细菌群体感应信号分子——酰基高丝氨酸内酯的检测

革兰氏阴性菌根据信号分子N-酰基高丝氨酸内酯(AHLs)的浓度可以监测周围环境中自身或其他细菌的数量变化,当信号分子达到一定浓度阈值时,能启动相关基因的表达来适应环境的变化,这一调控系统被称为细菌的群体感应(quorumsensing,QS)系统。快速简便而有效地检测细菌是否以及产生何种信号分子成为深入研究和了解细菌群体感应的重要手段。现对信号分子AHLs敏感的用于检测不同的信号分子AHLs的微生物传感菌进行综述,并对其检测能力进行了讨论。     

根瘤菌与群体感应

细菌在高细胞密度下可以产生群体感应信号分子,调控细菌相关基因的表达,这种信号分子被称为自体诱导物。酰基高丝氨酸内酯类化合物(acyl-HSLs)是在根瘤菌中广泛存在的一类自体诱导物,该群体感应系统与根瘤菌和植物的共生作用密切相关。本文概述了AHLs介导的群体感应系统的组成及调控机制和不同根瘤菌中群体感应调节对根瘤菌生理行为及共生固氮的影响。     

凡纳滨对虾优势腐败菌鉴定及其群体感应现象

为了鉴定凡纳滨对虾的优势腐败菌并研究其是否存在以N-酰基高丝氨酸内酯类化合物(AHLs)介导的群体感应系统,采用16S rRNA序列鉴定凡纳滨对虾的优势腐败菌,并采用紫色杆菌CV026对优势腐败菌的AHLs活性进行检测.结果发现凡纳滨对虾优势腐败菌菌株1(Aci-1)和菌株2 (Aci-2)均为不动杆菌属,均存在以AHLs为信号分子的群体感应系统.添加外源信号分子AHLs能促进Aci-1菌株生物膜的形成,且呈浓度依赖性.在一定的贮藏范围内,凡纳滨对虾腐败菌信号分子AHLs浓度与细菌总数、挥发性盐基氮含量存在正相关性,其相关系数r分别为0.846 6和0.986 7,分别在P＜0.05与P＜0.01水平上显著,结论是凡纳滨对虾优势腐败菌不动杆菌菌株存在以AHLs介导的群体感应系统,且与凡纳滨对虾的腐败密切相关.     

黄河鲤水产细菌的分离及其毒力因子和群体感应信号分子的检测

【背景】水产细菌病害制约水产养殖业健康发展,群体感应与细菌毒力因子的产生密切相关,群体感应调控细菌的毒力因子特性值得进一步研究。【目的】探究群体感应与黄河鲤细菌病害的关系,明确群体感应对细菌毒力因子特性的影响。【方法】通过16S rRNA基因测序并构建系统进化树确定筛选菌株的进化地位,通过脱脂牛奶平板法和偶氮酪蛋白法检测菌株胞外蛋白酶活力,采用结晶紫染色法对菌株的生物膜形成能力进行测定,通过报告菌株BB170和CV026分别测定菌株产信号分子AI-2和高丝氨酸内酯的能力,外源添加高丝氨酸内酯检测信号分子对菌株胞外蛋白酶活力和生物膜形成能力的影响。【结果】哈夫尼亚菌(Hafnia sp.) Z11和气单胞菌(Aeromonas sp.) Z12具有高水平的胞外蛋白酶活力和生物膜形成能力,能够分泌AHLs信号分子且具有菌体密度依赖性。外源添加HSL对菌株毒力因子特性有不同程度的影响,外源添加高浓度的N-丁酰基高丝氨酸内酯(C4-HSL)和N-己酰基高丝氨酸内酯(C6-HSL)能够分别提高菌株Z11和Z12的胞外蛋白酶活力和生物膜形成能力。【结论】高浓度群体感应信号分子AHLs对哈夫尼亚菌和气单胞菌胞外蛋白酶活性有促进作用,说明该2种菌的群体感应现象可能会影响其毒力。     

