铜绿假单胞菌群体感应系统研究进展

群体感应系统是一种细胞密度依赖的基因表达系统,其广泛存在于细菌性病原体中,是细菌细胞通讯方式的一种。群体感应系统可利用细菌释放的信号分子不断监控周围细菌的密度。当细菌密度达到阈值时,群体感应系统网络将启动,参与调控生物被膜、细菌毒力等特定基因的表达,从而使临床抗感染治疗失败。而通过抑制群体感应系统,可一定程度上治疗铜绿假单胞菌引起的感染。本文通过查阅近年国内外相关文献,对铜绿假单胞菌群体感应系统研究进展进行总结,为临床铜绿假单胞菌治疗提供新的方向,即群体感应系统抑制剂有可能成为治疗铜绿假单胞菌感染的新策略。     

桦褐孔菌发酵鸭跖草提取物对铜绿假单胞菌群体感应系统的影响

【背景】群体感应在铜绿假单胞菌感染中常引起对抗生素的耐药性,因此迫切需要寻找新型抑制剂。【目的】研究桦褐孔菌发酵鸭跖草提取物(fermented Commelina communis extract,FCC)对铜绿假单胞菌(Pseudomonas aeruginosa,PA)群体感应系统的影响及其原因。【方法】采用微量稀释法测定FCC对PA的最小抑菌浓度(minimal inhibitory concentration,MIC),通过微量法测定FCC对生物膜形成、以及毒力因子(绿脓素、LasA蛋白酶及鼠李糖脂)合成的影响,利用高效液相色谱(high performance liquidchromatography,HPLC)法分析发酵前后化学物质的变化,采用福林酚(Folinand Ciocalteu's phenolreagent,Folin-Ciocalteu)法测量FCC和未发酵鸭跖草提取物(unfermented Commelina communis extract,UCC)的总酚含量。【结果】发酵前后提取物的MIC分别为16 g/L和64 g/L,FCC抑制了生物膜的形成,降低了毒力因子的产生,发酵提取物的化学组分在发酵过程中产生了很大的变化,发酵后提取物总酚含量相较于未发酵增加了219.97%。【结论】桦褐孔菌固体发酵具有增强鸭跖草提取物抗PA群体感应的作用,通过抑制群体感应系统产生抑菌作用。     

植物发酵液提取物对铜绿假单胞菌生物膜的作用

【目的】探讨植物发酵液提取物(plant fermentation extract,PFE)对铜绿假单胞菌生物膜的抑制作用,为临床上铜绿假单胞菌感染相关疾病的治疗提供参考。【方法】通过划线法分离临床标本中的铜绿假单胞菌并进行鉴定,通过报告菌株测定铜绿假单胞菌的毒力因子,采用试管法和激光共聚焦扫描显微镜测定生物膜的形成。【结果】在分离出的16株铜绿假单胞菌中,PFE对PA007菌株的作用效果最好,1%PFE显著降低PA007菌株生物膜、绿脓菌素和N-(3-oxododecanoyl)-HSL(3-oxo-C12-HSL)的产量(P0.05)。同时,也显著降低Las A蛋白酶的活性以及持留菌存活率(P0.05)。荧光定量PCR实验结果表明PFE能显著抑制las I和pqs A基因的表达(P0.05)。【结论】PFE具有抗铜绿假单胞菌感染能力,在临床上铜绿假单胞菌感染疾病的治疗中具有巨大的潜在价值。     

铜绿假单胞菌生物膜和浮游菌的毒力表达差异

目的探究铜绿假单胞菌生物膜和浮游菌状态下毒力因子的表达差异。方法使用铜绿假单胞菌标准菌株PAO1,分别在生物膜(静置)和浮游菌(摇床)状态下培养,收集上清液,检测总蛋白酶、LasA和LasB弹性蛋白酶、鼠李糖脂、绿脓素、溶血活性;通过荧光定量PCR检测群体感应(quorum sensing, QS)系统相关基因的表达;同时,通过活菌计数检测PAO1在生物膜和浮游菌状态下的生长曲线。结果生物膜状态下,铜绿假单胞菌PAO1的总蛋白酶、LasA、LasB弹性蛋白酶、鼠李糖脂、绿脓素表达均增高(均P0.05),溶血活性增高(P0.05),生物膜和浮游菌状态下细菌生长曲线差异无统计学意义,QS相关基因rhlI、rhlR、rhlA、lasI、lasR、pqsA、pqsR表达增高(均P0.05)。结论铜绿假单胞菌PAO1在生物膜状态下毒力因子表达较浮游菌状态下增高。     

