其中上调基因144个,下调基因168个。其中与表型变化相关的基因可分为细胞分裂基因，HSPs，转录调节因子，PTS，毒力相关代谢基因及毒力因子等。ΔluxS突变株添加DPD引起的79个差异表达基因中，29个被认为是受luxS/AI-2密度感应系统调控的靶基因，与细菌毒力及铁摄取相关。综上所述，本研究通过基因敲除获得SS2中国强毒株05ZYH33的luxS缺失突变株，luxS缺失引起一系列表型变化，包括荚膜变薄，AI-2活性显著降低，对H₂O₂耐受性增强，唾液酸含量减少和对上皮细胞的粘附能力增强，一些已知的毒力基因表达下调，这样就导致luxS缺失严重影响了SS2对实验仔猪的致病力，而其功能互补株能使致病性恢复到接近野生株的水平则是从另一侧面证实luxS的功能。基因芯片分析luxS缺失引起的差异表达基因占05ZYH33总基因数的14.5%，证明luxS在SS2基因组中发挥了全局性的调控功能。此外，与AI-2应答的29个差异基因可认为是luxS/AI-2密度感应系统调控的靶基因，为揭示AI-2在SS2密度感应中作用的下游分子提供了靶标。据此我们得出结论，luxS/AI-2在SS2中具有代谢和QS双重调控功能。更多还原

【Abstract】 Streptococcus suis serotype2(SS2) is a major swine pathogen for causing a widevariety of diseases in pigs including meningitis, septicemia and endocarditic andresponsible for important economic losses to the swine industry worldwide. It can alsoinfect human accidently through skin lesions, respiratory or the oral route to causemeningitis and septicemia etc. The human cases often occurred sporadically and have agood prognosis.Notably two major emerging infectious disease outbreaks of SS2occurredin China (one in Jiangsu Province,1998, and the other in Sichuan Province,2005)characterized by the prevalence of streptococcal toxic shock syndrome (STSS) manifestingitself as acute high fever, multiple organ failures, short course of disease and highlethality.(62.7%~81.3%).Our research group explore a detailed and complete study on theevents and firstly reported the new clinical symptom caused by SS2infection-streptococcaltoxic shock syndrome which indicated virulence variation occurred in the epidemic strains.Our published article received extensive attentions in the academic community home andabroad. Comparative genomics analyses have suggested that virulent Chinese strains of SS2feature a specific,89-kb-long DNA fragment showing preliminary evidence that89-kb-longDNA may function as a pathogenicity island. Our further studies revealed a uniquetwo-component signal transduction system (TCSTS) which is orthologous to the SalK/SalRregulatory system of Streptococcus salivarius locating in the candidate89K PAI is apositive molecular switch on virulence controlling. Furthermore, a GI type IV secretionsystem (T4SS) encoded in89K was verified to involve in not only the pathogenesis ofSTSS, but also in the excision, duplication and transfer of89K. Then we concluded that theepidemic strain obtain the PAI89K through horizontal transfer mediated by a GI-type T4SSand turn to be virulent strain.Since the recent recognition of the revalence of S. suis humandisease with high virulence and mortality in southeast and east Asia, the interest of the scientific community in the pathogenesis of this pathogen has significantly increased.Intensified research efforts have been made to explore the potential contribution of differentdescribed S. suis virulence factors at each step of the pathogenesis of the infection,including the process of SS2colonizing the host, breaching epithelial barriers, reaching andsurviving in the bloodstream, invading different organs, and causing exaggeratedinflammation. Amounts of classical virulence factors and virulence factor candidates havebeen described such as Cps, Suilysin, Muramidase-released protein, Extracellular proteinfactor, Dipeptidyl peptidase IV etc. Some new virulence-associated regulon includingtwo-component signal transduction system (TCSTS) SalK-SalR and CiaRH, an orphanregulator, CovR, Rgg-like regulators etc. have been identified by our group recently tointerpret their roles in genome transcription regulation and virulence of SS2.Quorum sensing is a widespread chemical communication system in response tofluctuation of bacterial population density and has been regarded as a transcription regutorysystem associated with bacterial biofilm formation and virulence etc. AI-2mediatedquorum sensing is one of the important QS in bacteria and has been known to exist in boththe Gram positive and Gram negative bacteria extensively. LuxS, an autoinducer-2(AI-2)synthase have been reported to have homologue in more than80bacteria species with highconservation, is determined to catalyze the last committed step