植物对细菌群体感应系统的反应

细菌的群体感应系统参与包括动植物病原细菌致病因子产生在内的许多生物学功能的调节。植物可以感知细菌群体感应系统及其信号分子,并作出复杂反应。植物可能受细菌群体感应信号分子诱导产生系统性防御反应,能够分泌细菌群体感应信号分子的类似物,可能产生降解细菌N-酰基高丝氨酸内酯信号分子的酶来阻断或干扰细菌群体感应系统。     

土壤中群体感应淬灭细菌的原位分离与筛选

【背景】许多革兰氏阴性细菌通常以N-酰基高丝氨酸内酯(N-acylhomoserine lactones,AHLs)作为群体感应主要的信号分子。【目的】从土壤中筛选和鉴定新型群体感应淬灭细菌。【方法】通过"垫圈法"从土壤中原位培养分离细菌,采用琼脂条法、报告菌平板法及β-半乳糖苷酶活性测定筛选群体感应淬灭细菌,根据16S rRNA基因序列同源性分析确定菌株系统发育地位。【结果】从不同地区土样中原位培养共分离获得细菌502株。以根癌土壤杆菌Agrobacterium tumefaciens NTL4 (pZLR4)作为报告菌,最终得到11株具有较强降解AHLs能力的细菌,包括假单胞菌5株、不动杆菌4株、变形杆菌和莱茵海默氏菌各1株。大部分细菌可完全降解N-3-羰基十二酰基高丝氨酸内酯(3OC12-HSL),部分细菌对N-(3-氧代己酰)高丝氨酸内酯(3OC6-HSL)和N-3-氧代辛酰高丝氨酸内酯(3OC8-HSL)具有一定降解活性。【结论】Proteus和Rheinheimera可降解AHLs,为今后防治依赖群体感应的植物细菌病害提供新型生防资源。     

细菌群体感应LuxR solos蛋白研究进展

N-酰基高丝氨酸内酯(N-acyl-L-homoserine lactones,AHLs)信号分子介导的群体感应(quorum sensing,QS)是一种普遍的革兰氏阴性细菌信息交流方式。AHL-QS系统包括Lux I型AHLs合成酶和LuxR型受体蛋白。然而,部分革兰氏阴性菌缺失1个或多个LuxI型AHLs合成酶,仅有未配对的LuxR型受体蛋白,该LuxR型受体蛋白称为LuxR solo或Orphan蛋白。LuxR solos蛋白在细菌窃听、种间和种内的信号交流中起重要作用,为群体感应研究领域的热点。本文主要综述细菌LuxR solos蛋白的发现、基本概念、蛋白结构及类型,阐述感应AHLs和非AHLs信号分子的重要LuxR solos蛋白及功能,并对群体感应LuxR solos蛋白的研究前景和意义进行了展望。     

不动杆菌属中aidE基因编码高丝氨酸内酯酶

群体感应(Quorum sensing,QS)是细菌在进化过程中形成的依赖于群体密度的细菌间交流方式。许多革兰氏阴性细菌以N-酰基高丝氨酸内酯(AHL)为信号分子,感应自身群体密度并调控致病基因表达。因此,淬灭AHLs信号分子可防治此类细菌引起的植物病害。本实验室前期已筛选得到了一株具有AHLs信号降解能力的不动杆菌菌株Acinetobacter sp.77,本研究通过基因组文库筛选,自菌株77中克隆得到具有AHLs降解活性的基因aidE。该基因编码268个氨基酸。序列一致性比较发现aidE的氨基酸序列与吉伦伯不动杆菌Acinetobacter gyllenbergii CIP110306中β-内酰胺酶一致性高达95%,但与已知的AHLs降解酶序列一致性较低,最高为缓黄分支杆菌Mycobacterium lentiflavum中AHL内酯酶Att M/Aii B家族蛋白(CQD23908.1),一致性仅为33%。通过高压液相色谱(HPLC)分析Aid E蛋白处理N-己酰基高丝氨酸内酯(C6-HSL)的反应产物,证明aidE为AHL内酯酶。序列比对研究发现,aidE基因在不动杆菌属中并不保守,其在菌株77基因组中的上下游的基因排列存在菌株水平的特异性,且aidE基因下游存在疑似IS插入序列,上述证据表明aidE基因有可能是通过水平转移进入Acinetobacter sp.77基因组中,或其在基因组中的位置发生过重排。表达aidE的软腐果胶杆菌Z3-3中完全检测不到AHLs信号产生,且致病力明显降低。综上所述,aidE为新发现的AHL内酯酶。在防治依赖QS系统表达致病性的细菌病害中具有应用潜力。     