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。     

植物精油对铜绿假单胞菌群体感应系统的抑制作用研究进展

群体感应（quorum sensing,QS）是细菌个体与个体之间的一种交流机制,广泛存在于细菌中。铜绿假单胞菌是人类的一种条件致病菌,它具有至少3种QS系统,即las、rhl和pqs系统,且各系统之间存在着级联调控关系,它们共同作用调控着该菌众多毒力基因的表达和毒力因子的产生。近年来,通过抑制铜绿假单胞菌的QS系统以控制其毒力和致病力,成为一种新型的铜绿假单胞菌感染防控策略。植物精油是一种天然的群体感应抑制剂（quorum sensing inhibitors, QSI）,多种精油活性化合物都能抑制铜绿假单胞菌的QS系统,而且尚未发现细菌对其产生耐药性。基于此,梳理了铜绿假单胞菌QS系统的组成及其级联调控关系,简要介绍了植物精油的QS抑制机制和抑制活性,并重点综述了萜烯类化合物、芳香族化合物、脂肪族化合物、含硫含氮化合物4类精油化合物对铜绿假单胞菌QS系统抑制作用的研究进展,以期为从天然化合物中发现和筛选安全、高效的细菌QSI的相关研究提供参考,并为致病菌的防控奠定理论基础。     

铜绿假单胞菌群体感应系统的研究进展

细菌的群体感应也称自身诱导,是指细菌通过产生和感应信号分子浓度的变化来监测其群体密度,协调群体行为的过程.自身诱导物随着细菌密度增高而增高,当自身诱导物达到某一阈值后,会与一些转录调节子结合,从而诱导或抑制多种基因的表达.群体感应系统内由多种信号分子和效应蛋白组成复杂的调节网络,调控包括细菌毒力因子产生与释放、生物膜形成、接合反应等,从而影响细菌的致病过程.本文主要对铜绿假单胞菌的群体感应系统及其与宿主关系、群体感应抑制剂等方面的研究进展进行综述.     

川白芷抑菌活性及对铜绿假单胞菌群体感应的抑制作用

为寻找更加有效的抑菌杀菌药物,本研究利用琼脂平板打孔法和倍比稀释法评价川白芷不同溶剂萃取物对金黄色葡萄球菌、大肠杆菌、铜绿假单胞菌以及肺炎克雷伯氏菌的抗菌活性;特别是通过对铜绿假单胞菌毒力表型的影响研究考察该药物对病菌群体感应是否具有抑制作用。研究结果表明:川白芷提取液对四种细菌均有不同程度的抑制效果,且随提取物浓度增大抑制效果增强;进一步分离发现,三氯甲烷、乙酸乙酯和正丁醇提取物对四种细菌均有一定的抑制效果,其中乙酸乙酯萃取物的抑菌效果最佳;对铜绿假单胞菌的四种毒力表型的影响研究结果表明川白芷具有抑制铜绿假单胞菌群体感应的能力。试验表明川白芷的活性成分可以作为一种新型的群体感应抑制剂,其具有抑制多种细菌能力的同时不易产生耐药性,表明白芷这一传统中草药在现代医疗中具有较好的应用潜力。     