of AI-2biosyntheticpathway, producing AI-2with similar structure which can be recognized by differentbacteria and acts as universal language among intraspecies and interspecies communication.In addition, LuxS is also an important RH cleavage enzyme in activated methyl cycle andacts an important role in metabolic balance. After decades of efforts, LuxS/AI-2systemhave been confirmed to play multiple/varied roles in different bacterial species, such asrelated with growth, biofilm formation, virulence, antibiotics susceptibility and motility.Besides, LuxS/AI-2seemed to affect global transcription response in some microorganism.The pleiotropic roles of luxS and AI-2function in S suis2remained unclear. Here weidentified a functional member of the LuxS protein family from a Chinese SS2isolate,05ZYH33and tried to explore if it would be part of the regulatory network in SS2thatsenses changing surroundings and responds to changed environment for a better survival.The following experiments are conducted and results obtained:1. Bioinformatics analysis, Cloning and prokaryotic expression of the luxS gene in 05ZYH33:A luxS ortholog encoding160aa of polypeptide (05SSU0420) is found to locate on thereverse strand of S. suis05ZYH33. This LuxS homologue exhibits highly similarity tothose known LuxS proteins from other Streptococcus species (>80%aa identity). Not onlyare three highly conserved sites critical for zinc binding (His58, His61&Cys127) observedclearly in this suspected LuxS from05ZYH33, but also a recently-reported amino acid(Gly82) is present that was reported to be required for AI-2production. Structural modelingshowed that LuxS homologue adopts similar folding mode of tetranary structure, i.e., fourβ-sheets plus three α-helixs, indicating its possibility of being a functional member of LuxSfamily protein. In addition, bacterial genome annotation-based analyses showed that thisputative luxS gene is opposite to its two neighboring genes, suggesting that it might betranscribed in an independent operon. In addition, we prokaryoticly expressed genesegment including the luxS OFR plus its upstream putative promoter and downstreamsequence using the pET-32a system. SDS-PAGE results showed that the proteinfunctionally expressed in E.coli BL21with the expected molecular size.2. Knockout of luxS and functional complementation:The luxS gene in strain05ZYH33(WT) was inactivated by allelic replacement with aspectinomycin resistance (SpcR) cassette. The applified upstream and downstream DNAfragments adjacent to luxS (~1kb) were cloned into pUC18vector (Takara), and then theintermediate vector was inserted with the spcRgene, giving the luxS knockout vectordesignated pUC::luxS. The knockout plasmid was electroporated into S. suis competentcells. The expected mutant in which a double-crossover event has been undergone wasconfirmed by series of approaches including clony PCR, Southern blot, RT-PCR as well asdirect DNA sequencing.Reverse transcription-PCR (RT-PCR) detection was carried out toconfirm the successful deletion of the luxS gene in the mutant, designated ΔluxS. Forfunctional complementation, the DNA fragment covering the luxS coding region plus itsputative upstream promoter and downstream sequence was amplified from thechromosomal DNA of05ZYH33and cloned into an E. coli-S. suis shuttle vector, pVA838yielding plasmid pVA838::luxS. The resulting plasmid was introduced into the ΔluxSmutant to make the complementary strain (C-ΔluxS).3. The effects of luxS deletion on the phenotypes and virulence of05ZYH33: Different phenotypic properties of the wild type strain05ZYH33and the ΔluxS mutantwere compared under the same conditions. Firstly, Gram staining analyses showed that theΔluxS mutant tends to aggregate into clusters without apparent formation of chains, andexhibit abnormal morphology relative to the wild type05ZYH33. Transmission electronmicroscopy-based observation revealed that the capsule of ΔluxS is significantly thinnerthan that of its parental strain. These phenotypes can be restored in part by functionalcomplementation (C-ΔluxS). Secondly, we