细菌群体感应信号分子N-酰基高丝氨酸内酯的检测

群体感应是细菌生长到一定密度时相互感应,并进行基因表达及调控产生的独特、多样的群体行为现象。N-酰基高丝氨酸内酯(AHL)类化合物是革兰阴性菌群体感应中最重要的一类信号分子,调控许多生理特性基因的表达。快速、简便、有效地检测细菌能否产生AHL或产生何种信号分子,成为深入研究和了解细菌群体感应的重要手段。我们就细菌群体感应信号分子AHL检测的基本原理和方法及国内外研究进展进行了总结。     

食源假单胞菌群体感应信号分子的研究

从市售鲜鱼中分离的3株革兰氏阴性菌,经16S rDNA鉴定为假单胞菌属,该菌是一种导致食品腐败的重要腐败细菌。N-酰基-高丝氨酸内酯(AHLs)是革兰氏阴性菌群体感应(QS)系统中一类重要的信号分子,以密度依赖的方式调控某些生理性状的表达。利用AHLs检测菌株对3株假单胞菌进行检测发现,均产生AHLs类信号分子,且FML05-1和FML05-2至少产生两种AHLs,主要的信号分子是N-3-氧代-辛酰基-高丝氨酸内酯(N- 3-oxo-C_8-HSL)。同时对菌株FML05-2在生长过程中所产生的AHLs的活性变化进行研究,发现AHLs活性在菌体生长至12h时达到最大。首次对食源假单胞菌所产生的AHLs进行了研究,为以干扰腐败细菌群体感应为靶点的食品防腐保鲜策略提供研究基础。     

细菌群体感应信号网络及其应用

群体感应（Quorum sensing,QS）是一种细菌细胞与细胞间的通讯系统,即细菌通过分泌扩散性小分子信号感知细菌群体的密度,从而引起一组特定基因在转录水平协调表达。大量研究已表明,群体感应系统控制细菌多种生理行为和过程,以及与真核宿主（寄主）的互作。参与群体感应调控的信号分子多种多样,QS系统所调控的功能也具有多样性,甚至菌株专化性。通过聚焦同一细菌中由多个QS系统组成的信号网络,综合评述了不同QS系统之间如何相互作用全局调控基因表达,以及QS系统如何通过与其它全局调控系统整合精细调节细菌的社会行为以及环境适应性及其应用前景。     

Development of a novel series of non-natural triaryl agonists and antagonists of the Pseudomonas aeruginosa LasR quorum sensing receptor

Bacterial chemical communication, through a process called quorum sensing (QS), plays a central role in infection in numerous bacterial pathogens. Quorum sensing in Pseudomonas aeruginosa employs a series of small molecule receptors including the master QS regulator, LasR. In this study we investigate a non-natural triaryl series of LasR ligands using a combination of structure activity relationship studies and computational modeling. These studies have enabled the identification of key structural requirements for ligand binding and have revealed a new strategy for inducing the therapeutically relevant antagonism of LasR.     

