环二鸟苷酸——新型的细菌第二信使

环二鸟苷酸(cyclic diguanylate,c-di-GMP)是新近发现的在细菌中普遍存在的第二信使分子,参与调节多种生理功能,包括细胞分化、从运动状态到生物被膜状态的转变、致病因子产生等.基于其对细菌抗生素耐药的物理屏障—生物被膜形成的影响,c-di-GMP的研究越来越受到人们的关注.细胞内c-di-GMP的产生受二鸟苷酸环化酶(diguanylate cyclase,DGC)合成和磷酸二酯酶(phosphodiesterase,PDE)降解两条途径调控.在结构上,通常DGC含有GGDEF结构域,PDE含有EAL结构域.c-di-GMP的作用靶点包括PilZ结构域和GEMM 核开关两种类型.本文综述了c-di-GMP的代谢途径、调控机理、生物学功能等方面的最新研究进展,并对c-di-GMP在今后研究中的应用和发展趋势进行展望.     

绿原酸通过增强RsmA的表达抑制铜绿假单胞菌生物被膜的形成

铜绿假单胞菌是常见的人类条件致病菌,其生物被膜的形成会增强菌体的耐药性。已有文献报道绿原酸可抑制铜绿假单胞菌生物被膜的形成,本研究在此基础上主要探究了其对全局性次级代谢调控系统Gac-Rsm表达的影响。结果显示,绿原酸可抑制铜绿假单胞菌生物被膜形成的能力,降低胞外总多糖合成量,但关键胞外多糖psl的合成酶基因pslA转录未受影响,还可增强Gac-Rsm系统中关键调控因子RsmA的表达水平,降低细胞内关键信使分子环二鸟苷酸(cyclic dimeric guanosine monophosphate,c-di-GMP)水平。结果表明,绿原酸可通过增强RsmA的表达来抑制铜绿假单胞菌生物被膜的形成。     

环二鸟苷酸信号分子抑制剂的研究进展

环二鸟苷酸(Bis-(3′-5′)cyclic diguanylic acid,c-di-GMP)是细菌所特有的一类核酸类第二信使,参与并调节细菌多种生理功能,包括细胞分化、生物被膜的形成以及致病因子的产生等。阻断c-di-GMP信号的传导对于发展新型抗菌药物具有重要的意义。现有研究结果表明,基于c-di-GMP调控的信号通路开发新型抗菌药物具有3类潜在的靶点,分别是c-di-GMP合成酶(DGCs)、c-di-GMP降解酶(PDEs)以及c-di-GMP受体。文中根据上述3类关键靶点,介绍了相关小分子抑制剂的研究进展,并展望了c-di-GMP信号分子抑制剂的发展方向。     

鉴定cyclic di-GMP效应蛋白：高通量筛选策略与实验验证方法

环二鸟苷单磷酸(cyclic di-GMP或c-di-GMP)是细菌细胞中广泛存在的第二信使,调控细菌生物被膜发育、致病力、运动性、胞外多糖产生及细胞周期在内的诸多重要生理表型。c-di-GMP通过结合多种类型的效应子(包括核糖开关或效应蛋白)来发挥调控功能。由于c-di-GMP分子在构象上具有多变性,其结合的效应子同样具有多样性。新型效应蛋白的筛选、鉴定是当前细菌信号转导领域的研究热点和难点,也是解析c-di-GMP调控机制的首要环节。本文在阐述c-di-GMP结合不同类型的效应蛋白并调控细菌生物被膜发育的基础上,综述了目前筛选c-di-GMP效应蛋白的方法,包括遗传筛选、亲和色谱结合质谱鉴定、DRa CALA系统鉴定以及基于分子对接的预测等。同时,对验证c-di-GMP效应蛋白的技术,如等温微量热滴定、表面等离子共振、微量热泳动在内的多种验证方法进行了总结,对比了这些策略和方法在应用上的优、缺点,为在细菌及其真核宿主基因组水平鉴定c-di-GMP效应蛋白的研究提供参考。     

