嗜水气单胞菌群体感应信号分子AI-2的细胞外生物 合成及活性检测

【目的】对嗜水气单胞菌群体感应信号分子AI-2进行细胞外生物合成及活性检测。【方法】对LuxS、MtnN-1、MtnN-2蛋白进行氨基酸序列分析、表达及纯化。以S-腺苷同型半胱氨酸(SAH)为底物,利用纯化的LuxS分别与MtnN-1及MtnN-2蛋白共同作用合成AI-2,并利用哈维氏弧菌报告菌株BB170检测AI-2活性。【结果】嗜水气单胞菌培养液上清中AI-2活性在8 h达到空白对照的16.96倍。氨基酸序列分析表明,嗜水气单胞菌与水生病原菌哈维式弧菌和迟钝爱德华氏LuxS一致性达到76%以上,MtnN-1与MtnN-2氨基酸序列一致性为26.37%,其中MtnN-2与哈维氏弧菌和迟钝爱德华氏菌Pfs一致性达到53%以上。成功表达及纯化了LuxS、MtnN-1和MtnN-2蛋白,细胞外LuxS和MtnN-1共同作用合成的AI-2活性是空白对照的45.04倍,LuxS和MtnN-2共同作用合成的AI-2活性是空白对照的63.62倍。【结论】嗜水气单胞菌能够合成信号分子AI-2。MtnN-1和MtnN-2氨基酸序列尽管存在较大差异,但两者均能与LuxS共同催化AI-2的细胞外生物合成。     

鸭疫里默氏杆菌Mtan对底物SAH的催化活性

【目的】分析鸭疫里默氏杆菌(Riemerella anatipestifer,RA)不同血清型pfs基因的序列差异,并开展其编码蛋白S-腺苷高半胱氨酸核苷酶(Mtan,又称Pfs)的催化活性研究。【方法】PCR扩增9株不同血清型RA的pfs基因,分析其核苷酸序列的同源性;构建该基因的重组表达载体p Cold-RA-pfs,表达、纯化RA的重组蛋白Mtan(RA-Mtan);测定RA-Mtan对底物S-腺苷同型半胱氨酸(S-adenosylhomocysteine,SAH)的催化活性,运用哈维弧菌报告菌株BB170检测催化底物的自诱导物2(Autoinducer-2,AI-2)活性。【结果】对RA的pfs序列分析结果表明,不同血清型RA的核苷酸一致性在93.9%-100%之间;SDS-PAGE检测结果表明,RA-Mtan呈可溶性表达;酶活测定表明RA-Mtan和禽致病性大肠杆菌(Avain pathogenic Escherichia coli,APEC)的Lux S蛋白共同作用于底物时,可产生浓度为176.7μmol/L的同型半胱氨酸(Homocysteine,HCY);AI-2活性检测结果表明,产生的AI-2具有生物学活性。【结论】RA不同血清型的pfs高度保守,RA pfs基因的编码产物RA-Mtan在体外具有催化SAH的活性,RA-Mtan和禽致病性大肠杆菌的Lux S蛋白共同作用于底物SAH时,能产生有活性的AI-2,为进一步研究pfs对RA的调控作用提供参考。     

高产信号分子AI-2乳酸菌的筛选及其Pfs基因的克隆和表达

【目的】从锡盟地区酸马奶酒分离的乳酸菌中筛选出高产信号分子自体诱导物2(Autoinducer-2,AI-2)的乳酸菌,通过优化其重组蛋白Pfs的诱导条件体外合成信号分子AI-2。【方法】利用生物学发光法对不同乳酸菌产信号分子AI-2的产量进行比较,以高产信号分子AI-2乳酸菌基因组DNA为模板,扩增其S-腺苷高半胱氨酸核苷酶(S-adenosylhomocysteine nucleosidase,Pfs)基因,构建原核表达载体。利用异丙基-β-D-硫代吡喃半乳糖苷(IPTG)进行重组蛋白的诱导表达,通过优化培养基、诱导温度、诱导前菌体密度、IPTG浓度以及诱导时间得到高表达的Pfs蛋白,使其与底物作用最终体外合成信号分子AI-2。【结果】10株乳酸菌均可产信号分子AI-2,其中屎肠球菌8-3分泌信号分子AI-2的产量明显高于其他菌株;重组蛋白的最佳诱导条件为:选取SOC(Super optimal broth with catabolite repression)作为诱导表达培养基,菌液OD600为0.5–0.7时加入终浓度为0.1 mmol/L的IPTG,37°C诱导12 h;利用最优诱导条件获得了浓度为4.08 g/L的纯化Pfs蛋白,体外合成了信号分子AI-2。【结论】酸马奶酒中分离出的10株乳酸菌均可产生信号分子AI-2,且屎肠球菌8-3可通过Pfs基因的作用生成信号分子AI-2。     