Combinational quorum sensing devices for dynamic control in cross-feeding cocultivation

Quorum sensing (QS) offers cell density dependent dynamic regulations in cell culture through devices such as synchronized lysis circuit (SLC) and metabolic toggle switch (MTS). However, there is still a lack of studies on cocultivation with a combination of different QS-based devices. Taking the production of isopropanol and salidroside as case studies, we have mathematically modeled a comprehensive set of QS-regulated cocultivation schemes and constructed experimental combinations of QS devices, respectively, to evaluate their feasibility and optimality for regulating growth competition and corporative production. Glucose split ratio is proposed for the analysis of competition between cell growth and targeted production. Results show that the combination of different QS devices across multiple members offers a new tool with the potential to effectively coordinate synthetic microbial consortia for achieving high product titer in cross-feeding cocultivation. It is also evident that the performance of such systems is significantly affected by dynamic characteristics of chosen QS devices, carbon source control and the operational settings. This study offers insights for future applications of combinational QS devices in synthetic microbial consortia.     

群体感应系统在合成生物学中的应用*

群体感应(quorum sensing, QS)是一种广泛存在于多种微生物中的胞间通信系统,细菌产生的自诱导物随着种群密度的增加而积累,诱导细菌对种群密度的响应,调节生物膜的形成或特定基因的表达。近年来,随着群体感应系统原理与关键元件的逐渐清晰,应用合成生物学手段进行多技术联合以及多系统间正交性设计具有极大的发展潜力,群体感应系统已成为合成生物学家动态调控胞间通信常用的重要手段之一。在群体感应是细胞-细胞间通信系统的基础上,对多种群体感应系统的联合设计在生物基化学品生产中自动化调控的研究进展进行综述;并针对群体感应系统在生物电化学转化领域实现双向生物信息交流的应用进行总结;同时归纳了医学领域中群体感应系统的动态调控功能与多种疾病诊断及治疗结合的研究进展,讨论了群体感应系统在多细胞通信和实际应用等方面的发展前景。     

Synthesis and biological activity of a potent optically pure autoinducer-2 quorum sensing agonist

Quorum sensing (QS) regulates population-dependent bacterial behaviours, such as toxin production, biofilm formation and virulence. Autoinducer-2 (AI-2) is to date the only signalling molecule known to foster inter-species bacterial communication across distantly related bacterial species. In this work, the synthesis of pure enantiomers of C4-propoxy-HPD and C4-ethoxy-HPD, known AI-2 analogues, has been developed. The optimised synthesis is efficient, reproducible and short. The (4S) enantiomer of C4-propoxy-HPD was the most active compound being approximately twice as efficient as (4S)-DPD and ten-times more potent than the (4R) enantiomer. Additionally, the specificity of this analogue to bacteria with LuxP receptors makes it a good candidate for clinical applications, because it is not susceptible to scavenging by LsrB-containing bacteria that degrade the natural AI-2. All in all, this study provides a new brief and effective synthesis of isomerically pure analogues for QS modulation that include the most active AI-2 agonist described so far.     

Developing next generation antimicrobials by intercepting AI-2 mediated quorum sensing

Bacteria have been evolving antibiotic resistance since their discovery in the early twentieth century. Most new antibiotics are derivatives of older generations and there are now bacteria that are virtually resistant to almost all antibiotics. This poses a global threat to human health and has been classified as a clinical “super-challenge”, which has necessitated research into new antimicrobials that inhibit bacterial virulence while minimizing selective pressures that lead to the emergence of resistant strains. Quorum sensing (QS), the process of population dependent bacterial cell-cell signaling, can accelerate bacterial virulence and is an increasingly interesting target for developing next generation antimicrobials. Most QS inhibitors target species-specific signals, such as acylhomoserine lactones (AHLs) and oligopeptides. Methodologies for intercepting the cross-species signal, autoinducer-2 (AI-2), have only recently emerged. We review these strategies to prevent the relay of the AI-2 signal amongst pathogens, including Escherichia coli, Salmonella enterica serovar Typhimurium, Vibrio cholerae and Pseudomonas aeruginosa. Inhibition mechanisms are categorized based on the target (i.e., enzymes for signal generation, the signal molecule itself, or the various components of the signal transduction process). The universal nature of the AI-2 signal imparts on its inhibitors the potential for broad spectrum use.     