环二鸟苷酸(c-di-GMP)对大肠杆菌生物学功能的调控作用及机制

环二鸟苷酸(cyclic diguanosine monophosphate, c-di-GMP)是在细菌中发现的第二信使之一,参与大肠杆菌运动性、生物被膜形成及毒力等众多功能的调节。合成与水解c-di-GMP的二鸟苷酸环化酶(diguanylate cyclases, DGCs)和特异性的磷酸二酯酶(phosphodiesterases, PDEs)在大肠杆菌中分布广泛且丰富度较高。同时,DGCs和PDEs的蛋白结构不仅包括与c-di-GMP相关的酶活性中心,而且还具有多种感受环境变化和接收信号分子调节的结构域。鉴于c-di-GMP的调控复杂且值得深入探索,本文综述了c-di-GMP对大肠杆菌生物学特性的调控作用以及c-di-GMP在大肠杆菌中的两种调控模型,并对最新研究进展和应用进行展望。     

c-di-GMP对大肠杆菌生物膜调控的研究进展

大肠杆菌生物膜是由聚集于特定介质上的大肠杆菌菌体细胞相互黏附并分泌胞外基质聚合物(extracellular polymeric substances,EPS)而产生的一种结构复杂的膜状聚集物。感染宿主后的致病性大肠杆菌在形成生物膜后会极大地逃避免疫系统以及环境中各种有害因素对其的影响,对宿主造成持续甚至致命的伤害。环二鸟苷酸(cyclic diguanosine monophosphate,c-di-GMP)是广泛存在于细菌中的第二信使,在调节生物膜形成过程中起到至关重要的作用。基于此,本文对近些年来有关c-di-GMP对大肠杆菌生物膜形成过程中菌体的运动、黏附以及EPS产生机制的研究进行了综述,以期为从c-di-GMP角度抑制大肠杆菌生物膜提供依据和思路。     

c-di-GMP对细菌胞外多糖合成与运输的调控

环二鸟苷酸(Cyclic diguanylate,c-di-GMP)的发现已有29年。作为重要的细菌第二信使,c-di-GMP可参与调节细菌生物膜的合成与降解、运动、毒性、细胞周期、细胞分化等多种活动过程。胞外多糖(EPS)是细菌生物膜的主要组成成分,其合成和运输主要受c-di-GMP调控。目前细菌胞外多糖在医药、食品、农业、工业和环保等多个领域均有广泛的应用,其相关研究备受关注。本文旨在论述细菌中c-di-GMP合成与降解的调控,部分合成酶(Diguanylate cyclase,DGC)与降解酶(Phosphodiesterase,PDE)及其受体分子(Receptor)晶体结构等研究成果,并结合我们研究农杆菌ATCC31749中c-di-GMP对可德胶合成调控的基础上,重点阐述c-di-GMP对纤维素、藻酸盐、多聚氮乙酰葡萄糖胺(PNAG)和可德胶等EPS合成与运输的调控机制。     

检测大肠杆菌内c-di-GMP水平的双荧光素报告质粒的构建及应用

细菌通过调控第二信使环二鸟苷酸(cyclic diguanylate, c-di-GMP)而促进其适应环境、存活及致病。【目的】本研究旨在建立有效的c-di-GMP水平检测方法,为大肠杆菌内c-di-GMP水平检测提供便利条件。【方法】根据c-di-GMP核糖开关受体的调控方式、荧光报告基因等设计引物,通过重叠聚合酶链反应(overlap polymerase chain reaction, overlap PCR)和同源重组酶构成基于核糖开关的双荧光素报告质粒pAmCherry-Vc2EGFP(pACVcE),然后构建c-di-GMP代谢基因过表达菌株和缺失菌株,利用pACVcE检测大肠杆菌内c-di-GMP水平。【结果】OverlapPCR扩增产物与目的靶序列一致,测序结果证明pACVcE序列正确。表达c-di-GMP合成酶DgcZ的大肠杆菌胞内c-di-GMP水平显著升高,而表达c-di-GMP降解酶PdeK的大肠杆菌胞内c-di-GMP水平显著降低。禽致病性大肠杆菌的胞内c-di-GMP水平检测发现c-di-GMP降解酶基因pdeK缺失后胞内的c-di-GMP水平显著升高。【结...     

