2型猪链球菌Al-2合成酶LuxS的功能鉴定及其在毒力中的作用
|
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H_2O_2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H_2O_2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。
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.
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H_2O_2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H_2O_2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。
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.
引文
1. Berthelot-Herault, F., et al., Production of muraminidase-released protein (MRP),extracellular factor (EF) and suilysin by field isolates of Streptococcus suis capsulartypes2,1/2,9,7and3isolated from swine in France. Vet Res,2000.31(5): p.473-9.
2. Huang, Y.T., et al., Streptococcus suis infection. J Microbiol Immunol Infect,2005.38(5): p.306-13.
3. Wisselink, H.J., J.J. Joosten, and H.E. Smith, Multiplex PCR assays for simultaneousdetection of six major serotypes and two virulence-associated phenotypes ofStreptococcus suis in tonsillar specimens from pigs. J Clin Microbiol,2002.40(8): p.2922-9.
4. Vecht, U., et al., Differences in virulence between two strains of Streptococcus suis typeII after experimentally induced infection of newborn germ-free pigs. Am J Vet Res,
1989.50(7): p.1037-43.
5. Quessy, S., et al., Discrimination of virulent and avirulent Streptococcus suis capsulartype2isolates from different geographical origins. Infect Immun,1995.63(5): p.1975-9.
6. Silva, L.M., et al., Virulence-associated gene profiling of Streptococcus suis isolates byPCR. Vet Microbiol,2006.115(1-3): p.117-27.
7. Gottschalk, M., et al., Streptococcus suis: a new emerging or an old neglected zoonoticpathogen? Future Microbiol，2010.5(3): p.371-91.
8. Perch, B., P. Kristjansen, and K. Skadhauge, Group R streptococci pathogenic for man.Two cases of meningitis and one fatal case of sepsis. Acta Pathol Microbiol Scand,
1968.74(1): p.69-76.
9. Vilaichone, R.K., et al., Streptococcus suis infection in Thailand. J Med Assoc Thai,
2002.85Suppl1: p. S109-17.
10. Tang, J., et al., Streptococcal toxic shock syndrome caused by Streptococcus suisserotype2. PLoS Med,2006.3(5): p. e151.
11.唐家琪，朱进，郭恒彬，等,一起猪链球菌暴发流行的流行病学及病原学研究[J].解放军预防医学杂志,.2000,18(4): p.4.
12. Wei, Z., et al., Characterization of Streptococcus suis isolates from the diseased pigs inChina between2003and2007. Vet Microbiol,2009.137(1-2): p.196-201.
13. Ma, E., et al., Streptococcus suis infection in Hong Kong: an emerging infectiousdisease? Epidemiol Infect,2008.136(12): p.1691-7.
14. Hui, A.C., et al., Bacterial meningitis in Hong Kong:10-years' experience. Clin NeurolNeurosurg,2005.107(5): p.366-70.
15. Mai, N.T., et al., Streptococcus suis meningitis in adults in Vietnam. Clin Infect Dis,
2008.46(5): p.659-67.
16. Ip, M., et al., Streptococcus suis in Hong Kong. Diagn Microbiol Infect Dis,2007.57(1): p.15-20.
17. Fittipaldi, N., et al., Virulence factors involved in the pathogenesis of the infectioncaused by the swine pathogen and zoonotic agent Streptococcus suis. FutureMicrobiol,2012.7(2): p.259-79.
18. Smith, H.E., et al., Mutants of Streptococcus suis types1and2impaired in expressionof muramidase-released protein and extracellular protein induce disease in newborngermfree pigs. Infect Immun,1996.64(10): p.4409-12.
19. Wisselink, H.J., et al., Distribution of capsular types and production ofmuramidase-released protein (MRP) and extracellular factor (EF) of Streptococcussuis strains isolated from diseased pigs in seven European countries. Vet Microbiol,
2000.74(3): p.237-48.
20. Allen, A.G., et al., Generation and characterization of a defined mutant ofStreptococcus suis lacking suilysin. Infect Immun,2001.69(4): p.2732-5.
21. Lun, S., et al., Role of suilysin in pathogenesis of Streptococcus suis capsular serotype
2. Microb Pathog,2003.34(1): p.27-37.
22. Smith, H.E., et al., Identification and characterization of the cps locus of Streptococcussuis serotype2: the capsule protects against phagocytosis and is an importantvirulence factor. Infect Immun,1999.67(4): p.1750-6.
23. Gottschalk, M. and M. Segura, The pathogenesis of the meningitis caused byStreptococcus suis: the unresolved questions. Vet Microbiol,2000.76(3): p.259-72.
24. Li, J., et al., The two-component regulatory system CiaRH contributes to the virulenceof Streptococcus suis2. Vet Microbiol,2010.148(1): p.99-104.
25. Pan, X., et al., The orphan response regulator CovR: a globally negative modulator ofvirulence in Streptococcus suis serotype2. J Bacteriol,2009.191(8): p.2601-12.
26. Chen, C., et al., A glimpse of streptococcal toxic shock syndrome from comparativegenomics of S. suis2Chinese isolates. PLoS One,2007.2(3): p. e315.
27. Li, M., et al., SalK/SalR, a two-component signal transduction system, is essential forfull virulence of highly invasive Streptococcus suis serotype2. PLoS One,2008.3(5): p.e2080.
28. Li, M., et al., GI-type T4SS-mediated horizontal transfer of the89K pathogenicityisland in epidemic Streptococcus suis serotype2. Mol Microbiol,2011.79(6): p.1670-83.
29. Zheng, F., et al., Contribution of the Rgg transcription regulator to metabolism andvirulence of Streptococcus suis serotype2. Infect Immun,2011.79(3): p.1319-28.
30. Sun, J., et al., Is autoinducer-2a universal signal for interspecies communication: acomparative genomic and phylogenetic analysis of the synthesis and signaltransduction pathways. BMC Evol Biol,2004.4: p.36.
31. Bassler, B.L. and R. Losick, Bacterially speaking. Cell,2006.125(2): p.237-46.
32. Henke, J.M. and B.L. Bassler, Three parallel quorum-sensing systems regulate geneexpression in Vibrio harveyi. J Bacteriol,2004.186(20): p.6902-14.
33. Taga, M.E., S.T. Miller, and B.L. Bassler, Lsr-mediated transport and processing ofAI-2in Salmonella typhimurium. Mol Microbiol,2003.50(4): p.1411-27.
34. Xue, T., et al., LsrR-binding site recognition and regulatory characteristics inEscherichia coli AI-2quorum sensing. Cell Res,2009.19(11): p.1258-68.
35. Winzer, K., K.R. Hardie, and P. Williams, Bacterial cell-to-cell communication: sorry,can't talk now-gone to lunch! Curr Opin Microbiol,2002.5(2): p.216-22.
36. Heurlier, K., et al., Growth deficiencies of Neisseria meningitidis pfs and luxS mutantsare not due to inactivation of quorum sensing. J Bacteriol,2009.191(4): p.1293-302.
37. McNab, R., et al., LuxS-based signaling in Streptococcus gordonii: autoinducer2controls carbohydrate metabolism and biofilm formation with Porphyromonasgingivalis. J Bacteriol,2003.185(1): p.274-84.
38. Wen, Z.T., et al., Transcriptome analysis of LuxS-deficient Streptococcus mutans grownin biofilms. Mol Oral Microbiol,2011.26(1): p.2-18.
39. Jesudhasan, P.R., et al., Transcriptome analysis of genes controlled byluxS/autoinducer-2in Salmonella enterica serovar Typhimurium. Foodborne PathogDis,2009.7(4): p.399-410.
40. Novak, E.A., et al., Autoinducer-2and QseC control biofilm formation and in vivovirulence of Aggregatibacter actinomycetemcomitans. Infect Immun,2010.78(7): p.2919-26.
41. Reeser, R.J., et al., Characterization of Campylobacter jejuni biofilms under definedgrowth conditions. Appl Environ Microbiol,2007.73(6): p.1908-13.
42. Bobrov, A.G., et al., Functional quorum sensing systems affect biofilm formation andprotein expression in Yersinia pestis. Adv Exp Med Biol,2007.603: p.178-91.
43. De Araujo, C., et al., Quorum sensing affects biofilm formation throughlipopolysaccharide synthesis in Klebsiella pneumoniae. Res Microbiol,2010.161(7): p.595-603.
44. Stroeher, U.H., et al., Mutation of luxS of Streptococcus pneumoniae affects virulencein a mouse model. Infect Immun,2003.71(6): p.3206-12.
45. Plummer, P., et al., Critical role of LuxS in the virulence of Campylobacter jejuni in aguinea pig model of abortion. Infect Immun,2011.80(2): p.585-93.
46. Osaki, T., et al., Mutation of luxS affects motility and infectivity of Helicobacter pyloriin gastric mucosa of a Mongolian gerbil model. J Med Microbiol,2006.55(Pt11): p.1477-85.
47. Kim, S.Y., et al., Regulation of Vibrio vulnificus virulence by the LuxS quorum-sensingsystem. Mol Microbiol,2003.48(6): p.1647-64.
48. Winzer, K., et al., Role of Neisseria meningitidis luxS in cell-to-cell signaling andbacteremic infection. Infect Immun,2002.70(4): p.2245-8.
49. Choi, J., D. Shin, and S. Ryu, Implication of quorum sensing in Salmonella entericaserovar typhimurium virulence: the luxS gene is necessary for expression of genes inpathogenicity island1. Infect Immun,2007.75(10): p.4885-90.
50. Coulthurst, S.J., C.L. Kurz, and G.P. Salmond, luxS mutants of Serratia defective inautoinducer-2-dependent 'quorum sensing' show strain-dependent impacts on virulenceand production of carbapenem and prodigiosin. Microbiology,2004.150(Pt6): p.1901-10.
51. Xu, L., et al., Role of the luxS quorum-sensing system in biofilm formation andvirulence of Staphylococcus epidermidis. Infect Immun,2006.74(1): p.488-96.
52. Daines, D.A., et al., Haemophilus influenzae luxS mutants form a biofilm and haveincreased virulence. Microb Pathog,2005.39(3): p.87-96.
53. Ahmed, N.A., F.C. Petersen, and A.A. Scheie, AI-2quorum sensing affects antibioticsusceptibility in Streptococcus anginosus. J Antimicrob Chemother,2007.60(1): p.49-53.
54. Jeon, B., et al., Effects of quorum sensing on flaA transcription and autoagglutinationin Campylobacter jejuni. Microbiol Immunol,2003.47(11): p.833-9.
55. Rader, B.A., et al., The quorum-sensing molecule autoinducer2regulates motility andflagellar morphogenesis in Helicobacter pylori. J Bacteriol,2007.189(17): p.6109-17.
56. DeLisa, M.P., et al., DNA microarray-based identification of genes controlled byautoinducer2-stimulated quorum sensing in Escherichia coli. J Bacteriol,2001.183(18): p.5239-47.