Anti-pathogenic Potential of Coral Associated Bacteria Isolated from Gulf of Mannar Against Pseudomonas aeruginosa

Infections of Pseudomonas aeruginosa are of great concern because of its increasing resistance towards conventional antibiotics. Quorum sensing system of P. aeruginosa acts as a global regulator of almost all the virulence factors and majorly its biofilm formation. In the present study, quenching of QS system of P. aeruginosa has been explained with bioactives from bacteria associated with the coral Acropora digitifera. Isolated bioactives inhibited the expression of various virulence traits of P. aeruginosa like biofilm formation, and the production of extracellular enzymes like protease and elastase. This study also emphasises the potential of coral associated bacteria in producing bioactive agents with anti-pathogenic properties.     

细菌群体感应调控多样性及群体感应淬灭

群体感应(Quorum sensing, QS)是细菌通过信号分子分泌、识别,从而调控基因水平转移、毒力因子分泌、芽孢产生及生物膜形成等群体行为的细胞交流机制。干扰信号分子的分泌、识别,可以阻断群体感应,实现群体淬灭。群体淬灭(Quorum quenching, QQ)是目前致病性控制、致腐性预防以及生物膜污染削减的重要策略之一。本文以群体感应信号分泌-识别-响应为主线,将群体感应分为等级、平行及竞争型三类调控方式,并对其特征进行了详细阐述;同时,探讨了信号分子类似物、信号分子降解酶剂、信号受体激活剂/抑制剂等策略在不同调控方式淬灭中的适用性;最后,对群体感应调控及淬灭进行了展望,以期为丰富细菌群体感应认知、促进群体淬灭应用提供参考。     

群体感应(QS)在牙菌斑形成中的研究进展

形成牙菌斑的生物膜中存在大量微生物,各种微生物通过高丝氨酸内酯(AHL)或寡肽等不同的信号分子产生群体感应(QS),形成的群体感应使各种微生物建立了区系平衡,对龋齿及牙周炎等口腔疾病的治疗产生严重影响。因组成生物膜的各种微生物对抗生素的敏感性和耐受性有显著差异,因此在治疗中增加了对宿主免疫的相应要求。为进一步探讨QS系统在牙菌斑形成中的作用特点,本文就牙菌斑的形成与抗药机制、群体感应系统及其信号分子、群体感应系统的抑制因子和研究展望进行综述。     

Food color ‘Azorubine’ interferes with quorum sensing regulated functions and obliterates biofilm formed by food associated bacteria: An in vitro and in silico approach

Quorum sensing (QS) plays a crucial role in different stages of biofilm development, virulence production, and subsequently to the growth of bacteria in food environments. Biofilm mediated spoilage of food is one of the ongoing challenge faced by the food industry worldwide as it incurs substantial economic losses and leads to various health issues. In the present investigation, we studied the interference of quorum sensing, its regulated virulence functions, and biofilm in food-associated bacteria by colorant azorubine. In vitro bioassays demonstrated significant inhibition of QS and its coordinated virulence functions in Chromobacterium violaceum 12472 (violacein) and Pseudomonas aeruginosa PAO1 (elastase, protease, pyocyanin, and alginate). Further, the decrease in the production EPS (49–63%) and swarming motility (61–83%) of the pathogens was also recorded at sub-MICs. Azorubine demonstrated broad-spectrum biofilm inhibitory potency (50–65%) against Chromobacterium violaceum, Pseudomonas aeruginosa, E. coli O157:H7, Serratia marcescens, and Listeria monocytogenes. ROS generation due to the interaction between bacteria and azorubine could be responsible for the biofilm inhibitory action of the food colorant. Findings of the in vitro studies were well supported by molecular docking and simulation analysis of azorubine and QS virulence proteins. Azorubine showed strong binding to PqsA as compared to other virulent proteins (LasR, Vfr, and QscR). Thus, it is concluded that azorubine is a promising candidate to ensure food safety by curbing the menace of bacterial QS and biofilm-based spoilage of food and reduce economic losses.