noted growth defect in the ΔluxS mutant of S.suis exhibiting the lagged logarithmic phase relative to the wild type. Thirdly, we foundthat AI-2activity was apparently present in strain05ZYH33while in ΔluxS mutant it ismuch lower than that of wild type. Fourthly, Cellular adhesion assays using two differentcell lines (Hep-2and HUVEC cells) demonstrated that the deletion of luxS significantlyweakened the capability of S. suis adherence on its host cells. In addition, the disruption ofluxS gene from S. suis seemed to improve slightly its resistance to H2O2challenge (18~36mM). Phenotype alteration above except AI-2activity cannot be restored by the addition ofDPD, a precursor for AI-2production, indicating that these phenotypes are related to someother unknown function of LuxS rather than its putative role in quorum sensing ofStreptococcus suis. We also noticed the decreased concentration of sialic acid. Notably, weobserved that ΔluxS had significantly lower lethality than the WT strain in an experimentalpiglet infection model. Pathological examination of the two groups of sacrificed pigletsrevealed that many organs suffered less damage in the mutant-infected group.Co-colonization experiment results showed that the ΔluxS mutant infected the specifictissues less effectively than the wild type strain. Therefore, we can conclude that loss ofluxS in SS2attenuates its pathogenicity in the piglet infection model. In addition, all thesephenotype alterations of SS2were restored after complementation.4. Analysis by microarray on the altered global gene transcription profile ofΔluxS and AI-2regulated genes:By Agilent DNA microarray, we compared the differentially expressed genes betweenΔluxS strain and the WT strain grown to middle-exponential phase and verified the thereliability of microarray data by qPCR assays. Totally,14.5%of all the putative genesencoded in SS2genome are affected by the luxS mutation, of which144genes areup-regulated, and168are down-regulated. These genes were categorized into the following various functions: metabolism, transcription regulators, virulence-related factors etc. Wefocus on the following genes potentially involving in the phenotpe alterations.(i) Someknown virulence-related determinants: CPS biosynthesis locus, sialic acid synthase; MRP;EPF are less abundant in luxS mutant, which is somewhat consistent with the attenuation ofvirulence by luxS mutation.(ii) Three transcription factors down-regulated in the ΔluxSmutant, which have ever been determined to be related with pathogenicity of S. suis andother pathogens including RevS orphan response regulator,a homolog of PadR regulator ofphenolic acid metabolism and may be regulated by quorum sensing plus a catabolite controlprotein A (CcpA);(iii)A conserved cell division related FtsA homologue and a cellshape-determining protein (MreC) both were downregulated by luxS deletion, whichvalidates the morphological changes observed with the ΔluxS mutant;(iv) A collection ofheat shock proteins (HSP)-encoding genes are elevated significantly due to luxS disruption,such as Hsp33, ClpL,and ClpE, which may agree with the increased oxidative tolerance andattenuated virulence of the luxS mutant strain.We also observed that71genes in the ΔluxSmutant are differentially expressed in response to the addition of DPD, among them29genes are thought to be regulated by AI-2associated QS.To sum up, inactivation of luxS gene led to a wide range of phenotypic changesincluding thinner capsular walls, increased tolerance to H2O2, reduced adherence capacityto epithelial cells and attenuated virulence in experimental model of piglets, functionalcomplementation restored these phenotypes nearly to the level of parent strain.Genome-wide transcriptome analyses suggested the global effects of luxS on thetranscription profiles. Simultaneously,29of71genes with differentially expression levelare proposed to be targets candidate regulated by LuxS/AI-2-dependent quorum sensing.Wethen draw a conclusion that LuxS/AI-2plays an important role in both the metabolic andQS function in SS2.更多还原