57. Li, M., et al., AI-2-dependent gene regulation in Staphylococcus epidermidis. BMCMicrobiol,2008.8: p.4.
58. Sperandio, V., et al., Quorum sensing is a global regulatory mechanism inenterohemorrhagic Escherichia coli O157:H7. J Bacteriol,2001.183(17): p.5187-97.
59. Malladi, V.L., et al., Inhibition of LuxS by S-ribosylhomocysteine analogues containinga [4-aza]ribose ring. Bioorg Med Chem,2011.19(18): p.5507-19.
60. Hilgers, M.T. and M.L. Ludwig, Crystal structure of the quorum-sensing protein LuxSreveals a catalytic metal site. Proc Natl Acad Sci U S A,2001.98(20): p.11169-74.
61. Plummer, P., et al., Identification of a key amino acid of LuxS involved in AI-2production in Campylobacter jejuni. PLoS One,2011.6(1): p. e15876.
62. Pei, D. and J. Zhu, Mechanism of action of S-ribosylhomocysteinase (LuxS). Curr OpinChem Biol,2004.8(5): p.492-7.
63. Zhu, J., R. Patel, and D. Pei, Catalytic mechanism of S-ribosylhomocysteinase (LuxS):stereochemical course and kinetic isotope effect of proton transfer reactions.Biochemistry,2004.43(31): p.10166-72.
64. Das, S.K., et al., Cloning, purification, crystallization and preliminary crystallographicanalysis of Bacillus subtilis LuxS. Acta Crystallogr D Biol Crystallogr,2001.57(Pt9):p.1324-5.
65. Lewis, H.A., et al., A structural genomics approach to the study of quorum sensing:crystal structures of three LuxS orthologs. Structure,2001.9(6): p.527-37.
66. Rajan, R., et al., Crystal structure of S-ribosylhomocysteinase (LuxS) in complex with acatalytic2-ketone intermediate. Biochemistry,2005.44(10): p.3745-53.
67. Li, H., et al., Crystallization and preliminary X-ray analysis ofS-ribosylhomocysteinase from Streptococcus mutans. Acta Crystallogr Sect F StructBiol Cryst Commun,2012.68(Pt2): p.199-202.
68. Bhattacharyya, M. and S. Vishveshwara, Elucidation of the conformational free energylandscape in H.pylori LuxS and its implications to catalysis. BMC Struct Biol,2010.10:p.27.
69. Bhattacharyya, M. and S. Vishveshwara, Functional correlation of bacterial LuxS withtheir quaternary associations: interface analysis of the structure networks. BMC StructBiol,2009.9: p.8.
70. De Keersmaecker S C, S.K., Vanderleyden J, Let LuxS speak up in AI-2signaling.Trends Microbiol,2006.14(3): p.１１４－１１９.
71. Takamatsu, D., M. Osaki, and T. Sekizaki, Construction and characterization ofStreptococcus suis-Escherichia coli shuttle cloning vectors. Plasmid,2001.45(2): p.101-13.
72. Macrina, F.L., et al., Novel shuttle plasmid vehicles for Escherichia-Streptococcustransgeneric cloning. Gene,1983.25(1): p.145-50.
73. Smith, H.E., et al., High-efficiency transformation and gene inactivation inStreptococcus suis type2. Microbiology,1995.141(Pt1): p.181-8.
74. Ota, K., et al., Expression of histone acetyltransferases was down-regulated inpoly(ADP-ribose) polymerase-1-deficient murine cells. Biochem Biophys ResCommun,2003.310(2): p.312-7.
75. Plasterk, R.H., RNA silencing: the genome's immune system. Science,2002.296(5571):p.1263-5.
76. Court, D.L., J.A. Sawitzke, and L.C. Thomason, Genetic engineering usinghomologous recombination. Annu Rev Genet,2002.36: p.361-88.
77.李明，胡福泉，王长军，等.,环境条件对猪链球菌2型电转化效率的影响.中国人兽共患病学报,,2007.23(8): p.760-3.
78. Iacono, V.J., et al., Selective antibacterial properties of lysozyme for oralmicroorganisms. Infect Immun,1980.29(2): p.623-32.
79. Turovskiy, Y., et al., Quorum sensing: fact, fiction, and everything in between. AdvAppl Microbiol,2007.62: p.191-234.
80. Bassler, B.L., et al., Intercellular signalling in Vibrio harveyi: sequence and function ofgenes regulating expression of luminescence. Mol Microbiol,1993.9(4): p.773-86.
81. Tischler, A.S., J.C. Riseberg, and V. Cherington, Multiple mitogenic signallingpathways in chromaffin cells: a model for cell cycle regulation in the nervous system.Neurosci Lett,1994.168(1-2): p.181-4.
82. Surette, M.G., M.B. Miller, and B.L. Bassler, Quorum sensing in Escherichia coli,Salmonella typhimurium, and Vibrio harveyi: a new family of genes responsible forautoinducer production. Proc Natl Acad Sci U S A,1999.96(4): p.1639-44.
83. Chen, X., et al., Structural identification of a bacterial quorum-sensing signalcontaining boron. Nature,2002.415(6871): p.545-9.
84. Miller, S.T., et al., Salmonella typhimurium recognizes a chemically distinct form of thebacterial quorum-sensing signal AI-2. Mol Cell,2004.15(5): p.677-87.
85. Surette, M.G. and B.L. Bassler, Quorum sensing in Escherichia coli and Salmonellatyphimurium. Proc Natl Acad Sci U S A,1998.95(12): p.7046-50.
86. Lyon, W.R., et al., Mutation of luxS affects growth and virulence factor expression inStreptococcus pyogenes. Mol Microbiol,2001.42(1): p.145-57.
87. Yuan, L., J.D. Hillman, and A. Progulske-Fox, Microarray analysis ofquorum-sensing-regulated genes in Porphyromonas gingivalis. Infect Immun,2005.73(7): p.4146-54.
88.欧阳谦，马文丽，刘翠华，石嵘，郑文岭, ΔluxS的表型分析及其在GBS毒力调控机制研究中的应用.南方医科大学学报,2006.26(1): p.117-121.
89. Tavender, T.J., et al., LuxS-independent formation of AI-2from ribulose-5-phosphate.BMC Microbiol,2008.8: p.98.
90. Jacques, M., et al., Ultrastructural study of surface components of Streptococcus suis. JBacteriol,1990.172(6): p.2833-8.
91. Verneuil, N., et al., Contribution of a PerR-like regulator to the oxidative-stressresponse and virulence of Enterococcus faecalis. Microbiology,2005.151(Pt12): p.3997-4004.
92. Charland, N., et al., Role of capsular sialic acid in virulence and resistance tophagocytosis of Streptococcus suis capsular type2. FEMS Immunol Med Microbiol,
1996.14(4): p.195-203.
93. Jones, M.B. and M.J. Blaser, Detection of a luxS-signaling molecule in Bacillusanthracis. Infect Immun,2003.71(7): p.3914-9.
94. Jordan, D.M., et al., Colonization of gnotobiotic piglets by a luxS mutant strain ofEscherichia coli O157:H7. Infect Immun,2005.73(2): p.1214-6.
95. Wen, Z.T. and R.A. Burne, LuxS-mediated signaling in Streptococcus mutans isinvolved in regulation of acid and oxidative stress tolerance and biofilm formation. JBacteriol,2004.186(9): p.2682-91.
96. Krin, E., et al., Pleiotropic role of quorum-sensing autoinducer2in Photorhabdusluminescens. Appl Environ Microbiol,2006.72(10): p.6439-51.
97. Unnikrishnan, M., J. Cohen, and S. Sriskandan, Growth-phase-dependent expression ofvirulence factors in an M1T1clinical isolate of Streptococcus pyogenes. Infect Immun,
1999.67(10): p.5495-9.
98. Maganti, S., et al., The role of sialic acid in opsonin-dependent andopsonin-independent adhesion of Listeria monocytogenes to murine peritonealmacrophages. Infect Immun,1998.66(2): p.620-6.
99. Sztajer, H., et al., Autoinducer-2-regulated genes in Streptococcus mutans UA159andglobal metabolic effect of the luxS mutation. J Bacteriol,2008.190(1): p.401-15.
100.Schmittgen, T.D. and B.A. Zakrajsek, Effect of experimental treatment onhousekeeping gene expression: validation by real-time, quantitative RT-PCR. JBiochem Biophys Methods,2000.46(1-2): p.69-81.
101.Livak, K.J. and T.D. Schmittgen, Analysis of relative gene expression data usingreal-time quantitative PCR and the2(-Delta Delta C(T)) Method. Methods,2001.25(4):p.402-8.
102.Geissler, B., D. Shiomi, and W. Margolin, The ftsA*gain-of-function allele ofEscherichia coli and its effects on the stability and dynamics of the Z ring.Microbiology,2007.153(Pt3): p.814-25.
103.Divakaruni, A.V., et al., The cell shape proteins MreB and MreC control cellmorphogenesis by positioning cell wall synthetic complexes. Mol Microbiol,2007.66(1): p.174-88.
104.Kumsta, C. and U. Jakob, Redox-regulated chaperones. Biochemistry,2009.48(22): p.4666-76.
105.Schauer, K., et al., The Helicobacter pylori GroES cochaperonin HspA functions as aspecialized nickel chaperone and sequestration protein through its unique C-terminalextension. J Bacteriol,2010.192(5): p.1231-7.
106.Ishikawa, M., et al., Cloning and characterization of grpE in Acetobacter pasteurianusNBRC3283. J Biosci Bioeng,2012.109(1): p.25-31.
107.Bolean, M., et al., Photodynamic therapy with rose bengal induces GroEL expressionin Streptococcus mutans. Photomed Laser Surg,2010.28Suppl1: p. S79-84.
108.Di Pasqua, R., et al., Changes in the proteome of Salmonella enterica serovarThompson as stress adaptation to sublethal concentrations of thymol. Proteomics,2010.10(5): p.1040-9.
109.Hickey, T.B., et al., Mycobacterium tuberculosis employs Cpn60.2as an adhesin thatbinds CD43on the macrophage surface. Cell Microbiol,2010.12(11): p.1634-47.
110. Suokko, A., et al., ClpL is essential for induction of thermotolerance and is potentiallypart of the HrcA regulon in Lactobacillus gasseri. Proteomics,2008.8(5): p.1029-41.
111. Kajfasz, J.K., et al., Role of Clp proteins in expression of virulence properties ofStreptococcus mutans. J Bacteriol,2009.191(7): p.2060-8.
112. Tu le, N., et al., Modulation of adherence, invasion, and tumor necrosis factor alphasecretion during the early stages of infection by Streptococcus pneumoniae ClpL. InfectImmun,2007.75(6): p.2996-3005.
