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.     

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。     

鼠疫耶尔森氏菌环二鸟苷酸代谢及生物被膜形成调控研究进展

鼠疫耶尔森氏菌(Yersinia pestis,以下简称"鼠疫菌")是烈性传染病鼠疫的病原菌,以鼠蚤作为传播媒介。鼠疫菌在其传播媒介鼠蚤的前胃中形成生物被膜从而促进其在宿主间传播。鼠疫菌生物被膜的形成受第二信使分子环二鸟苷酸(c-di-GMP)的正向调控。鼠疫菌中c-di-GMP由二鸟苷酸环化酶(DGC)HmsT和HmsD合成,由磷酸二酯酶(PDE)HmsP降解。文中主要介绍影响鼠疫菌环二鸟苷酸代谢及生物被膜形成的调控因子,并对其作用机制进行讨论和总结。     

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。     

c-di-AMP调控细菌生物被膜的形成

细菌生物被膜是细菌持续性致病的重要机制。研究细菌生物被膜的形成和发展可为顽固性细菌感染防治提供新的思路与策略。环二腺苷酸c-di-AMP(Cyclic diadenosine monophosphate)是继c-di-GMP之后在细菌中新发现的一种核苷酸第二信使分子。研究发现,c-di-AMP参与调节细菌多种生理功能,包括细菌生长代谢、生物被膜形成、细胞壁的合成以及细菌毒力因子等。本文综述了c-di-AMP参与调控细菌生物被膜形成的不同方式及其分子机制。鉴于c-di-AMP在调控细菌生物被膜中的重要性,其可作为抗细菌生物被膜感染新药研发的潜在靶点。     

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。     

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。     

霍乱弧菌生物被膜发育与环境调控

生物被膜状态的霍乱弧菌具有极强的环境适应性和超高的感染性,生物被膜的发育调控研究对霍乱弧菌的宿主感染和环境适应非常重要。本文综述了近年来霍乱弧菌生物被膜研究结果,包括霍乱弧菌生物被膜的组成、发育和环境调控,尤其着重阐述了各种环境因子对霍乱弧菌生物被膜发育的影响,包括细菌自体信号分子、自然环境因子和宿主信号分子。     

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。     

环二鸟苷单磷酸核糖开关的结构与功能

环二鸟苷单磷酸(c-di-GMP)是细菌中广泛存在的一类核苷类第二信使分子,能够调控细菌的生物被膜形成、运动性、黏附、毒力以及胞外多糖的产生等众多生理活动。核糖开关是m RNA 5′-非翻译区(5′-Untranslational region,5′-UTR)的一段RNA序列,包含可以识别并结合配体的保守序列——适配体区(Aptamer domain,AD),以及结构多变、可以调控下游编码基因的表达平台区(Expression platform,EP)。当代谢物分子浓度比较高时,其与适配体区结合,引起下游的表达平台区发生构象变化,进而实现对下游基因的调节。目前已发现c-di-GMP-Ⅰ和c-di-GMP-Ⅱ两类c-di-GMP的核糖开关。它们通过特异性地结合c-di-GMP,调控种类繁多的下游基因的表达。c-di-GMP-I核糖开关分布广泛,尤其在厚壁菌门(Firmicutes)和变形菌门(Proteobacteria)的细菌中最为丰富。c-di-GMP-Ⅱ核糖开关具备变构核酶的功能,结合c-di-GMP后在其非典型剪切位点处发生结构变化,调节下游基因表达。文中围绕c-di-GMP核糖开关的发现、功能、分类以及下游调控基因的功能进行综述与分析。     

The RNA Domain Vc1 Regulates Downstream Gene Expression in Response to Cyclic Diguanylate in Vibrio cholerae