c-di-GMP的磷酸二酯酶PA4781在抗菌肽Merecidin抑制铜绿假单胞菌生物被膜中的作用

【背景】抗菌肽Merecidin可抑制临床菌株铜绿假单胞菌PA03生物被膜。PA4781基因是课题组通过生物信息学分析筛选出的差异表达基因,PA4781作为细菌第二信使分子环二鸟苷酸(cyclic diguanylate,c-di-GMP)的磷酸二酯酶具有降解c-di-GMP的作用,其在抗菌肽Merecidin抑制生物被膜中的作用机制尚不清楚。【目的】研究细菌第二信使分子c-di-GMP的磷酸二酯酶PA4781基因在抗菌肽Merecidin抑制铜绿假单胞菌生物被膜中的作用。【方法】利用单碱基突变技术敲除PA4781基因,Sanger测序方法检测敲除的正确性。采用结晶紫染色法观察PA03菌株、PA4781过表达菌株、PA4781敲除菌株24 h生物被膜生长情况,以及在抗菌肽Merecidin 24、48、72μmol/L作用下各菌株生物被膜的生长情况。采用对羟基联苯溶液显色法检测在抗菌肽Merecidin 48、72μmol/L作用下,PA03菌株、PA4781过表达菌株、PA4781敲除菌株生物被膜藻酸盐的变化情况。【结果】Sanger测序结果显示,用pnCasPABEC系统成功实现了靶点位置的单碱基突变,提前终止了PA4781的转录;结晶紫染色结果显示,培养24h时,在24μmol/L抗菌肽Merecidin作用下PA03菌株、PA4781过表达菌株、PA4781敲除菌株生物被膜形成情况无显著性差异(P0.05),在抗菌肽Merecidin 48、72μmol/L处理下,过表达株与正常株和敲除株有显著性差异(P0.05),生物被膜明显减少,敲除株生物被膜厚度高于PA03组(P0.05)。随着抗菌肽Merecidin浓度升高各组藻酸盐含量下降,其中过表达菌株在抗菌肽Merecidin作用下藻酸盐生成量抑制率最高,可达65%。【结论】抗菌肽Merecidin能够促进细菌第二信使分子磷酸二酯酶PA4781的表达,为抗菌肽Merecidin抑制铜绿假单胞菌生物被膜的作用机制可能通过细菌第二信使分子这一信号途径提供新的研究思路。     

SadC合成的c-di-GMP信号通过PilZ和FlgZ调节铜绿假单胞菌的泳动能力

【目的】探究铜绿假单胞菌(Pseudomonas aeruginosa)鸟苷酸环化酶(diguanylate cyclase,DGC)SadC合成的环二鸟苷酸(cyclicdi-GMP,c-di-GMP)信号与PilZ结构域受体间的信号传递关系,分析鉴定出特定PilZ结构域受体的调控功能和机制。【方法】SadC突变株和过表达菌株的构建及泳动能力分析;SadC过表达背景下,PilZ结构域受体突变各菌株的泳动表型分析和筛选;基因敲除和过表达解析筛选出的PilZ结构域受体功能;定点突变和遗传互补检测筛选出的PilZ结构域受体是否参与SadC合成c-di-GMP对泳动能力的调控。【结果】SadC通过影响鞭毛功能而非鞭毛形成抑制铜绿假单胞菌的泳动能力;PilZ结构域受体突变菌株筛选发现PilZ、FlgZ这2个受体参与了SadC介导的泳动能力抑制;功能分析发现ΔpilZ或ΔflgZ的泳动能力相比野生型PA14显著增强,而过表达PilZ或FlgZ则抑制了泳动能力;定点突变和回补实验发现PilZ第10位和FlgZ第140位氨基酸R对其介导SadC负调控泳动能力至关重要,多序列比对分析表明这些位点是其保...     

Systematic analysis of cyclic di-GMP signalling enzymes and their role in biofilm formation and virulence in Yersinia pestis

Cyclic di-GMP (c-di-GMP) is a signalling molecule that governs the transition between planktonic and biofilm states. Previously, we showed that the diguanylate cyclase HmsT and the putative c-di-GMP phosphodiesterase HmsP inversely regulate biofilm formation through control of HmsHFRS-dependent poly-β-1,6-N-acetylglucosamine synthesis. Here, we systematically examine the functionality of the genes encoding putative c-di-GMP metabolic enzymes in Yersinia pestis. We determine that, in addition to hmsT and hmsP, only the gene y3730 encodes a functional enzyme capable of synthesizing c-di-GMP. The seven remaining genes are pseudogenes or encode proteins that do not function catalytically or are not expressed. Furthermore, we show that HmsP has c-di-GMP-specific phosphodiesterase activity. We report that a mutant incapable of c-di-GMP synthesis is unaffected in virulence in plague mouse models. Conversely, an hmsP mutant, unable to degrade c-di-GMP, is defective in virulence by a subcutaneous route of infection due to poly-β-1,6-N-acetylglucosamine overproduction. This suggests that c-di-GMP signalling is not only dispensable but deleterious for Y. pestis virulence. Our results show that a key event in the evolution of Y. pestis from the ancestral Yersinia pseudotuberculosis was a significant reduction in the complexity of its c-di-GMP signalling network likely resulting from the different disease cycles of these human pathogens.     