113. Zhang, Q., et al., Contribution of ClpE to virulence of Streptococcus pneumoniae. CanJ Microbiol,2009.55(10): p.1187-94.
114. Joseph, B., et al., Identification of Listeria monocytogenes genes contributing tointracellular replication by expression profiling and mutant screening. J Bacteriol,
2006.188(2): p.556-68.
115. Lamarche, M.G., et al., Inactivation of the pst system reduces the virulence of an avianpathogenic Escherichia coli O78strain. Infect Immun,2005.73(7): p.4138-45.
116. Daigle, F., J.M. Fairbrother, and J. Harel, Identification of a mutation in the pst-phoUoperon that reduces pathogenicity of an Escherichia coli strain causing septicemia inpigs. Infect Immun,1995.63(12): p.4924-7.
117. de Greeff, A., et al., Response regulator important in pathogenesis of Streptococcussuis serotype2. Microb Pathog,2002.33(4): p.185-92.
118. Kovacikova, G. and K. Skorupski, Regulation of virulence gene expression in Vibriocholerae by quorum sensing: HapR functions at the aphA promoter. Mol Microbiol,
2002.46(4): p.1135-47.
119. Iyer, R., N.S. Baliga, and A. Camilli, Catabolite control protein A (CcpA) contributesto virulence and regulation of sugar metabolism in Streptococcus pneumoniae. JBacteriol,2005.187(24): p.8340-9.
120.Shelburne, S.A.,3rd, et al., A direct link between carbohydrate utilization andvirulence in the major human pathogen group A Streptococcus. Proc Natl Acad Sci U SA,2008.105(5): p.1698-703.
121.Kietzman, C.C. and M.G. Caparon, CcpA and LacD.1affect temporal regulation ofStreptococcus pyogenes virulence genes. Infect Immun,2009.78(1): p.241-52.
122.Willenborg, J., et al., Role of glucose and CcpA in capsule expression and virulence ofStreptococcus suis. Microbiology,2011.157(Pt6): p.1823-33.
123.Charland, N., et al., Streptococcus suis serotype2mutants deficient in capsularexpression. Microbiology,1998.144(Pt2): p.325-32.
124.Wilson, T.L., et al., A novel signature-tagged mutagenesis system for Streptococcussuis serotype2. Vet Microbiol,2007.122(1-2): p.135-45.
125.董瑞萍，王长军，程功，李明，王晶，潘秀珍，唐家琪,猪链球菌2型唾液酸合成酶neuB基因敲除突变株的构建及其生物学特性.微生物学通报，2009.36(2): p.238-244.
126.Vecht, U., et al., Identification of two proteins associated with virulence ofStreptococcus suis type2. Infect Immun,1991.59(9): p.3156-62.
127.Terao, Y., et al., Novel laminin-binding protein of Streptococcus pyogenes, Lbp, isinvolved in adhesion to epithelial cells. Infect Immun,2002.70(2): p.993-7.
128.王伟,肖.明.,微生物嗜铁素介导的铁摄取.生物学杂志,2005.22(4): p.4.
129.Kim, C.M. and S.H. Shin, Modulation of iron-uptake systems by a mutation of luxSencoding an autoinducer-2synthase in Vibrio vulnificus. Biol Pharm Bull,2011.34(5):p.632-7.
130.James, C.E., et al., LuxS involvement in the regulation of genes coding for hemin andiron acquisition systems in Porphyromonas gingivalis. Infect Immun,2006.74(7): p.3834-44.
131.Chung, W.O., et al., Signaling system in Porphyromonas gingivalis based on a LuxSprotein. J Bacteriol,2001.183(13): p.3903-9.
132.Walters, M. and V. Sperandio, Autoinducer3and epinephrine signaling in the kineticsof locus of enterocyte effacement gene expression in enterohemorrhagic Escherichiacoli. Infect Immun,2006.74(10): p.5445-55.
1. Fuqua, W.C., S.C. Winans, and E.P. Greenberg, Quorum sensing in bacteria: theLuxR-LuxI family of cell density-responsive transcriptional regulators. J Bacteriol,
1994.176(2): p.269-75.
2. Xavier, K.B. and B.L. Bassler, LuxS quorum sensing: more than just a numbers game.Curr Opin Microbiol,2003.6(2): p.191-7.
3. Plummer, P., et al., Critical role of LuxS in the virulence of Campylobacter jejuni in aguinea pig model of abortion. Infect Immun,2011.80(2): p.585-93.
4. Choi, J., D. Shin, and S. Ryu, Implication of quorum sensing in Salmonella entericaserovar typhimurium virulence: the luxS gene is necessary for expression of genes inpathogenicity island1. Infect Immun,2007.75(10): p.4885-90.
5. Osaki, T., et al., Mutation of luxS affects motility and infectivity of Helicobacter pyloriin gastric mucosa of a Mongolian gerbil model. J Med Microbiol,2006.55(Pt11): p.1477-85.
6. Hao, Y., et al., Identification and characterization of new LuxR/LuxI-type quorumsensing systems from metagenomic libraries. Environ Microbiol,2009.12(1): p.105-17.
7. Thoendel, M. and A.R. Horswill, Identification of Staphylococcus aureus AgrD residuesrequired for autoinducing peptide biosynthesis. J Biol Chem,2009.284(33): p.21828-38.
8. Sun, J., et al., Is autoinducer-2a universal signal for interspecies communication: acomparative genomic and phylogenetic analysis of the synthesis and signaltransduction pathways. BMC Evol Biol,2004.4: p.36.
9. Bassler, B.L. and R. Losick, Bacterially speaking. Cell,2006.125(2): p.237-46.
10. Khajanchi, B.K., et al., The two-component QseBC signalling system regulates in vitroand in vivo virulence of Aeromonas hydrophila. Microbiology,2011.158(Pt1): p.259-71.
11. Walters, M., M.P. Sircili, and V. Sperandio, AI-3synthesis is not dependent on luxS inEscherichia coli. J Bacteriol,2006.188(16): p.5668-81.
12. Bassler, B.L., et al., Intercellular signalling in Vibrio harveyi: sequence and function ofgenes regulating expression of luminescence. Mol Microbiol,1993.9(4): p.773-86.
13. Tischler, A.S., J.C. Riseberg, and V. Cherington, Multiple mitogenic signallingpathways in chromaffin cells: a model for cell cycle regulation in the nervous system.Neurosci Lett,1994.168(1-2): p.181-4.
14. Bassler, B.L., E.P. Greenberg, and A.M. Stevens, Cross-species induction ofluminescence in the quorum-sensing bacterium Vibrio harveyi. J Bacteriol,1997.179(12): p.4043-5.
15. Surette, M.G., M.B. Miller, and B.L. Bassler, Quorum sensing in Escherichia coli,Salmonella typhimurium, and Vibrio harveyi: a new family of genes responsible forautoinducer production. Proc Natl Acad Sci U S A,1999.96(4): p.1639-44.
16. Winzer, K., K.R. Hardie, and P. Williams, Bacterial cell-to-cell communication: sorry,can't talk now-gone to lunch! Curr Opin Microbiol,2002.5(2): p.216-22.
17. Winzer, K., et al., LuxS: its role in central metabolism and the in vitro synthesis of4-hydroxy-5-methyl-3(2H)-furanone. Microbiology,2002.148(Pt4): p.909-22.
18. Sauer, U., et al., The soluble and membrane-bound transhydrogenases UdhA and PntABhave divergent functions in NADPH metabolism of Escherichia coli. J Biol Chem,2004.279(8): p.6613-9.
19. Tavender, T.J., et al., LuxS-independent formation of AI-2from ribulose-5-phosphate.BMC Microbiol,2008.8: p.98.
20. Lyon, W.R., et al., Mutation of luxS affects growth and virulence factor expression inStreptococcus pyogenes. Mol Microbiol,2001.42(1): p.145-57.
21. Cao, M., et al., Functional definition of LuxS, an autoinducer-2(AI-2) synthase and itsrole in full virulence of Streptococcus suis serotype2. J Microbiol,2011.49(6): p.1000-11.
22. Vendeville, A., et al., Making 'sense' of metabolism: autoinducer-2, LuxS andpathogenic bacteria. Nat Rev Microbiol,2005.3(5): p.383-96.
23. Chen, X., et al., Structural identification of a bacterial quorum-sensing signalcontaining boron. Nature,2002.415(6871): p.545-9.
24. Miller, S.T., et al., Salmonella typhimurium recognizes a chemically distinct form of thebacterial quorum-sensing signal AI-2. Mol Cell,2004.15(5): p.677-87.
25. Das, S.K., et al., Cloning, purification, crystallization and preliminary crystallographicanalysis of Bacillus subtilis LuxS. Acta Crystallogr D Biol Crystallogr,2001.57(Pt9): p.1324-5.
26. Hilgers, M.T. and M.L. Ludwig, Crystal structure of the quorum-sensing protein LuxSreveals a catalytic metal site. Proc Natl Acad Sci U S A,2001.98(20): p.11169-74.
27. Lewis, H.A., et al., A structural genomics approach to the study of quorum sensing:crystal structures of three LuxS orthologs. Structure,2001.9(6): p.527-37.
28. Rajan, R., et al., Crystal structure of S-ribosylhomocysteinase (LuxS) in complex with acatalytic2-ketone intermediate. Biochemistry,2005.44(10): p.3745-53.
29. Li, H., et al., Crystallization and preliminary X-ray analysis of S-ribosylhomocysteinasefrom Streptococcus mutans. Acta Crystallogr Sect F Struct Biol Cryst Commun,2012.68(Pt2): p.199-202.
30. Bhattacharyya, M. and S. Vishveshwara, Elucidation of the conformational free energylandscape in H.pylori LuxS and its implications to catalysis. BMC Struct Biol,2010.10:p.27.
31. De Keersmaecker S C, S.K., Vanderleyden J, Let LuxS speak up in AI-2signaling.Trends Microbiol,2006.14(3): p.１１４－１１９.
32. Pei, D. and J. Zhu, Mechanism of action of S-ribosylhomocysteinase (LuxS). Curr OpinChem Biol,2004.8(5): p.492-7.
33. Zhu, J., R. Patel, and D. Pei, Catalytic mechanism of S-ribosylhomocysteinase (LuxS):stereochemical course and kinetic isotope effect of proton transfer reactions.Biochemistry,2004.43(31): p.10166-72.
34. Plummer, P., et al., Identification of a key amino acid of LuxS involved in AI-2production in Campylobacter jejuni. PLoS One,2011.6(1): p. e15876.
35. Bhattacharyya, M. and S. Vishveshwara, Functional correlation of bacterial LuxS withtheir quaternary associations: interface analysis of the structure networks. BMC StructBiol,2009.9: p.8.
36. Henke, J.M. and B.L. Bassler, Three parallel quorum-sensing systems regulate geneexpression in Vibrio harveyi. J Bacteriol,2004.186(20): p.6902-14.