信号分子AI-2的体外合成及其对副溶血性弧菌四环素耐药性的调控作用

【背景】抗菌药的过度使用引起细菌耐药性日益严重,作为重要的食源性致病菌,副溶血性弧菌也表现出一定程度的耐药性。群体感应系统可以调控细菌的耐药性,为研究副溶血性弧菌的耐药机制和控制技术提供新的途径。【目的】探讨群体感应信号分子AI-2 (autoinducer-2)对海产品中分离的副溶血性弧菌四环素耐药性的调控作用。【方法】通过原核表达制备AI-2合成关键酶——S-核糖同型半胱氨酸酶(S-ribosylhomocysteinase, LuxS)和S-腺苷同型半胱氨酸核苷酶(S-adenosylhomocysteinenucleosidase,Pfs),体外合成AI-2,通过菌落计数法分析AI-2对副溶血性弧菌在四环素亚抑菌浓度下耐受性的影响,采用逆转录实时荧光定量PCR法测定不同浓度AI-2对副溶血性弧菌四环素耐药基因转录水平的影响。【结果】通过原核表达获得LuxS和Pfs,作用于底物S-腺苷同型半胱氨酸能合成具有生物活性的AI-2,其荧光强度约为阳性对照的6倍。在四环素亚抑菌浓度下,AI-2能显著促进副溶血性弧菌的生长,6、15、30μmol/L浓度AI-2能不同程度地提高副溶血性弧菌四环素耐药基因的转录水平。【结论】AI-2能增强副溶血性弧菌对四环素的耐受作用,为解析副溶血性弧菌的耐药机制、研制以AI-2为靶点的副溶血性弧菌耐药性控制技术提供基础。     

长双歧杆菌NCC2705群体感应系统信号分子AI-2的检测

目的:用生物学方法检测长双歧杆菌NCC2705是否产生群体感应系统信号分子AI-2。方法:将长双歧杆菌NCC2705不同时间点的培养上清分别加至AI-2特异报告系统哈氏弧菌BB170中,以空白培养基上清为对照,用荧光光度计对哈氏弧菌发光强度进行计量,推测出长双歧杆菌NCC2705上清中是否含有分泌的AI-2,并由此推断AI-2的活性。结果:通过微孔板检测系统对加入长双歧杆菌NCC2705培养上清的哈氏弧菌BB170进行检测,发现双歧杆菌上清的加入增强了哈氏弧菌BB170发出的荧光强度。结论:长双歧杆菌NCC2705中存在依赖于luxS/AI-2的群体感应系统,并能够分泌有活性的AI-2,为进一步研究长双歧杆菌NCC2705AI-2及luxS基因的功能打下基础。     

罗氏沼虾中副溶血弧菌群体感应的检测

利用3种不同的哈维氏弧菌报告菌株V.harveyi JMH612、V.harveyi JMH597和V.harveyi JAF375检测了罗氏沼虾体内分离的副溶血弧菌VIB461和VIB800的群体感应信号分子,同时用PCR扩增了两株菌调控AI-2型信号分子分泌的LuxS基因。结果表明,两株菌均能产生3种类型的信号分子:HAI-1、AI-2和CAI-1。3种信号分子的活性均具有生长阶段依赖性,在对数生长初期出现并在对数生长后期或稳定期前期达到最高水平,然后随着培养时间的延长呈现一定的下降。两株副溶血弧菌的PCR扩增片段的测序结果与已上传的副溶血弧菌的LuxS基因序列的相似度均在95%以上,说明两株副溶血弧菌都含有LuxS基因。     