铜绿假单胞菌和白假丝酵母的跨界相互作用

铜绿假单胞菌和白假丝酵母(又称白念珠菌)这两种条件致病菌可共存于人体。两者间存在复杂的相互关系,即跨界相互作用。铜绿假单胞菌抑制白念珠菌形态转换,抑制其生物膜形成并毒杀其菌丝;白念珠菌则抑制铜绿假单胞菌绿脓素形成并抑制其丛集运动。跨界相互作用可能存在3种机制:信号转导通路、生物膜和毒力因子。铜绿假单胞菌通过信号分子N-3-氧代十二烷酰-L-同型丝氨酸内酯(3-oxo-C12-HSL)抑制白念珠菌形态转换,而白念珠菌通过信号分子法呢醇抑制铜绿假单胞菌绿脓素生成和丛集运动,即存在信号分子介导的跨界相互作用。跨界相互作用影响铜绿假单胞菌和白念珠菌各自的致病性。如能充分利用跨界相互作用,将有助于优化治疗的选择。     

铜绿假单胞菌群体感应抑制剂研究进展

铜绿假单胞菌是一种常见的机会致病菌,可在人群中引起严重的急性和慢性感染,是病人在医院期间发生感染的第三大致病菌。铜绿假单胞菌多种毒力因子的分泌以及生物被膜的形成都是受一种被称为群体感应(Quorum Sensing,QS)的胞间信号传导系统调控的。QS使细菌能够根据细胞密度变化进行基因表达的调控。通过抑制QS来治疗铜绿假单胞菌感染是一个很有前景的发展方向。本文将就近年来铜绿假单胞菌群体感应抑制剂的研究进展进行综述。     

Diversity and polymorphism in AHL-lactonase gene (aiiA) of Bacillus

To explore bacterial diversity for elucidating genetic variability in acylhomoserine lactone (AHL) lactonase structure, we screened 800 bacterial strains. It revealed the presence of a quorum quenching (QQ) AHL-lactonase gene (aiiA) in 42 strains. These 42 strains were identified using rrs (16S rDNA) sequencing as Bacillus strains, predominantly B. cereus. An in silico restriction endonuclease (RE) digestion of 22 AHL lactonase gene (aiiA) sequences (from NCBI database) belonging to 9 different genera, along with 42 aiiA gene sequences from different Bacillus spp. (isolated here) with 14 type II REs, revealed distinct patterns of fragments (nucleotide length and order) with four REs; AluI, DpnII, RsaI, and Tru9I. Our study reflects on the biodiversity of aiiA among Bacillus species. Bacillus sp. strain MBG11 with polymorphism (115Alanine > Valine) may confer increased stability to AHL lactonase, and can be a potential candidate for heterologous expression and mass production. Microbes with ability to produce AHL-lactonases degrade quorum sensing signals such as AHL by opening of the lactone ring. The naturally occurring diversity of QQ molecules provides opportunities to use them for preventing bacterial infections, spoilage of food, and bioremediation.     

AiiA lactonase disrupts N-acylhomoserine lactone and attenuates quorum-sensing-related virulence in Pectobacterium carotovorum EMPCC

Enzymatic degradation of N-acyl homoserine lactone (NAHL) was found to interfere with the quorum sensing (QS) system and related functions in several soil bacteria. In this research, the NAHL lactonase gene aiiA was amplified using aiiA-7F/aiiA7R PCR primers from the quorum sensing inhibitor rhizobacterium Bacillus sp. strain DMS133, and cloned. The plasmid pME7075, carrying the DMS133 aiiA gene under the constitutive lac promoter, was introduced into the plant pathogen Pectobacterium carotovorum EMPCC, creating strain EMPCC/aiiA. Heterologous expression of the DMS133 aiiA gene in EMPCC severely reduced the accumulation of the NAHL throughout growth, and completely prevented pigmentation of the CV026 bioreporter strain. Virulence analysis revealed that the P. carotovorum strain EMPCC/aiiA expressing AiiA lactonase had drastically reduced tissue maceration activity compared with the wild type EMPCC strain. These results provide evidence that AiiA plays an important role in the quorum quenching ability of Bacillus sp. DMS133 whose AHL degradation capacity was investigated previously. In addition, the communication signal-inactivation approach represents a promising strategy for the prevention of diseases in which virulence is regulated by QS signal molecules.     