37. Turovskiy, Y., et al., Quorum sensing: fact, fiction, and everything in between. AdvAppl Microbiol,2007.62: p.191-234.
38. Miller, M.B., et al., Parallel quorum sensing systems converge to regulate virulence inVibrio cholerae. Cell,2002.110(3): p.303-14.
39. Taga, M.E., S.T. Miller, and B.L. Bassler, Lsr-mediated transport and processing ofAI-2in Salmonella typhimurium. Mol Microbiol,2003.50(4): p.1411-27.
40. Xue, T., et al., LsrR-binding site recognition and regulatory characteristics inEscherichia coli AI-2quorum sensing. Cell Res,2009.19(11): p.1258-68.
41. Novak, E.A., et al., Autoinducer-2and QseC control biofilm formation and in vivovirulence of Aggregatibacter actinomycetemcomitans. Infect Immun,2010.78(7): p.2919-26.
42. Rader, B.A., et al., Helicobacter pylori perceives the quorum-sensing molecule AI-2asa chemorepellent via the chemoreceptor TlpB. Microbiology,2011.157(Pt9): p.2445-55.
43. Widmer, K.W., P. Jesudhasan, and S.D. Pillai, Fatty Acid Modulation of Autoinducer(AI-2) Influenced Growth and Macrophage Invasion by Salmonella Typhimurium.Foodborne Pathog Dis,2012.9(3): p.211-7.
44. Walters, M. and V. Sperandio, Autoinducer3and epinephrine signaling in the kineticsof locus of enterocyte effacement gene expression in enterohemorrhagic Escherichiacoli. Infect Immun,2006.74(10): p.5445-55.
45. Merritt, J., et al., Mutation of luxS affects biofilm formation in Streptococcus mutans.Infect Immun,2003.71(4): p.1972-9.
46. Wen, Z.T. and R.A. Burne, LuxS-mediated signaling in Streptococcus mutans isinvolved in regulation of acid and oxidative stress tolerance and biofilm formation. JBacteriol,2004.186(9): p.2682-91.
47. Hasona, A., et al., Membrane composition changes and physiological adaptation byStreptococcus mutans signal recognition particle pathway mutants. J Bacteriol,2007.189(4): p.1219-30.
48. Shemesh, M., et al., DNA-microarrays identification of Streptococcus mutans genesassociated with biofilm thickness. BMC Microbiol,2008.8: p.236.
49. Wen, Z.T., et al., Transcriptome analysis of LuxS-deficient Streptococcus mutans grownin biofilms. Mol Oral Microbiol,2011.26(1): p.2-18.
50. McNab, R., et al., LuxS-based signaling in Streptococcus gordonii: autoinducer2controls carbohydrate metabolism and biofilm formation with Porphyromonasgingivalis. J Bacteriol,2003.185(1): p.274-84.
51. Vidal, J.E., et al., The LuxS-dependent quorum-sensing system regulates early biofilmformation by Streptococcus pneumoniae strain D39. Infect Immun,2011.79(10): p.4050-60.
52. Bodor, A.M., et al., The luxS mutation causes loosely-bound biofilms in Shewanellaoneidensis. BMC Res Notes,2011.4: p.180.
53. De Araujo, C., et al., Quorum sensing affects biofilm formation throughlipopolysaccharide synthesis in Klebsiella pneumoniae. Res Microbiol,2010.161(7): p.595-603.
54. Jesudhasan, P.R., et al., Transcriptome analysis of genes controlled byluxS/autoinducer-2in Salmonella enterica serovar Typhimurium. Foodborne PathogDis,2009.7(4): p.399-410.
55. Ahmed, N.A., F.C. Petersen, and A.A. Scheie, Biofilm formation and autoinducer-2signaling in Streptococcus intermedius: role of thermal and pH factors. Oral MicrobiolImmunol,2008.23(6): p.492-7.
56. Armbruster, C.E., et al., LuxS promotes biofilm maturation and persistence ofnontypeable haemophilus influenzae in vivo via modulation of lipooligosaccharides onthe bacterial surface. Infect Immun,2009.77(9): p.4081-91.
57. Bobrov, A.G., et al., Functional quorum sensing systems affect biofilm formation andprotein expression in Yersinia pestis. Adv Exp Med Biol,2007.603: p.178-91.
58. Li, J., et al., Quorum sensing in Escherichia coli is signaled by AI-2/LsrR: effects onsmall RNA and biofilm architecture. J Bacteriol,2007.189(16): p.6011-20.
59. Reeser, R.J., et al., Characterization of Campylobacter jejuni biofilms under definedgrowth conditions. Appl Environ Microbiol,2007.73(6): p.1908-13.
60. Li, L., et al., Analysis on Actinobacillus pleuropneumoniae LuxS regulated genesreveals pleiotropic roles of LuxS/AI-2on biofilm formation, adhesion ability and ironmetabolism. Microb Pathog,2011.50(6): p.293-302.
61. Kozlova, E.V., et al., Mutation in the S-ribosylhomocysteinase (luxS) gene involved inquorum sensing affects biofilm formation and virulence in a clinical isolate ofAeromonas hydrophila. Microb Pathog,2008.45(5-6): p.343-54.
62. Joyce, E.A., et al., LuxS is required for persistent pneumococcal carriage andexpression of virulence and biosynthesis genes. Infect Immun,2004.72(5): p.2964-75.
63. Jones, M.B., et al., Role of luxS in Bacillus anthracis growth and virulence factorexpression. Virulence,2010.1(2): p.72-83.
64. Zhu, J., et al., Involvement of quorum sensing and heat-stable enterotoxin a in celldamage caused by a porcine enterotoxigenic Escherichia coli strain. InfectImmun,2011.79(4): p.1688-95.
65. Ohtani, K.,[Analysis of regulatory system of toxin production by cell-cell signaling inClostridium perfringens]. Nihon Saikingaku Zasshi,2011.66(2-3): p.169-74.
66. Stroeher, U.H., et al., Mutation of luxS of Streptococcus pneumoniae affects virulence ina mouse model. Infect Immun,2003.71(6): p.3206-12.
67. Coulthurst, S.J., C.L. Kurz, and G.P. Salmond, luxS mutants of Serratia defective inautoinducer-2-dependent 'quorum sensing' show strain-dependent impacts on virulenceand production of carbapenem and prodigiosin. Microbiology,2004.150(Pt6): p.1901-10.
68. Kim, S.Y., et al., Regulation of Vibrio vulnificus virulence by the LuxS quorum-sensingsystem. Mol Microbiol,2003.48(6): p.1647-64.
69. Krin, E., et al., Pleiotropic role of quorum-sensing autoinducer2in Photorhabdusluminescens. Appl Environ Microbiol,2006.72(10): p.6439-51.
70. Winzer, K., et al., Role of Neisseria meningitidis luxS in cell-to-cell signaling andbacteremic infection. Infect Immun,2002.70(4): p.2245-8.
71. Xu, L., et al., Role of the luxS quorum-sensing system in biofilm formation andvirulence of Staphylococcus epidermidis. Infect Immun,2006.74(1): p.488-96.
72. Daines, D.A., et al., Haemophilus influenzae luxS mutants form a biofilm and haveincreased virulence. Microb Pathog,2005.39(3): p.87-96.
73. Doherty, N., et al., Functional analysis of luxS in Staphylococcus aureus reveals a rolein metabolism but not quorum sensing. J Bacteriol,2006.188(8): p.2885-97.
74. Rader, B.A., et al., The quorum-sensing molecule autoinducer2regulates motility andflagellar morphogenesis in Helicobacter pylori. J Bacteriol,2007.189(17): p.6109-17.
75. Jeon, B., et al., Effects of quorum sensing on flaA transcription and autoagglutinationin Campylobacter jejuni. Microbiol Immunol,2003.47(11): p.833-9.
76. Kim, C.M. and S.H. Shin, Modulation of iron-uptake systems by a mutation of luxSencoding an autoinducer-2synthase in Vibrio vulnificus. Biol Pharm Bull,2011.34(5):p.632-7.
77. James, C.E., et al., LuxS involvement in the regulation of genes coding for hemin andiron acquisition systems in Porphyromonas gingivalis. Infect Immun,2006.74(7): p.3834-44.
78. Chung, W.O., et al., Signaling system in Porphyromonas gingivalis based on a LuxSprotein. J Bacteriol,2001.183(13): p.3903-9.
79. Ahmed, N.A., F.C. Petersen, and A.A. Scheie, AI-2quorum sensing affects antibioticsusceptibility in Streptococcus anginosus. J Antimicrob Chemother,2007.60(1): p.49-53.
80. Yu, D.N., et al.,[Function of luxS gene in sulfurmetabolism of Streptococcus mutans].Zhonghua Kou Qiang Yi Xue Za Zhi,2011.46(4): p.210-3.
81. Siller, M., et al., Functional analysis of the group A streptococcal luxS/AI-2system inmetabolism, adaptation to stress and interaction with host cells. BMC Microbiol,2008.8: p.188.
82. Learman, D.R., et al., Involvement of Shewanella oneidensis MR-1LuxS in biofilmdevelopment and sulfur metabolism. Appl Environ Microbiol,2009.75(5): p.1301-7.
83. Lebeer, S., et al., Functional analysis of luxS in the probiotic strain Lactobacillusrhamnosus GG reveals a central metabolic role important for growth and biofilmformation. J Bacteriol,2007.189(3): p.860-71.
84. Rezzonico, F. and B. Duffy, The role of luxS in the fire blight pathogen Erwiniaamylovora is limited to metabolism and does not involve quorum sensing. Mol PlantMicrobe Interact,2007.20(10): p.1284-97.
85. Tannock, G.W., et al., Ecological behavior of Lactobacillus reuteri100-23is affected bymutation of the luxS gene. Appl Environ Microbiol,2005.71(12): p.8419-25.
86. Wilson, C.M., et al., Transcriptional and metabolomic consequences of luxSinactivation reveal a metabolic rather than quorum sensing role for LuxS inLactobacillus reuteri100-23. J Bacteriol.2012
87. Heurlier, K., et al., Growth deficiencies of Neisseria meningitidis pfs and luxS mutantsare not due to inactivation of quorum sensing. J Bacteriol,2009.191(4): p.1293-302.
88. Malladi, V.L., et al., Inhibition of LuxS by S-ribosylhomocysteine analogues containinga [4-aza]ribose ring. Bioorg Med Chem,2011.19(18): p.5507-19.
89. Lonn-Stensrud, J., et al., Synthetic bromated furanone inhibits autoinducer-2-mediatedcommunication and biofilm formation in oral streptococci. Oral Microbiol Immunol,
2007.22(5): p.340-6.
90. Ren, D., J.J. Sims, and T.K. Wood, Inhibition of biofilm formation and swarming ofEscherichia coli by (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone.Environ Microbiol,2001.3(11): p.731-6.