信号分子AI-2合成关键基因luxS对副溶血性弧菌生物学特性的影响

[背景]副溶血性弧菌是全球范围重要的食源性病原菌,能引起急性肠胃炎。群体感应系统LuxS/AI-2影响细菌的生物学特性,为研究副溶血性弧菌的传播机制和控制技术提供了新的途径。[目的]探讨群体感应信号分子AI-2合成关键基因luxS对海产品中分离的副溶血性弧菌Vp2009027生物学特性的影响。[方法]利用自杀质粒同源重组技术敲除信号分子AI-2合成关键基因luxS,构建副溶血性弧菌Vp2009027的luxS基因缺失株,通过比较野生株与luxS基因缺失株的生长曲线、AI-2活性、运动能力、生物膜形成能力和耐药性,分析LuxS/AI-2系统对副溶血性弧菌生物学特性的影响。[结果]构建了副溶血性弧菌Vp2009027的luxS基因缺失株,野生株和luxS基因缺失株的生长无明显差异,luxS基因的缺失导致AI-2合成受阻、运动能力和生物膜形成能力增强、四环素耐药性降低。[结论]luxS基因对副溶血性弧菌的生物学特性具有重要的调控作用,为进一步研究副溶血性弧菌的传播机制和研发控制技术提供基础。     

AI-2通过甲基化趋化受体McpU调控恶臭假单胞菌KT2440的趋化运动及生物膜形成

【背景】广泛存在于革兰氏阴性菌和革兰氏阳性菌中的自诱导物autoinducer-2(AI-2)能够介导细菌种内和种间通讯,并调节细菌的多种生理过程。然而恶臭假单胞菌KT2440能否感知AI-2信号还未见报道。【目的】挖掘介导恶臭假单胞菌KT2440对AI-2趋化反应的趋化受体,检测AI-2信号通过趋化受体对恶臭假单胞菌KT2440生物膜形成的调控作用。【方法】本研究首先检测恶臭假单胞菌KT2440对AI-2信号的趋化反应,随后表达纯化了与铜绿假单胞菌AI-2受体高度同源的甲基化趋化受体McpU的配体结合结构域(ligand-binding domain,LBD),利用哈维氏弧菌的生物发光实验和等温滴定量热法(ITC)分析McpU-LBD与AI-2的相互作用;软琼脂平板法和毛细管定量分析法分析KT2440及mcpU敲除菌株(ΔmcpU)对AI-2的趋化反应;结晶紫染色法检测AI-2对KT2440及ΔmcpU生物膜形成能力的影响。【结果】软琼脂平板法和毛细管定量分析发现KT2440对AI-2信号表现出明显的正趋向性。哈维氏弧菌的生物发光实验和ITC分析发现AI-2与McpU-LBD具有高亲...     

AI-2通过调节c-di-GMP代谢酶DosC影响类志贺邻单胞菌生物膜形成及运动性

细菌中广泛分布的群体感应信号分子autoinducer-2 (AI-2)会影响细菌的生物膜形成及运动性等生理过程,然而该信号对类志贺邻单胞菌相关表型的调控作用及其分子机制尚未有所报道。【目的】揭示AI-2通过影响胞内环二鸟苷单磷酸(c-di-GMP)水平调控类志贺邻单胞菌生物膜形成及运动性的内在机制,为类志贺邻单胞菌感染的防治提供新思路。【方法】首先利用同源重组方法构建luxS基因敲除菌株(ΔluxS),通过软琼脂平板法和结晶紫染色法分别检测其与野生型泳动能力和生物膜形成水平的差异;之后通过序列比对找到AI-2的潜在受体蛋白DosC(SAMEA2665130＿2180),利用哈维氏弧菌生物发光实验及等温滴定量热实验(ITC)研究DosC的配体结合结构域(ligand-binding domain,LBD)与AI-2的结合能力;通过体外酶活实验、胞内c-di-GMP定量分析研究AI-2对DosC受体活性的影响;最后参照前述方法构建受体DosC编码基因敲除菌株(ΔdosC)并检测其与野生型相比泳动能力和生物膜形成水平的变化。【结果】AI-2与DosC-LBD显示出高亲和作用力;通过高效液相...     