Loss of a biofilm-inhibiting glycosyl hydrolase during the emergence of Yersinia pestis

Yersinia pestis, the bacterial agent of plague, forms a biofilm in the foregut of its flea vector to produce a transmissible infection. The closely related Yersinia pseudotuberculosis, from which Y. pestis recently evolved, can colonize the flea midgut but does not form a biofilm in the foregut. Y. pestis biofilm in the flea and in vitro is dependent on an extracellular matrix synthesized by products of the hms genes; identical genes are present in Y. pseudotuberculosis. The Yersinia Hms proteins contain functional domains present in Escherichia coli and Staphylococcus proteins known to synthesize a poly-beta-1,6-N-acetyl-D-glucosamine biofilm matrix. In this study, we show that the extracellular matrices (ECM) of Y. pestis and staphylococcal biofilms are antigenically related, indicating a similar biochemical structure. We also characterized a glycosyl hydrolase (NghA) of Y. pseudotuberculosis that cleaved beta-linked N-acetylglucosamine residues and reduced biofilm formation by staphylococci and Y. pestis in vitro. The Y. pestis nghA ortholog is a pseudogene, and overexpression of functional nghA reduced ECM surface accumulation and inhibited the ability of Y. pestis to produce biofilm in the flea foregut. Mutational loss of this glycosidase activity in Y. pestis may have contributed to the recent evolution of flea-borne transmission.     

GGDEF proteins YeaI, YedQ, and YfiN reduce early biofilm formation and swimming motility in Escherichia coli

The second messenger 3′–5′-cyclic diguanylic acid (c-di-GMP) promotes biofilm formation, and c-di-GMP is synthesized by diguanylate cyclases (characterized by a GGDEF domain) and degraded by phosphodiesterases. Here, we evaluated the effect of the 12 E. coli GGDEF-only proteins on biofilm formation and motility. Deletions of the genes encoding the GGDEF proteins YeaI, YedQ, YfiN, YeaJ, and YneF increased swimming motility as expected for strains with reduced c-di-GMP. Alanine substitution in the EGEVF motif of YeaI abolished its impact on swimming motility. In addition, extracellular DNA (eDNA) was increased as expected for the deletions of yeaI (tenfold), yedQ (1.8-fold), and yfiN (3.2-fold). As a result of the significantly enhanced motility, but contrary to current models of decreased biofilm formation with decreased diguanylate cyclase activity, early biofilm formation increased dramatically for the deletions of yeaI (30-fold), yedQ (12-fold), and yfiN (18-fold). Our results indicate that YeaI, YedQ, and YfiN are active diguanylate cyclases that reduce motility, eDNA, and early biofilm formation and contrary to the current paradigm, the results indicate that c-di-GMP levels should be reduced, not increased, for initial biofilm formation so c-di-GMP levels must be regulated in a temporal fashion in biofilms.     

Serotype differences and lack of biofilm formation characterize Yersinia pseudotuberculosis infection of the Xenopsylla cheopis flea vector of Yersinia pestis

Yersinia pestis, the agent of plague, is usually transmitted by fleas. To produce a transmissible infection, Y. pestis colonizes the flea midgut and forms a biofilm in the proventricular valve, which blocks normal blood feeding. The enteropathogen Yersinia pseudotuberculosis, from which Y. pestis recently evolved, is not transmitted by fleas. However, both Y. pestis and Y. pseudotuberculosis form biofilms that adhere to the external mouthparts and block feeding of Caenorhabditis elegans nematodes, which has been proposed as a model of Y. pestis-flea interactions. We compared the ability of Y. pestis and Y. pseudotuberculosis to infect the rat flea Xenopsylla cheopis and to produce biofilms in the flea and in vitro. Five of 18 Y. pseudotuberculosis strains, encompassing seven serotypes, including all three serotype O3 strains tested, were unable to stably colonize the flea midgut. The other strains persisted in the flea midgut for 4 weeks but did not increase in numbers, and none of the 18 strains colonized the proventriculus or produced a biofilm in the flea. Y. pseudotuberculosis strains also varied greatly in their ability to produce biofilms in vitro, but there was no correlation between biofilm phenotype in vitro or on the surface of C. elegans and the ability to colonize or block fleas. Our results support a model in which a genetic change in the Y. pseudotuberculosis progenitor of Y. pestis extended its pre-existing ex vivo biofilm-forming ability to the flea gut environment, thus enabling proventricular blockage and efficient flea-borne transmission.     