91. Ren, D., et al., Differential gene expression shows natural brominated furanonesinterfere with the autoinducer-2bacterial signaling system of Escherichia coli.Biotechnol Bioeng,2004.88(5): p.630-42.
2. Huang, Y.T., et al., Streptococcus suis infection. J Microbiol Immunol Infect,2005.38(5): p.306-13.
3. Wisselink, H.J., J.J. Joosten, and H.E. Smith, Multiplex PCR assays for simultaneousdetection of six major serotypes and two virulence-associated phenotypes ofStreptococcus suis in tonsillar specimens from pigs. J Clin Microbiol,2002.40(8): p.2922-9.
4. Vecht, U., et al., Differences in virulence between two strains of Streptococcus suis typeII after experimentally induced infection of newborn germ-free pigs. Am J Vet Res,
1989.50(7): p.1037-43.
5. Quessy, S., et al., Discrimination of virulent and avirulent Streptococcus suis capsulartype2isolates from different geographical origins. Infect Immun,1995.63(5): p.1975-9.
6. Silva, L.M., et al., Virulence-associated gene profiling of Streptococcus suis isolates byPCR. Vet Microbiol,2006.115(1-3): p.117-27.
7. Gottschalk, M., et al., Streptococcus suis: a new emerging or an old neglected zoonoticpathogen? Future Microbiol，2010.5(3): p.371-91.
8. Perch, B., P. Kristjansen, and K. Skadhauge, Group R streptococci pathogenic for man.Two cases of meningitis and one fatal case of sepsis. Acta Pathol Microbiol Scand,
1968.74(1): p.69-76.
9. Vilaichone, R.K., et al., Streptococcus suis infection in Thailand. J Med Assoc Thai,
2002.85Suppl1: p. S109-17.
10. Tang, J., et al., Streptococcal toxic shock syndrome caused by Streptococcus suisserotype2. PLoS Med,2006.3(5): p. e151.
11.唐家琪，朱进，郭恒彬，等,一起猪链球菌暴发流行的流行病学及病原学研究[J].解放军预防医学杂志,.2000,18(4): p.4.
12. Wei, Z., et al., Characterization of Streptococcus suis isolates from the diseased pigs inChina between2003and2007. Vet Microbiol,2009.137(1-2): p.196-201.
13. Ma, E., et al., Streptococcus suis infection in Hong Kong: an emerging infectiousdisease? Epidemiol Infect,2008.136(12): p.1691-7.
14. Hui, A.C., et al., Bacterial meningitis in Hong Kong:10-years' experience. Clin NeurolNeurosurg,2005.107(5): p.366-70.
15. Mai, N.T., et al., Streptococcus suis meningitis in adults in Vietnam. Clin Infect Dis,
2008.46(5): p.659-67.
16. Ip, M., et al., Streptococcus suis in Hong Kong. Diagn Microbiol Infect Dis,2007.57(1): p.15-20.
17. Fittipaldi, N., et al., Virulence factors involved in the pathogenesis of the infectioncaused by the swine pathogen and zoonotic agent Streptococcus suis. FutureMicrobiol,2012.7(2): p.259-79.
18. Smith, H.E., et al., Mutants of Streptococcus suis types1and2impaired in expressionof muramidase-released protein and extracellular protein induce disease in newborngermfree pigs. Infect Immun,1996.64(10): p.4409-12.
19. Wisselink, H.J., et al., Distribution of capsular types and production ofmuramidase-released protein (MRP) and extracellular factor (EF) of Streptococcussuis strains isolated from diseased pigs in seven European countries. Vet Microbiol,
2000.74(3): p.237-48.
20. Allen, A.G., et al., Generation and characterization of a defined mutant ofStreptococcus suis lacking suilysin. Infect Immun,2001.69(4): p.2732-5.
21. Lun, S., et al., Role of suilysin in pathogenesis of Streptococcus suis capsular serotype
2. Microb Pathog,2003.34(1): p.27-37.
22. Smith, H.E., et al., Identification and characterization of the cps locus of Streptococcussuis serotype2: the capsule protects against phagocytosis and is an importantvirulence factor. Infect Immun,1999.67(4): p.1750-6.
23. Gottschalk, M. and M. Segura, The pathogenesis of the meningitis caused byStreptococcus suis: the unresolved questions. Vet Microbiol,2000.76(3): p.259-72.
24. Li, J., et al., The two-component regulatory system CiaRH contributes to the virulenceof Streptococcus suis2. Vet Microbiol,2010.148(1): p.99-104.
25. Pan, X., et al., The orphan response regulator CovR: a globally negative modulator ofvirulence in Streptococcus suis serotype2. J Bacteriol,2009.191(8): p.2601-12.
26. Chen, C., et al., A glimpse of streptococcal toxic shock syndrome from comparativegenomics of S. suis2Chinese isolates. PLoS One,2007.2(3): p. e315.
27. Li, M., et al., SalK/SalR, a two-component signal transduction system, is essential forfull virulence of highly invasive Streptococcus suis serotype2. PLoS One,2008.3(5): p.e2080.
28. Li, M., et al., GI-type T4SS-mediated horizontal transfer of the89K pathogenicityisland in epidemic Streptococcus suis serotype2. Mol Microbiol,2011.79(6): p.1670-83.
29. Zheng, F., et al., Contribution of the Rgg transcription regulator to metabolism andvirulence of Streptococcus suis serotype2. Infect Immun,2011.79(3): p.1319-28.
30. Sun, J., et al., Is autoinducer-2a universal signal for interspecies communication: acomparative genomic and phylogenetic analysis of the synthesis and signaltransduction pathways. BMC Evol Biol,2004.4: p.36.
31. Bassler, B.L. and R. Losick, Bacterially speaking. Cell,2006.125(2): p.237-46.
32. Henke, J.M. and B.L. Bassler, Three parallel quorum-sensing systems regulate geneexpression in Vibrio harveyi. J Bacteriol,2004.186(20): p.6902-14.
33. Taga, M.E., S.T. Miller, and B.L. Bassler, Lsr-mediated transport and processing ofAI-2in Salmonella typhimurium. Mol Microbiol,2003.50(4): p.1411-27.
34. Xue, T., et al., LsrR-binding site recognition and regulatory characteristics inEscherichia coli AI-2quorum sensing. Cell Res,2009.19(11): p.1258-68.
35. Winzer, K., K.R. Hardie, and P. Williams, Bacterial cell-to-cell communication: sorry,can't talk now-gone to lunch! Curr Opin Microbiol,2002.5(2): p.216-22.
36. Heurlier, K., et al., Growth deficiencies of Neisseria meningitidis pfs and luxS mutantsare not due to inactivation of quorum sensing. J Bacteriol,2009.191(4): p.1293-302.
37. McNab, R., et al., LuxS-based signaling in Streptococcus gordonii: autoinducer2controls carbohydrate metabolism and biofilm formation with Porphyromonasgingivalis. J Bacteriol,2003.185(1): p.274-84.
38. Wen, Z.T., et al., Transcriptome analysis of LuxS-deficient Streptococcus mutans grownin biofilms. Mol Oral Microbiol,2011.26(1): p.2-18.
39. Jesudhasan, P.R., et al., Transcriptome analysis of genes controlled byluxS/autoinducer-2in Salmonella enterica serovar Typhimurium. Foodborne PathogDis,2009.7(4): p.399-410.
40. Novak, E.A., et al., Autoinducer-2and QseC control biofilm formation and in vivovirulence of Aggregatibacter actinomycetemcomitans. Infect Immun,2010.78(7): p.2919-26.
41. Reeser, R.J., et al., Characterization of Campylobacter jejuni biofilms under definedgrowth conditions. Appl Environ Microbiol,2007.73(6): p.1908-13.
42. Bobrov, A.G., et al., Functional quorum sensing systems affect biofilm formation andprotein expression in Yersinia pestis. Adv Exp Med Biol,2007.603: p.178-91.
43. De Araujo, C., et al., Quorum sensing affects biofilm formation throughlipopolysaccharide synthesis in Klebsiella pneumoniae. Res Microbiol,2010.161(7): p.595-603.
44. Stroeher, U.H., et al., Mutation of luxS of Streptococcus pneumoniae affects virulencein a mouse model. Infect Immun,2003.71(6): p.3206-12.
45. Plummer, P., et al., Critical role of LuxS in the virulence of Campylobacter jejuni in aguinea pig model of abortion. Infect Immun,2011.80(2): p.585-93.
46. Osaki, T., et al., Mutation of luxS affects motility and infectivity of Helicobacter pyloriin gastric mucosa of a Mongolian gerbil model. J Med Microbiol,2006.55(Pt11): p.1477-85.
47. Kim, S.Y., et al., Regulation of Vibrio vulnificus virulence by the LuxS quorum-sensingsystem. Mol Microbiol,2003.48(6): p.1647-64.
48. Winzer, K., et al., Role of Neisseria meningitidis luxS in cell-to-cell signaling andbacteremic infection. Infect Immun,2002.70(4): p.2245-8.
49. Choi, J., D. Shin, and S. Ryu, Implication of quorum sensing in Salmonella entericaserovar typhimurium virulence: the luxS gene is necessary for expression of genes inpathogenicity island1. Infect Immun,2007.75(10): p.4885-90.
50. Coulthurst, S.J., C.L. Kurz, and G.P. Salmond, luxS mutants of Serratia defective inautoinducer-2-dependent 'quorum sensing' show strain-dependent impacts on virulenceand production of carbapenem and prodigiosin. Microbiology,2004.150(Pt6): p.1901-10.
51. Xu, L., et al., Role of the luxS quorum-sensing system in biofilm formation andvirulence of Staphylococcus epidermidis. Infect Immun,2006.74(1): p.488-96.
52. Daines, D.A., et al., Haemophilus influenzae luxS mutants form a biofilm and haveincreased virulence. Microb Pathog,2005.39(3): p.87-96.
53. Ahmed, N.A., F.C. Petersen, and A.A. Scheie, AI-2quorum sensing affects antibioticsusceptibility in Streptococcus anginosus. J Antimicrob Chemother,2007.60(1): p.49-53.
54. Jeon, B., et al., Effects of quorum sensing on flaA transcription and autoagglutinationin Campylobacter jejuni. Microbiol Immunol,2003.47(11): p.833-9.
55. Rader, B.A., et al., The quorum-sensing molecule autoinducer2regulates motility andflagellar morphogenesis in Helicobacter pylori. J Bacteriol,2007.189(17): p.6109-17.
56. DeLisa, M.P., et al., DNA microarray-based identification of genes controlled byautoinducer2-stimulated quorum sensing in Escherichia coli. J Bacteriol,2001.183(18): p.5239-47.