迟缓爱德华菌luxS基因在不同生长时期表达水平的差异

摘要:【目的】探索迟缓爱德华菌LuxS/AI-2型密度感应系统关键基因luxS的分布及在不同生长时期的表达特征和生物功能。【方法】克隆迟缓爱德华菌luxS基因,利用生物信息学工具和网络数据库分析该基因序列的特征及编码蛋白的基本特征和保守结构;原核表达LuxS蛋白,纯化后制备抗LuxS的抗体,运用Western-blot技术分析LuxS在不同毒力、不同来源迟缓爱德华菌中的分布情况及在不同生长时期的表达水平;利用抗体中和方法,分析抗LuxS抗体对迟缓爱德华菌生长的影响,探索LuxS是否为信号分子AI-2的特 异依赖模式。【结果】克隆到迟缓爱德华菌luxS基因,长度为516 bp,序列分析结果表明,该基因在迟缓爱德华菌属中高度保守;对多株迟缓爱德华菌LuxS蛋白的检测结果表明,该基因在该菌属中普遍存在;对不同生长时期LuxS蛋白的检测结果表明,LuxS蛋白的表达量在迟缓期较低,进入对数生长期逐渐增加,在对数生长后期最大,稳定后期逐渐减少;抗体中和生长试验结果表明,1%抗血清(效价1:40000)能延长迟缓爱德华菌生长的平台期,但对细菌生长无显著影响。【结论】LuxS/AI-2介导的密度感应系统在迟缓爱德华菌中普遍存在;关键基因luxS的序列高度保守,在不同生长时期表达量不一致,在对数生长后期达到峰值。     

In vitro biosynthesis of autoinducer 2 of Steptococcus suis Serotype 2 using recombinant LuxS and Pfs

Quorum sensing is the cell population density-dependent regulation of gene expression by small signaling molecules, called autoinducers. LuxS and Pfs catalyze synthesis of the quorum-sensing signaling molecule autoinducer 2 (AI-2), which has been shown to control a variety of cellular processes. We studied the cloning, expression, and purification of LuxS and Pfs from Streptococcus suis Serotype 2 strain HA9801 (SS2); the two enzymes gave an apparent single protein band, and revealed a molecular mass of 21.74 and 28.44 kDa on an SDS-PAGE, respectively. Expressed and purified LuxS and Pfs were incubated with S-ribosylhomocysteine (SAH). The reaction products were able to induce luminescence of Vibrio harveyi BB170, clearly demonstrating that recombinant Pfs and LuxS synthesize AI-2 in vitro from SAH. Optimum pH and temperature for biosynthesis AI-2 in vitro were 8.0 and 37 °C, respectively. Biosynthesis AI-2 in vitro was stimulated by Cr³⁺, Al³⁺, and Ba²⁺and was inhibited by Fe²⁺ and Ni²⁺, respectively. It was strongly inhibited by Hg²⁺, Cu²⁺, and Mn²⁺, while enzyme activity was not affected by Li⁺, Mg²⁺, and Zn²⁺. In this study, we cloned, expressed, and purified LuxS and Pfs, identified the pathway of AI-2 synthesis in SS2, and analyzed the impact factor of AI-2 synthesis in vitro, which provided a solid basis for future research concerning the role of AI-2 in SS2.     

Biological activity and identification of a peptide inhibitor of LuxS from Streptococcus suis serotype 2

The virulence of bacterial communities may be regulated by mechanisms involving the synthesis of the quorum-sensing signal autoinducer 2 (AI-2), which allows both intra- and interspecies communication. AI-2 is produced in bacteria that express the gene luxS . In the present study, expressed and purified LuxS from Streptococcus suis serotype 2 (SS2) was used to catalyze the substrate S -ribosylhomocysteine in a reaction that leads to the production of AI-2. The biological activity of the in vitro synthesized AI-2 was demonstrated in a Vibrio harveyi strain BB170 bioassay; real-time PCR results showed that biosynthesis of AI-2 can increase the virulence of SS2. Phage-encoded peptides that specifically interact with the LuxS enzyme were selected following three rounds of phage display. One such peptide inhibitor (TNRHNPHHLHHV) of LuxS was shown to partially inhibit the activity of the enzyme. Furthermore, 14 peptides containing the consensus sequence HSIR showed high affinity with LuxS. The selected and characterized specific inhibitor as well as the high-affinity ligands may facilitate the identification of new vaccination targets, opening up new approaches to the development of therapeutic drugs.     