Epizootological role of fleas in the Gorno-Altai natural plague focus (a review)

Epizootological role of fleas in the Gorno-Altai natural plague focus (Sailugemsk focus) and numerous data on the flea viability are analyzed and generalized. Information concerning the flea natural infectivity with Yersinia pestis altaica is represented. Ecological peculiarities of some flea species parasitizing the main host, Mongolian pika Ochotona pallasi, and nature of their interrelations with Y. pestis are investigated. It is shown that the flea taxocenosis provides the permanent all year-round circulation of Y. pestis in the Gorno-Altai natural focus. Certain combinations of structural elements of the flea taxocenosis have a dominant significance in determination the circulation process at different phases of the annual epizootic cycle.     

A staphylococcal GGDEF domain protein regulates biofilm formation independently of cyclic dimeric GMP

Cyclic dimeric GMP (c-di-GMP) is an important biofilm regulator that allosterically activates enzymes of exopolysaccharide biosynthesis. Proteobacterial genomes usually encode multiple GGDEF domain-containing diguanylate cyclases responsible for c-di-GMP synthesis. In contrast, only one conserved GGDEF domain protein, GdpS (for GGDEF domain protein from Staphylococcus), and a second protein with a highly modified GGDEF domain, GdpP, are present in the sequenced staphylococcal genomes. Here, we investigated the role of GdpS in biofilm formation in Staphylococcus epidermidis. Inactivation of gdpS impaired biofilm formation in medium supplemented with NaCl under static and flow-cell conditions, whereas gdpS overexpression complemented the mutation and enhanced wild-type biofilm development. GdpS increased production of the icaADBC-encoded exopolysaccharide, poly-N-acetyl-glucosamine, by elevating icaADBC mRNA levels. Unexpectedly, c-di-GMP synthesis was found to be irrelevant for the ability of GdpS to elevate icaADBC expression. Mutagenesis of the GGEEF motif essential for diguanylate cyclase activity did not impair GdpS, and the N-terminal fragment of GdpS lacking the GGDEF domain partially complemented the gdpS mutation. Furthermore, heterologous diguanylate cyclases expressed in trans failed to complement the gdpS mutation, and the purified GGDEF domain from GdpS possessed no diguanylate cyclase activity in vitro. The gdpS gene from Staphylococcus aureus exhibited similar characteristics to its S. epidermidis ortholog, suggesting that the GdpS-mediated signal transduction is conserved in staphylococci. Therefore, GdpS affects biofilm formation through a novel c-di-GMP-independent mechanism involving increased icaADBC mRNA levels and exopolysaccharide biosynthesis. Our data raise the possibility that staphylococci cannot synthesize c-di-GMP and have only remnants of a c-di-GMP signaling pathway.     

High-frequency conjugative transfer of antibiotic resistance genes to Yersinia pestis in the flea midgut

The acquisition of foreign DNA by horizontal transfer from unrelated organisms is a major source of variation leading to new strains of bacterial pathogens. The extent to which this occurs varies widely, due in part to lifestyle factors that determine exposure to potential donors. Yersinia pestis, the plague bacillus, infects normally sterile sites in its mammalian host, but forms dense aggregates in the non-sterile digestive tract of its flea vector to produce a transmissible infection. Here we show that unrelated co-infecting bacteria in the flea midgut are readily incorporated into these aggregates, and that this close physical contact leads to high-frequency conjugative genetic exchange. Transfer of an antibiotic resistance plasmid from an Escherichia coli donor to Y. pestis occurred in the flea midgut at a frequency of 10-3 after only 3 days of co-infection, and after 4 weeks 95% of co-infected fleas contained an average of 103 antibiotic-resistant Y. pestis transconjugants. Thus, transit in its arthropod vector exposes Y. pestis to favourable conditions for efficient genetic exchange with microbial flora of the flea gut. Horizontal gene transfer in the flea may be the source of antibiotic-resistant Y. pestis strains recently isolated from plague patients in Madagascar.