57. Li, M., et al., AI-2-dependent gene regulation in Staphylococcus epidermidis. BMCMicrobiol,2008.8: p.4.
58. Sperandio, V., et al., Quorum sensing is a global regulatory mechanism inenterohemorrhagic Escherichia coli O157:H7. J Bacteriol,2001.183(17): p.5187-97.
59. Malladi, V.L., et al., Inhibition of LuxS by S-ribosylhomocysteine analogues containinga [4-aza]ribose ring. Bioorg Med Chem,2011.19(18): p.5507-19.
60. Hilgers, M.T. and M.L. Ludwig, Crystal structure of the quorum-sensing protein LuxSreveals a catalytic metal site. Proc Natl Acad Sci U S A,2001.98(20): p.11169-74.
61. Plummer, P., et al., Identification of a key amino acid of LuxS involved in AI-2production in Campylobacter jejuni. PLoS One,2011.6(1): p. e15876.
62. Pei, D. and J. Zhu, Mechanism of action of S-ribosylhomocysteinase (LuxS). Curr OpinChem Biol,2004.8(5): p.492-7.
63. Zhu, J., R. Patel, and D. Pei, Catalytic mechanism of S-ribosylhomocysteinase (LuxS):stereochemical course and kinetic isotope effect of proton transfer reactions.Biochemistry,2004.43(31): p.10166-72.
64. Das, S.K., et al., Cloning, purification, crystallization and preliminary crystallographicanalysis of Bacillus subtilis LuxS. Acta Crystallogr D Biol Crystallogr,2001.57(Pt9):p.1324-5.
65. Lewis, H.A., et al., A structural genomics approach to the study of quorum sensing:crystal structures of three LuxS orthologs. Structure,2001.9(6): p.527-37.
66. Rajan, R., et al., Crystal structure of S-ribosylhomocysteinase (LuxS) in complex with acatalytic2-ketone intermediate. Biochemistry,2005.44(10): p.3745-53.
67. Li, H., et al., Crystallization and preliminary X-ray analysis ofS-ribosylhomocysteinase from Streptococcus mutans. Acta Crystallogr Sect F StructBiol Cryst Commun,2012.68(Pt2): p.199-202.
68. Bhattacharyya, M. and S. Vishveshwara, Elucidation of the conformational free energylandscape in H.pylori LuxS and its implications to catalysis. BMC Struct Biol,2010.10:p.27.
69. Bhattacharyya, M. and S. Vishveshwara, Functional correlation of bacterial LuxS withtheir quaternary associations: interface analysis of the structure networks. BMC StructBiol,2009.9: p.8.
70. De Keersmaecker S C, S.K., Vanderleyden J, Let LuxS speak up in AI-2signaling.Trends Microbiol,2006.14(3): p.１１４－１１９.
71. Takamatsu, D., M. Osaki, and T. Sekizaki, Construction and characterization ofStreptococcus suis-Escherichia coli shuttle cloning vectors. Plasmid,2001.45(2): p.101-13.
72. Macrina, F.L., et al., Novel shuttle plasmid vehicles for Escherichia-Streptococcustransgeneric cloning. Gene,1983.25(1): p.145-50.
73. Smith, H.E., et al., High-efficiency transformation and gene inactivation inStreptococcus suis type2. Microbiology,1995.141(Pt1): p.181-8.
74. Ota, K., et al., Expression of histone acetyltransferases was down-regulated inpoly(ADP-ribose) polymerase-1-deficient murine cells. Biochem Biophys ResCommun,2003.310(2): p.312-7.
75. Plasterk, R.H., RNA silencing: the genome's immune system. Science,2002.296(5571):p.1263-5.
76. Court, D.L., J.A. Sawitzke, and L.C. Thomason, Genetic engineering usinghomologous recombination. Annu Rev Genet,2002.36: p.361-88.
77.李明，胡福泉，王长军，等.,环境条件对猪链球菌2型电转化效率的影响.中国人兽共患病学报,,2007.23(8): p.760-3.
78. Iacono, V.J., et al., Selective antibacterial properties of lysozyme for oralmicroorganisms. Infect Immun,1980.29(2): p.623-32.
79. Turovskiy, Y., et al., Quorum sensing: fact, fiction, and everything in between. AdvAppl Microbiol,2007.62: p.191-234.
80. Bassler, B.L., et al., Intercellular signalling in Vibrio harveyi: sequence and function ofgenes regulating expression of luminescence. Mol Microbiol,1993.9(4): p.773-86.
81. Tischler, A.S., J.C. Riseberg, and V. Cherington, Multiple mitogenic signallingpathways in chromaffin cells: a model for cell cycle regulation in the nervous system.Neurosci Lett,1994.168(1-2): p.181-4.
82. Surette, M.G., M.B. Miller, and B.L. Bassler, Quorum sensing in Escherichia coli,Salmonella typhimurium, and Vibrio harveyi: a new family of genes responsible forautoinducer production. Proc Natl Acad Sci U S A,1999.96(4): p.1639-44.
83. Chen, X., et al., Structural identification of a bacterial quorum-sensing signalcontaining boron. Nature,2002.415(6871): p.545-9.
84. Miller, S.T., et al., Salmonella typhimurium recognizes a chemically distinct form of thebacterial quorum-sensing signal AI-2. Mol Cell,2004.15(5): p.677-87.
85. Surette, M.G. and B.L. Bassler, Quorum sensing in Escherichia coli and Salmonellatyphimurium. Proc Natl Acad Sci U S A,1998.95(12): p.7046-50.
86. Lyon, W.R., et al., Mutation of luxS affects growth and virulence factor expression inStreptococcus pyogenes. Mol Microbiol,2001.42(1): p.145-57.
87. Yuan, L., J.D. Hillman, and A. Progulske-Fox, Microarray analysis ofquorum-sensing-regulated genes in Porphyromonas gingivalis. Infect Immun,2005.73(7): p.4146-54.
88.欧阳谦，马文丽，刘翠华，石嵘，郑文岭, ΔluxS的表型分析及其在GBS毒力调控机制研究中的应用.南方医科大学学报,2006.26(1): p.117-121.
89. Tavender, T.J., et al., LuxS-independent formation of AI-2from ribulose-5-phosphate.BMC Microbiol,2008.8: p.98.
90. Jacques, M., et al., Ultrastructural study of surface components of Streptococcus suis. JBacteriol,1990.172(6): p.2833-8.
91. Verneuil, N., et al., Contribution of a PerR-like regulator to the oxidative-stressresponse and virulence of Enterococcus faecalis. Microbiology,2005.151(Pt12): p.3997-4004.
92. Charland, N., et al., Role of capsular sialic acid in virulence and resistance tophagocytosis of Streptococcus suis capsular type2. FEMS Immunol Med Microbiol,
1996.14(4): p.195-203.
93. Jones, M.B. and M.J. Blaser, Detection of a luxS-signaling molecule in Bacillusanthracis. Infect Immun,2003.71(7): p.3914-9.
94. Jordan, D.M., et al., Colonization of gnotobiotic piglets by a luxS mutant strain ofEscherichia coli O157:H7. Infect Immun,2005.73(2): p.1214-6.
95. Wen, Z.T. and R.A. Burne, LuxS-mediated signaling in Streptococcus mutans isinvolved in regulation of acid and oxidative stress tolerance and biofilm formation. JBacteriol,2004.186(9): p.2682-91.
96. Krin, E., et al., Pleiotropic role of quorum-sensing autoinducer2in Photorhabdusluminescens. Appl Environ Microbiol,2006.72(10): p.6439-51.
97. Unnikrishnan, M., J. Cohen, and S. Sriskandan, Growth-phase-dependent expression ofvirulence factors in an M1T1clinical isolate of Streptococcus pyogenes. Infect Immun,
1999.67(10): p.5495-9.
98. Maganti, S., et al., The role of sialic acid in opsonin-dependent andopsonin-independent adhesion of Listeria monocytogenes to murine peritonealmacrophages. Infect Immun,1998.66(2): p.620-6.
99. Sztajer, H., et al., Autoinducer-2-regulated genes in Streptococcus mutans UA159andglobal metabolic effect of the luxS mutation. J Bacteriol,2008.190(1): p.401-15.
100.Schmittgen, T.D. and B.A. Zakrajsek, Effect of experimental treatment onhousekeeping gene expression: validation by real-time, quantitative RT-PCR. JBiochem Biophys Methods,2000.46(1-2): p.69-81.
101.Livak, K.J. and T.D. Schmittgen, Analysis of relative gene expression data usingreal-time quantitative PCR and the2(-Delta Delta C(T)) Method. Methods,2001.25(4):p.402-8.
102.Geissler, B., D. Shiomi, and W. Margolin, The ftsA*gain-of-function allele ofEscherichia coli and its effects on the stability and dynamics of the Z ring.Microbiology,2007.153(Pt3): p.814-25.
103.Divakaruni, A.V., et al., The cell shape proteins MreB and MreC control cellmorphogenesis by positioning cell wall synthetic complexes. Mol Microbiol,2007.66(1): p.174-88.
104.Kumsta, C. and U. Jakob, Redox-regulated chaperones. Biochemistry,2009.48(22): p.4666-76.
105.Schauer, K., et al., The Helicobacter pylori GroES cochaperonin HspA functions as aspecialized nickel chaperone and sequestration protein through its unique C-terminalextension. J Bacteriol,2010.192(5): p.1231-7.
106.Ishikawa, M., et al., Cloning and characterization of grpE in Acetobacter pasteurianusNBRC3283. J Biosci Bioeng,2012.109(1): p.25-31.
107.Bolean, M., et al., Photodynamic therapy with rose bengal induces GroEL expressionin Streptococcus mutans. Photomed Laser Surg,2010.28Suppl1: p. S79-84.
108.Di Pasqua, R., et al., Changes in the proteome of Salmonella enterica serovarThompson as stress adaptation to sublethal concentrations of thymol. Proteomics,2010.10(5): p.1040-9.
109.Hickey, T.B., et al., Mycobacterium tuberculosis employs Cpn60.2as an adhesin thatbinds CD43on the macrophage surface. Cell Microbiol,2010.12(11): p.1634-47.
110. Suokko, A., et al., ClpL is essential for induction of thermotolerance and is potentiallypart of the HrcA regulon in Lactobacillus gasseri. Proteomics,2008.8(5): p.1029-41.
111. Kajfasz, J.K., et al., Role of Clp proteins in expression of virulence properties ofStreptococcus mutans. J Bacteriol,2009.191(7): p.2060-8.
112. Tu le, N., et al., Modulation of adherence, invasion, and tumor necrosis factor alphasecretion during the early stages of infection by Streptococcus pneumoniae ClpL. InfectImmun,2007.75(6): p.2996-3005.
113. Zhang, Q., et al., Contribution of ClpE to virulence of Streptococcus pneumoniae. CanJ Microbiol,2009.55(10): p.1187-94.
114. Joseph, B., et al., Identification of Listeria monocytogenes genes contributing tointracellular replication by expression profiling and mutant screening. J Bacteriol,
2006.188(2): p.556-68.