Growth Deficiencies of Neisseria meningitidis pfs and luxS Mutants Are Not Due to Inactivation of Quorum Sensing

The activated methyl cycle (AMC) is a central metabolic pathway used to generate (and recycle) several important metabolites and enable methylation. Pfs and LuxS are considered integral components of this pathway because they convert S-adenosylhomocysteine (SAH) to S-ribosylhomocysteine (SRH) and S-ribosylhomocysteine to homocysteine (HCY), respectively. The latter reaction has a second function since it also generates the precursor of the quorum-sensing molecule autoinducer 2 (AI-2). By demonstrating that there was a complete lack of AI-2 production in pfs mutants of the causative agent of meningitis and septicemia, Neisseria meningitidis, we showed that the Pfs reaction is the sole intracellular source of the AI-2 signal. Analysis of lacZ reporters and real-time PCR experiments indicated that pfs is expressed constitutively from a promoter immediately upstream, and careful study of the pfs mutants revealed a growth defect that could not be attributed to a lack of AI-2. Metabolite profiling of the wild type and of a pfs mutant under various growth conditions revealed changes in the concentrations of several AMC metabolites, particularly SRH and SAH and under some conditions also HCY. Similar studies established that an N. meningitidis luxS mutant also has metabolite pool changes and growth defects in line with the function of LuxS downstream of Pfs in the AMC. Thus, the observed growth defect of N. meningitidis pfs and luxS mutants is not due to quorum sensing but is probably due to metabolic imbalance and, in the case of pfs inactivation, is most likely due to toxic accumulation of SAH.     

AI-3 synthesis is not dependent on luxS in Escherichia coli

The quorum-sensing (QS) signal autoinducer-2 (AI-2) has been proposed to promote interspecies signaling in a broad range of bacterial species. AI-2 is spontaneously derived from 4,5-dihydroxy-2,3-pentanedione that, along with homocysteine, is produced by cleavage of S-adenosylhomocysteine (SAH) and S-ribosylhomocysteine by the Pfs and LuxS enzymes. Numerous phenotypes have been attributed to AI-2 QS signaling using luxS mutants. We have previously reported that the luxS mutation also affects the synthesis of the AI-3 autoinducer that activates enterohemorrhagic Escherichia coli virulence genes. Here we show that several species of bacteria synthesize AI-3, suggesting a possible role in interspecies bacterial communication. The luxS mutation leaves the cell with only one pathway, involving oxaloacetate and l-glutamate, for de novo synthesis of homocysteine. The exclusive use of this pathway for homocysteine production appears to alter metabolism in the luxS mutant, leading to decreased levels of AI-3. The addition of aspartate and expression of an aromatic amino acid transporter, as well as a tyrosine-specific transporter, restored AI-3-dependent phenotypes in an luxS mutant. The defect in AI-3 production, but not in AI-2 production, in the luxS mutant was restored by expressing the Pseudomonas aeruginosa S-adenosylhomocysteine hydrolase that synthesizes homocysteine directly from SAH. Furthermore, phenotype microarrays revealed that the luxS mutation caused numerous metabolic deficiencies, while AI-3 signaling had little effect on metabolism. This study examines how AI-3 production is affected by the luxS mutation and explores the roles of the LuxS/AI-2 system in metabolism and QS.     

AI-2 biosynthesis module in a magnetic nanofactory alters bacterial response via localized synthesis and delivery

Nanofactories are nano-dimensioned and comprised of modules serving various functions that alter the response of targeted cells when deployed by locally synthesizing and delivering cargo to the surfaces of the targeted cells. In its basic form, a nanofactory consists of a minimum of two functional modules: a cell capture module and a synthesis module. In this work, magnetic nanofactories that alter the response of targeted bacteria by the localized synthesis and delivery of the "universal" bacterial quorum sensing signal molecule autoinducer AI-2 are demonstrated. The magnetic nanofactories consist of a cell capture module (chitosan-mag nanoparticles) and an AI-2 biosynthesis module that contains both AI-2 biosynthetic enzymes Pfs and LuxS on a fusion protein (His-LuxS-Pfs-Tyr, HLPT) assembled together. HLPT is hypothesized to be more efficient than its constituent enzymes (used separately) at conversion of the substrate SAH to product AI-2 on account of the proximity of the two enzymes within the fusion protein. HLPT is demonstrated to be more active than the constituent enzymes, Pfs and LuxS, over a wide range of experimental conditions. The magnetic nanofactories (containing bound HLPT) are also demonstrated to be more active than free, unbound HLPT. They are also shown to elicit an increased response in targeted Escherichia coli cells, due to the localized synthesis and delivery of AI-2, when compared to the response produced by the addition of AI-2 directly to the cells. Studies investigating the universality of AI-2 and unraveling AI-2 based quorum sensing in bacteria using magnetic nanofactories are envisioned. The prospects of using such multi-modular nanofactories in developing the next generation of antimicrobials based on intercepting and interrupting quorum sensing based signaling are discussed.     