The role of AiiA, a quorum-quenching enzyme from Bacillus thuringiensis, on the rhizosphere competence

Bacteria sense their population density and coordinate the expression of target genes, including virulence factors in Gram-negative bacteria, by the N-acylhomoserine lactones (AHLs)-dependent quorum-sensing (QS) mechanism. In contrast, several soil bacteria are able to interfere with QS by enzymatic degradation of AHLs, referred to as quorum quenching. A potent AHL-degrading enzyme, AiiA, of Bacillus thuringiensis has been reported to effectively attenuate the virulence of bacteria by quorum quenching. However, little is known about the role of AiiA in B. thuringiensis itself. In the present study, an aiiA-defective mutant was generated to investigate the role of AiiA in rhizosphere competence in the root system of pepper. The aiiA mutant showed no detectable AHL-degrading activity and was less effective for suppression of soft-rot symptom caused by Erwinia carotovora on the potato slice. On the pepper root, the survival rate of the aiiA mutant significantly decreased over time compared with that of wild type. Interestingly, viable cell count analysis revealed that the bacterial number and composition of E. carotovora were not different between treatments of wild type and the aiiA mutant, although root application of the aiiA mutant in pepper failed to protect the plant from root rot. These results provide evidence that AiiA can play an important role in rhizosphere competentce of B. thuringiensis and bacterial quorum quenching to Gram-negative bacteria without changing bacterial number or composition.     

Inhibition of Biofilm Formation by T7 Bacteriophages Producing Quorum-Quenching Enzymes

Bacterial growth in biofilms is the major cause of recalcitrant biofouling in industrial processes and of persistent infections in clinical settings. The use of bacteriophage treatment to lyse bacteria in biofilms has attracted growing interest. In particular, many natural or engineered phages produce depolymerases to degrade polysaccharides in the biofilm matrix and allow access to host bacteria. However, the phage-produced depolymerases are highly specific for only the host-derived polysaccharides and may have limited effects on natural multispecies biofilms. In this study, an engineered T7 bacteriophage was constructed to encode a lactonase enzyme with broad-range activity for quenching of quorum sensing, a form of bacterial cell-cell communication via small chemical molecules (acyl homoserine lactones [AHLs]) that is necessary for biofilm formation. Our results demonstrated that the engineered T7 phage expressed the AiiA lactonase to effectively degrade AHLs from many bacteria. Addition of the engineered T7 phage to mixed-species biofilms containing Pseudomonas aeruginosa and Escherichia coli resulted in inhibition of biofilm formation. Such quorum-quenching phages that can lyse host bacteria and express quorum-quenching enzymes to affect diverse bacteria in biofilm communities may become novel antifouling and antibiofilm agents in industrial and clinical settings.     

GacA对假单胞菌株M18两个phz基因簇和菌群传感的调控

摘要:【目的】假单胞菌株M18（Pseudomonas sp. M18）是从甜瓜根际土壤中分离获得的一株对多种植物病原菌具有显著拮抗作用的菌株,在菌群传感（quorum sensing）系统的调控下,能分泌吩嗪-1-羧酸（PCA）以及多种吩嗪（phz）类衍生物的抗真菌物质。全局性因子GacA是M18菌株吩嗪类物质的合成与菌群传感系统的重要调控因子,本文将就GacA对上述两者的调控做进一步研究。【方法】PCR基因扩增和测序研究M18菌株中PCA合成基因簇,运用RT-PCR及构建phzA-lacZ转录融合手段     

Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis

Gram-negative bacteria can communicate with each other by N-acyl homoserine lactones (AHLs), which are quorum-sensing autoinducers. Recently, the aiiA gene (encoding an enzyme catalyzing the degradation of AHL) has been cloned from Bacillus sp. strain 240B1. During investigations in the course of the ongoing Bacillus thuringiensis subsp. morrisoni genome project, an aiiA homologue gene in the genome sequence was found. These results led to consideration of the possibility of the widespread existence of the gene in B. thuringiensis. aiiA homologue genes were found in 16 subspecies of B. thuringiensis, and their sequences were determined. Comparison of the Bacillus sp. strain 240B1 aiiA gene with the B. thuringiensis aiiA homologue genes showed high homologies of 89 to 95% and 90 to 96% in the nucleotide sequence and deduced amino acid sequence, respectively. Among the subspecies of B. thuringiensis having an aiiA gene, the subspecies aizawai, galleriae, kurstaki, kyushuensis, ostriniae, and subtoxicus were shown to degrade AHL. It was observed that recombinant Escherichia coli producing AiiA proteins also had AHL-degrading activity and could also attenuate the plant pathogenicity of Erwinia carotovora. These results indicate that insecticidal B. thuringiensis strains might have potential to compete with gram-negative bacteria in natural ecosystems by autoinducer-degrading activity.     

青岛近海沉积物生物被膜中密度感应及密度感应淬灭细菌多样性

【背景】细菌密度感应(Quorum sensing,QS)是指细菌利用分泌的信号分子进行相互交流的现象,而密度感应淬灭(Quorumquenching,QQ)是指通过干扰信号分子的产生、释放、积累或应答从而阻抑密度感应通路。【目的】探究青岛近海沉积物生物被膜中密度感应和密度感应淬灭细菌的多样性。【方法】采用海水培养基2216E从青岛近海沉积物生物被膜中分离获取可培养细菌,采用平板交互划线和高通量筛选的方法分别筛选具有密度感应和密度感应淬灭的菌株。【结果】共分离获得83株共54种具有密度感应和密度感应淬灭的细菌,分属于四大细菌门类:变形菌门、拟杆菌门、厚壁菌门和放线菌门。其中,38株(45.8%)可以产生酰基高丝氨酸内酯(Acyl-homoserine lactone,AHL)类信号分子,它们分属于变形菌门(37株,15种)和拟杆菌门(1株,1种),优势属为弧菌属和鲁杰氏菌属;能够降解AHL类信号分子的有57株(68.7%),在变形菌门(41株,23种)、拟杆菌门(14株,10种)、厚壁菌门(5株,5种)以及放线菌门(1株,1种)中均有分布。【结论】在青岛近海沉积物生物被膜可培养细菌中,具有密度感应和密度感应淬灭现象的菌株具有很高的丰度和多样性,为后续生态学意义的研究与海洋微生物的开发提供了参考。     

蜡样芽胞杆菌磷脂酶C在乳酸克鲁维酵母中重组表达、纯化及酶学性质分析

【目的】构建蜡样芽胞杆菌(Bacillus cereus)磷脂酶C(Phospholipase C,PLC)的重组乳酸克鲁维酵母(Kluyveromyces lactis)菌株、纯化重组蛋白并对其进行酶学性质分析。【方法】以B.cereus基因组DNA为模板,PCR扩增得到磷脂酶C基因(bcplc),构建重组乳酸克鲁维酵母表达质粒并转化到乳酸克鲁维酵母中,实现bcplc基因的表达。利用镍柱亲和层析纯化和脱盐柱得到电泳纯的重组磷脂酶C(rbcPLC)。【结果】成功构建产磷脂酶C的重组乳酸克鲁维酵母并纯化了重组磷脂酶C,纯化后rbcPLC经SDS-PAGE分析在40 kDa附近出现显性条带。NPPC法测得rbcPLC酶活为19251 U/mg,最适反应温度为80°C,最适pH为9.0。在低于40°C时,pH 7.0-8.0时,rbcPLC重组酶较稳定。Cu~(2+)和Co~(2+)对其有明显的抑制作用;Zn~(2+)、Mn~(2+)、Ca~(2+)、Mg~(2+)对其有明显的促进作用。【结论】首次实现了对蜡样芽胞杆菌来源的磷脂酶C在乳酸克鲁维酵母中的重组表达、纯化及其酶学性质分析,为其它食品安全性微生物来源的磷脂酶C的研究提供了借鉴意义。