115. Lamarche, M.G., et al., Inactivation of the pst system reduces the virulence of an avianpathogenic Escherichia coli O78strain. Infect Immun,2005.73(7): p.4138-45.
116. Daigle, F., J.M. Fairbrother, and J. Harel, Identification of a mutation in the pst-phoUoperon that reduces pathogenicity of an Escherichia coli strain causing septicemia inpigs. Infect Immun,1995.63(12): p.4924-7.
117. de Greeff, A., et al., Response regulator important in pathogenesis of Streptococcussuis serotype2. Microb Pathog,2002.33(4): p.185-92.
118. Kovacikova, G. and K. Skorupski, Regulation of virulence gene expression in Vibriocholerae by quorum sensing: HapR functions at the aphA promoter. Mol Microbiol,
2002.46(4): p.1135-47.
119. Iyer, R., N.S. Baliga, and A. Camilli, Catabolite control protein A (CcpA) contributesto virulence and regulation of sugar metabolism in Streptococcus pneumoniae. JBacteriol,2005.187(24): p.8340-9.
120.Shelburne, S.A.,3rd, et al., A direct link between carbohydrate utilization andvirulence in the major human pathogen group A Streptococcus. Proc Natl Acad Sci U SA,2008.105(5): p.1698-703.
121.Kietzman, C.C. and M.G. Caparon, CcpA and LacD.1affect temporal regulation ofStreptococcus pyogenes virulence genes. Infect Immun,2009.78(1): p.241-52.
122.Willenborg, J., et al., Role of glucose and CcpA in capsule expression and virulence ofStreptococcus suis. Microbiology,2011.157(Pt6): p.1823-33.
123.Charland, N., et al., Streptococcus suis serotype2mutants deficient in capsularexpression. Microbiology,1998.144(Pt2): p.325-32.
124.Wilson, T.L., et al., A novel signature-tagged mutagenesis system for Streptococcussuis serotype2. Vet Microbiol,2007.122(1-2): p.135-45.
125.董瑞萍，王长军，程功，李明，王晶，潘秀珍，唐家琪,猪链球菌2型唾液酸合成酶neuB基因敲除突变株的构建及其生物学特性.微生物学通报，2009.36(2): p.238-244.
126.Vecht, U., et al., Identification of two proteins associated with virulence ofStreptococcus suis type2. Infect Immun,1991.59(9): p.3156-62.
127.Terao, Y., et al., Novel laminin-binding protein of Streptococcus pyogenes, Lbp, isinvolved in adhesion to epithelial cells. Infect Immun,2002.70(2): p.993-7.
128.王伟,肖.明.,微生物嗜铁素介导的铁摄取.生物学杂志,2005.22(4): p.4.
129.Kim, C.M. and S.H. Shin, Modulation of iron-uptake systems by a mutation of luxSencoding an autoinducer-2synthase in Vibrio vulnificus. Biol Pharm Bull,2011.34(5):p.632-7.
130.James, C.E., et al., LuxS involvement in the regulation of genes coding for hemin andiron acquisition systems in Porphyromonas gingivalis. Infect Immun,2006.74(7): p.3834-44.
131.Chung, W.O., et al., Signaling system in Porphyromonas gingivalis based on a LuxSprotein. J Bacteriol,2001.183(13): p.3903-9.
132.Walters, M. and V. Sperandio, Autoinducer3and epinephrine signaling in the kineticsof locus of enterocyte effacement gene expression in enterohemorrhagic Escherichiacoli. Infect Immun,2006.74(10): p.5445-55.
1. Fuqua, W.C., S.C. Winans, and E.P. Greenberg, Quorum sensing in bacteria: theLuxR-LuxI family of cell density-responsive transcriptional regulators. J Bacteriol,
1994.176(2): p.269-75.
2. Xavier, K.B. and B.L. Bassler, LuxS quorum sensing: more than just a numbers game.Curr Opin Microbiol,2003.6(2): p.191-7.
3. Plummer, P., et al., Critical role of LuxS in the virulence of Campylobacter jejuni in aguinea pig model of abortion. Infect Immun,2011.80(2): p.585-93.
4. Choi, J., D. Shin, and S. Ryu, Implication of quorum sensing in Salmonella entericaserovar typhimurium virulence: the luxS gene is necessary for expression of genes inpathogenicity island1. Infect Immun,2007.75(10): p.4885-90.
5. Osaki, T., et al., Mutation of luxS affects motility and infectivity of Helicobacter pyloriin gastric mucosa of a Mongolian gerbil model. J Med Microbiol,2006.55(Pt11): p.1477-85.
6. Hao, Y., et al., Identification and characterization of new LuxR/LuxI-type quorumsensing systems from metagenomic libraries. Environ Microbiol,2009.12(1): p.105-17.
7. Thoendel, M. and A.R. Horswill, Identification of Staphylococcus aureus AgrD residuesrequired for autoinducing peptide biosynthesis. J Biol Chem,2009.284(33): p.21828-38.
8. Sun, J., et al., Is autoinducer-2a universal signal for interspecies communication: acomparative genomic and phylogenetic analysis of the synthesis and signaltransduction pathways. BMC Evol Biol,2004.4: p.36.
9. Bassler, B.L. and R. Losick, Bacterially speaking. Cell,2006.125(2): p.237-46.
10. Khajanchi, B.K., et al., The two-component QseBC signalling system regulates in vitroand in vivo virulence of Aeromonas hydrophila. Microbiology,2011.158(Pt1): p.259-71.
11. Walters, M., M.P. Sircili, and V. Sperandio, AI-3synthesis is not dependent on luxS inEscherichia coli. J Bacteriol,2006.188(16): p.5668-81.
12. Bassler, B.L., et al., Intercellular signalling in Vibrio harveyi: sequence and function ofgenes regulating expression of luminescence. Mol Microbiol,1993.9(4): p.773-86.
13. Tischler, A.S., J.C. Riseberg, and V. Cherington, Multiple mitogenic signallingpathways in chromaffin cells: a model for cell cycle regulation in the nervous system.Neurosci Lett,1994.168(1-2): p.181-4.
14. Bassler, B.L., E.P. Greenberg, and A.M. Stevens, Cross-species induction ofluminescence in the quorum-sensing bacterium Vibrio harveyi. J Bacteriol,1997.179(12): p.4043-5.
15. Surette, M.G., M.B. Miller, and B.L. Bassler, Quorum sensing in Escherichia coli,Salmonella typhimurium, and Vibrio harveyi: a new family of genes responsible forautoinducer production. Proc Natl Acad Sci U S A,1999.96(4): p.1639-44.
16. Winzer, K., K.R. Hardie, and P. Williams, Bacterial cell-to-cell communication: sorry,can't talk now-gone to lunch! Curr Opin Microbiol,2002.5(2): p.216-22.
17. Winzer, K., et al., LuxS: its role in central metabolism and the in vitro synthesis of4-hydroxy-5-methyl-3(2H)-furanone. Microbiology,2002.148(Pt4): p.909-22.
18. Sauer, U., et al., The soluble and membrane-bound transhydrogenases UdhA and PntABhave divergent functions in NADPH metabolism of Escherichia coli. J Biol Chem,2004.279(8): p.6613-9.
19. Tavender, T.J., et al., LuxS-independent formation of AI-2from ribulose-5-phosphate.BMC Microbiol,2008.8: p.98.
20. Lyon, W.R., et al., Mutation of luxS affects growth and virulence factor expression inStreptococcus pyogenes. Mol Microbiol,2001.42(1): p.145-57.
21. Cao, M., et al., Functional definition of LuxS, an autoinducer-2(AI-2) synthase and itsrole in full virulence of Streptococcus suis serotype2. J Microbiol,2011.49(6): p.1000-11.
22. Vendeville, A., et al., Making 'sense' of metabolism: autoinducer-2, LuxS andpathogenic bacteria. Nat Rev Microbiol,2005.3(5): p.383-96.
23. Chen, X., et al., Structural identification of a bacterial quorum-sensing signalcontaining boron. Nature,2002.415(6871): p.545-9.
24. Miller, S.T., et al., Salmonella typhimurium recognizes a chemically distinct form of thebacterial quorum-sensing signal AI-2. Mol Cell,2004.15(5): p.677-87.
25. Das, S.K., et al., Cloning, purification, crystallization and preliminary crystallographicanalysis of Bacillus subtilis LuxS. Acta Crystallogr D Biol Crystallogr,2001.57(Pt9): p.1324-5.
26. Hilgers, M.T. and M.L. Ludwig, Crystal structure of the quorum-sensing protein LuxSreveals a catalytic metal site. Proc Natl Acad Sci U S A,2001.98(20): p.11169-74.
27. Lewis, H.A., et al., A structural genomics approach to the study of quorum sensing:crystal structures of three LuxS orthologs. Structure,2001.9(6): p.527-37.
28. Rajan, R., et al., Crystal structure of S-ribosylhomocysteinase (LuxS) in complex with acatalytic2-ketone intermediate. Biochemistry,2005.44(10): p.3745-53.
29. Li, H., et al., Crystallization and preliminary X-ray analysis of S-ribosylhomocysteinasefrom Streptococcus mutans. Acta Crystallogr Sect F Struct Biol Cryst Commun,2012.68(Pt2): p.199-202.
30. Bhattacharyya, M. and S. Vishveshwara, Elucidation of the conformational free energylandscape in H.pylori LuxS and its implications to catalysis. BMC Struct Biol,2010.10:p.27.
31. De Keersmaecker S C, S.K., Vanderleyden J, Let LuxS speak up in AI-2signaling.Trends Microbiol,2006.14(3): p.１１４－１１９.
32. Pei, D. and J. Zhu, Mechanism of action of S-ribosylhomocysteinase (LuxS). Curr OpinChem Biol,2004.8(5): p.492-7.
33. Zhu, J., R. Patel, and D. Pei, Catalytic mechanism of S-ribosylhomocysteinase (LuxS):stereochemical course and kinetic isotope effect of proton transfer reactions.Biochemistry,2004.43(31): p.10166-72.
34. Plummer, P., et al., Identification of a key amino acid of LuxS involved in AI-2production in Campylobacter jejuni. PLoS One,2011.6(1): p. e15876.
35. Bhattacharyya, M. and S. Vishveshwara, Functional correlation of bacterial LuxS withtheir quaternary associations: interface analysis of the structure networks. BMC StructBiol,2009.9: p.8.
36. Henke, J.M. and B.L. Bassler, Three parallel quorum-sensing systems regulate geneexpression in Vibrio harveyi. J Bacteriol,2004.186(20): p.6902-14.
37. Turovskiy, Y., et al., Quorum sensing: fact, fiction, and everything in between. AdvAppl Microbiol,2007.62: p.191-234.