luxS基因缺失对禽致病性大肠杆菌O1、O2和O78三种血清型分离株的生物学特性影响

【目的】LuxS/AI-2型密度群体感应系统产生的自诱导信号分子AI-2(AI-2的产生需要luxS基因编码的Lux S蛋白参与)参与对细菌众多生理功能的调控。探讨luxS对不同血清型禽致病性大肠杆菌(Avian Pathogenicity Escherichia coli,APEC)生物学特性的影响。【方法】本研究以APEC优势血清型APECO_1(O_1血清型)、DE17(O_2血清型)、E940(O_(78)血清型)及其相应luxS缺失株为研究对象,对野生株和缺失株的生长特性、生物被膜形成、rdar(red,dry and rough)形态、运动性和耐药性等特性进行分析。【结果】luxS基因的缺失不影响APEC生长特性,但导致APEC不能产生AI-2;此外,luxS基因的缺失显著降低APECO_1和E940的生物被膜形成(P0.05),而DE17的生物被膜形成无显著变化。对各菌株的rdar形态和运动性检测结果表明,luxS基因的缺失改变了APECO_1的rdar形态,对DE17和E940并无影响;显著降低了APECO_1和DE17运动能力,对E940并无影响。荧光定量PCR检测结果表明,luxS基因的缺失显著降低APECO_1、DE17和E940与细菌运动性相关的鞭毛基因fli G和fli I的转录水平(P0.05)。此外,对各菌株的耐药性检测结果表明,luxS基因缺失导致APECO_1对头孢吡肟和丁胺卡那由耐药变为高敏,同时对氯霉素与E940相同由高敏变为耐药,但对DE17的耐药性无显著改变。【结论】luxS对APEC的生物学特性具有重要的调控作用,且这种调控具有菌株特异性。     

Crystal Structure and Identification of Two Key Amino Acids Involved in AI-2 Production and Biofilm Formation in Streptococcus suis LuxS

Streptococcus suis has emerged as an important zoonotic pathogen that causes meningitis, arthritis, septicemia and even sudden death in pigs and humans. Quorum sensing is the signaling network for cell-to-cell communication that bacterial cells can use to monitor their own population density through production and exchange of signal molecules. S-Ribosylhomocysteinase (LuxS) is the key enzyme involved in the activated methyl cycle. Autoinducer 2 (AI-2) is the adduct of borate and a ribose derivative and is produced from S-adenosylhomocysteine (SAH). AI-2 can mediate interspecies communication and in some species facilitate the bacterial behavior regulation such as biofilm formation and virulence in both Gram-positive and Gram-negative bacteria. Here, we reported the overexpression, purification and crystallographic structure of LuxS from S. suis. Our results showed the catalytically active LuxS exists as a homodimer in solution. Inductively coupled plasma-mass spectrometry (ICP-MS) revealed the presence of Zn²⁺ in LuxS. Although the core structure shares the similar topology with LuxS proteins from other bacterial species, structural analyses and comparative amino acid sequence alignments identified two key amino acid differences in S. suis LuxS, Phe80 and His87, which are located near the substrate binding site. The results of site-directed mutagenesis and enzymology studies confirmed that these two residues affect the catalytic activity of the enzyme. These in vitro results were corroborated in vivo by expression of the LuxS variants in a S. suis ΔluxS strain. The single and two amino acid of LuxS variant decreased AI-2 production and biofilm formation significantly compared to that of the parent strain. Our findings highlight the importance of key LuxS residues that influence the AI-2 production and biofilm formation in S.suis.