38. Miller, M.B., et al., Parallel quorum sensing systems converge to regulate virulence inVibrio cholerae. Cell,2002.110(3): p.303-14.
39. Taga, M.E., S.T. Miller, and B.L. Bassler, Lsr-mediated transport and processing ofAI-2in Salmonella typhimurium. Mol Microbiol,2003.50(4): p.1411-27.
40. Xue, T., et al., LsrR-binding site recognition and regulatory characteristics inEscherichia coli AI-2quorum sensing. Cell Res,2009.19(11): p.1258-68.
41. Novak, E.A., et al., Autoinducer-2and QseC control biofilm formation and in vivovirulence of Aggregatibacter actinomycetemcomitans. Infect Immun,2010.78(7): p.2919-26.
42. Rader, B.A., et al., Helicobacter pylori perceives the quorum-sensing molecule AI-2asa chemorepellent via the chemoreceptor TlpB. Microbiology,2011.157(Pt9): p.2445-55.
43. Widmer, K.W., P. Jesudhasan, and S.D. Pillai, Fatty Acid Modulation of Autoinducer(AI-2) Influenced Growth and Macrophage Invasion by Salmonella Typhimurium.Foodborne Pathog Dis,2012.9(3): p.211-7.
44. Walters, M. and V. Sperandio, Autoinducer3and epinephrine signaling in the kineticsof locus of enterocyte effacement gene expression in enterohemorrhagic Escherichiacoli. Infect Immun,2006.74(10): p.5445-55.
45. Merritt, J., et al., Mutation of luxS affects biofilm formation in Streptococcus mutans.Infect Immun,2003.71(4): p.1972-9.
46. Wen, Z.T. and R.A. Burne, LuxS-mediated signaling in Streptococcus mutans isinvolved in regulation of acid and oxidative stress tolerance and biofilm formation. JBacteriol,2004.186(9): p.2682-91.
47. Hasona, A., et al., Membrane composition changes and physiological adaptation byStreptococcus mutans signal recognition particle pathway mutants. J Bacteriol,2007.189(4): p.1219-30.
48. Shemesh, M., et al., DNA-microarrays identification of Streptococcus mutans genesassociated with biofilm thickness. BMC Microbiol,2008.8: p.236.
49. Wen, Z.T., et al., Transcriptome analysis of LuxS-deficient Streptococcus mutans grownin biofilms. Mol Oral Microbiol,2011.26(1): p.2-18.
50. McNab, R., et al., LuxS-based signaling in Streptococcus gordonii: autoinducer2controls carbohydrate metabolism and biofilm formation with Porphyromonasgingivalis. J Bacteriol,2003.185(1): p.274-84.
51. Vidal, J.E., et al., The LuxS-dependent quorum-sensing system regulates early biofilmformation by Streptococcus pneumoniae strain D39. Infect Immun,2011.79(10): p.4050-60.
52. Bodor, A.M., et al., The luxS mutation causes loosely-bound biofilms in Shewanellaoneidensis. BMC Res Notes,2011.4: p.180.
53. De Araujo, C., et al., Quorum sensing affects biofilm formation throughlipopolysaccharide synthesis in Klebsiella pneumoniae. Res Microbiol,2010.161(7): p.595-603.
54. Jesudhasan, P.R., et al., Transcriptome analysis of genes controlled byluxS/autoinducer-2in Salmonella enterica serovar Typhimurium. Foodborne PathogDis,2009.7(4): p.399-410.
55. Ahmed, N.A., F.C. Petersen, and A.A. Scheie, Biofilm formation and autoinducer-2signaling in Streptococcus intermedius: role of thermal and pH factors. Oral MicrobiolImmunol,2008.23(6): p.492-7.
56. Armbruster, C.E., et al., LuxS promotes biofilm maturation and persistence ofnontypeable haemophilus influenzae in vivo via modulation of lipooligosaccharides onthe bacterial surface. Infect Immun,2009.77(9): p.4081-91.
57. Bobrov, A.G., et al., Functional quorum sensing systems affect biofilm formation andprotein expression in Yersinia pestis. Adv Exp Med Biol,2007.603: p.178-91.
58. Li, J., et al., Quorum sensing in Escherichia coli is signaled by AI-2/LsrR: effects onsmall RNA and biofilm architecture. J Bacteriol,2007.189(16): p.6011-20.
59. Reeser, R.J., et al., Characterization of Campylobacter jejuni biofilms under definedgrowth conditions. Appl Environ Microbiol,2007.73(6): p.1908-13.
60. Li, L., et al., Analysis on Actinobacillus pleuropneumoniae LuxS regulated genesreveals pleiotropic roles of LuxS/AI-2on biofilm formation, adhesion ability and ironmetabolism. Microb Pathog,2011.50(6): p.293-302.
61. Kozlova, E.V., et al., Mutation in the S-ribosylhomocysteinase (luxS) gene involved inquorum sensing affects biofilm formation and virulence in a clinical isolate ofAeromonas hydrophila. Microb Pathog,2008.45(5-6): p.343-54.
62. Joyce, E.A., et al., LuxS is required for persistent pneumococcal carriage andexpression of virulence and biosynthesis genes. Infect Immun,2004.72(5): p.2964-75.
63. Jones, M.B., et al., Role of luxS in Bacillus anthracis growth and virulence factorexpression. Virulence,2010.1(2): p.72-83.
64. Zhu, J., et al., Involvement of quorum sensing and heat-stable enterotoxin a in celldamage caused by a porcine enterotoxigenic Escherichia coli strain. InfectImmun,2011.79(4): p.1688-95.
65. Ohtani, K.,[Analysis of regulatory system of toxin production by cell-cell signaling inClostridium perfringens]. Nihon Saikingaku Zasshi,2011.66(2-3): p.169-74.
66. Stroeher, U.H., et al., Mutation of luxS of Streptococcus pneumoniae affects virulence ina mouse model. Infect Immun,2003.71(6): p.3206-12.
67. Coulthurst, S.J., C.L. Kurz, and G.P. Salmond, luxS mutants of Serratia defective inautoinducer-2-dependent 'quorum sensing' show strain-dependent impacts on virulenceand production of carbapenem and prodigiosin. Microbiology,2004.150(Pt6): p.1901-10.
68. Kim, S.Y., et al., Regulation of Vibrio vulnificus virulence by the LuxS quorum-sensingsystem. Mol Microbiol,2003.48(6): p.1647-64.
69. Krin, E., et al., Pleiotropic role of quorum-sensing autoinducer2in Photorhabdusluminescens. Appl Environ Microbiol,2006.72(10): p.6439-51.
70. Winzer, K., et al., Role of Neisseria meningitidis luxS in cell-to-cell signaling andbacteremic infection. Infect Immun,2002.70(4): p.2245-8.
71. Xu, L., et al., Role of the luxS quorum-sensing system in biofilm formation andvirulence of Staphylococcus epidermidis. Infect Immun,2006.74(1): p.488-96.
72. Daines, D.A., et al., Haemophilus influenzae luxS mutants form a biofilm and haveincreased virulence. Microb Pathog,2005.39(3): p.87-96.
73. Doherty, N., et al., Functional analysis of luxS in Staphylococcus aureus reveals a rolein metabolism but not quorum sensing. J Bacteriol,2006.188(8): p.2885-97.
74. Rader, B.A., et al., The quorum-sensing molecule autoinducer2regulates motility andflagellar morphogenesis in Helicobacter pylori. J Bacteriol,2007.189(17): p.6109-17.
75. Jeon, B., et al., Effects of quorum sensing on flaA transcription and autoagglutinationin Campylobacter jejuni. Microbiol Immunol,2003.47(11): p.833-9.
76. Kim, C.M. and S.H. Shin, Modulation of iron-uptake systems by a mutation of luxSencoding an autoinducer-2synthase in Vibrio vulnificus. Biol Pharm Bull,2011.34(5):p.632-7.
77. James, C.E., et al., LuxS involvement in the regulation of genes coding for hemin andiron acquisition systems in Porphyromonas gingivalis. Infect Immun,2006.74(7): p.3834-44.
78. Chung, W.O., et al., Signaling system in Porphyromonas gingivalis based on a LuxSprotein. J Bacteriol,2001.183(13): p.3903-9.
79. Ahmed, N.A., F.C. Petersen, and A.A. Scheie, AI-2quorum sensing affects antibioticsusceptibility in Streptococcus anginosus. J Antimicrob Chemother,2007.60(1): p.49-53.
80. Yu, D.N., et al.,[Function of luxS gene in sulfurmetabolism of Streptococcus mutans].Zhonghua Kou Qiang Yi Xue Za Zhi,2011.46(4): p.210-3.
81. Siller, M., et al., Functional analysis of the group A streptococcal luxS/AI-2system inmetabolism, adaptation to stress and interaction with host cells. BMC Microbiol,2008.8: p.188.
82. Learman, D.R., et al., Involvement of Shewanella oneidensis MR-1LuxS in biofilmdevelopment and sulfur metabolism. Appl Environ Microbiol,2009.75(5): p.1301-7.
83. Lebeer, S., et al., Functional analysis of luxS in the probiotic strain Lactobacillusrhamnosus GG reveals a central metabolic role important for growth and biofilmformation. J Bacteriol,2007.189(3): p.860-71.
84. Rezzonico, F. and B. Duffy, The role of luxS in the fire blight pathogen Erwiniaamylovora is limited to metabolism and does not involve quorum sensing. Mol PlantMicrobe Interact,2007.20(10): p.1284-97.
85. Tannock, G.W., et al., Ecological behavior of Lactobacillus reuteri100-23is affected bymutation of the luxS gene. Appl Environ Microbiol,2005.71(12): p.8419-25.
86. Wilson, C.M., et al., Transcriptional and metabolomic consequences of luxSinactivation reveal a metabolic rather than quorum sensing role for LuxS inLactobacillus reuteri100-23. J Bacteriol.2012
87. Heurlier, K., et al., Growth deficiencies of Neisseria meningitidis pfs and luxS mutantsare not due to inactivation of quorum sensing. J Bacteriol,2009.191(4): p.1293-302.
88. Malladi, V.L., et al., Inhibition of LuxS by S-ribosylhomocysteine analogues containinga [4-aza]ribose ring. Bioorg Med Chem,2011.19(18): p.5507-19.
89. Lonn-Stensrud, J., et al., Synthetic bromated furanone inhibits autoinducer-2-mediatedcommunication and biofilm formation in oral streptococci. Oral Microbiol Immunol,
2007.22(5): p.340-6.
90. Ren, D., J.J. Sims, and T.K. Wood, Inhibition of biofilm formation and swarming ofEscherichia coli by (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone.Environ Microbiol,2001.3(11): p.731-6.
91. Ren, D., et al., Differential gene expression shows natural brominated furanonesinterfere with the autoinducer-2bacterial signaling system of Escherichia coli.Biotechnol Bioeng,2004.88(5): p.630-42.