In many bacterial species, including the aquatic bacterium and human pathogen Vibrio cholerae, the second messenger cyclic diguanylate (c-di-GMP) modulates processes such as biofilm formation, motility, and virulence factor production. By interacting with various effectors, c-di-GMP regulates gene expression or protein function. One type of c-di-GMP receptor is the class I riboswitch, representatives of which have been shown to bind c-di-GMP in vitro. Herein, we examined the in vitro and in vivo function of the putative class I riboswitch in Vibrio cholerae, Vc1, which lies upstream of the gene encoding GbpA, a colonization factor that contributes to attachment of V. cholerae to environmental and host surfaces containing N-acetylglucosamine moieties. We provide evidence that Vc1 RNA interacts directly with c-di-GMP in vitro, and that nucleotides conserved among this class of riboswitch are important for binding. Yet the mutation of these conserved residues individually in the V. cholerae chromosome inconsistently affects the expression of gbpA and production of the GbpA protein. By isolating the regulatory function of Vc1, we show that the Vc1 element positively regulates downstream gene expression in response to c-di-GMP. Together these data suggest that the Vc1 element responds to c-di-GMP in vivo. Positive regulation of gbpA expression by c-di-GMP via Vc1 may influence the ability of V. cholerae to associate with chitin in the aquatic environment and the host intestinal environment.     

Structural characterization of a conserved, calcium-dependent periplasmic protease from Legionella pneumophila

The bacterial dinucleotide second messenger c-di-GMP has emerged as a central molecule in regulating bacterial behavior, including motility and biofilm formation. Proteins for the synthesis and degradation of c-di-GMP and effectors for its signal transmission are widely used in the bacterial domain. Previous work established the GGDEF-EAL domain-containing receptor LapD as a central switch in Pseudomonas fluorescens cell adhesion. LapD senses c-di-GMP inside the cytosol and relays this signal to the outside by the differential recruitment of the periplasmic protease LapG. Here we identify the core components of an orthologous system in Legionella pneumophila. Despite only moderate sequence conservation at the protein level, key features concerning the regulation of LapG are retained. The output domain of the LapD-like receptor from L. pneumophila, CdgS9, binds the LapG ortholog involving a strictly conserved surface tryptophan residue. While the endogenous substrate for L. pneumophila LapG is unknown, the enzyme processed the corresponding P. fluorescens substrate, indicating a common catalytic mechanism and substrate recognition. Crystal structures of L. pneumophila LapG provide the first atomic models of bacterial proteases of the DUF920 family and reveal a conserved calcium-binding site important for LapG function.     

c-di-GMP信号途径对细菌致病性的调控作用

环鸟苷二磷酸(c-di-GMP)是一种广泛存在于细菌中的新型第二信使。鸟苷酸环化酶(DGC)和磷酸二脂酶(PDE)分别控制了c-di-GMP的合成和降解, 其中DGC活性由GGDEF结构域决定, PDE活性由EAL和HD-GYP结构域决定。c-di-GMP调控着细菌多种生物学功能, 抑制毒性因子产生和运动性, 促进生物膜形成。c-di-GMP下游是一个包括转录、翻译以及翻译后等多层次的复杂调控网络。本文结合本室有关水稻白叶枯病菌的研究结果, 综述近年来国内外在c-di-GMP研究领域的最新进展。     

A chemical proteomics approach to identify c-di-GMP binding proteins in Pseudomonas aeruginosa

In many bacteria, high levels of the ubiquitous second messenger c-di-GMP have been demonstrated to suppress motility and to promote the establishment of surface-adherent biofilm communities. While molecular mechanisms underlying the synthesis and degradation of c-di-GMP have been comprehensively characterized, little is known about how c-di-GMP mediates its regulatory effects. In this study, we have established a chemical proteomics approach to identify c-di-GMP interacting proteins in the opportunistic pathogen Pseudomonas aeruginosa. A functionalized c-di-GMP analog, 2′-aminohexylcarbamoyl-c-di-GMP (2′-AHC-c-di-GMP), was chemically synthesized and following its immobilization used to perform affinity pull down experiments. Enriched proteins were subsequently identified by high-resolution mass spectrometry. 2′-AHC-c-di-GMP was also employed in surface plasmon resonance studies to evaluate and quantify the interaction of c-di-GMP with its potential target molecules in vitro. The biochemical tools presented here may serve the identification of novel classes of c-di-GMP effectors and thus contribute to a better characterization and understanding of the complex c-di-GMP signaling network.     

Elevated level of the second messenger c-di-GMP in <Emphasis Type="Italic">Comamonas testosteroni</Emphasis> enhances biofilm formation and biofilm-based biodegradation of 3-chloroaniline

The bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous second messenger that determines bacterial lifestyle between the planktonic and biofilm modes of life. Although the role of c-di-GMP signaling in biofilm development and dispersal has been extensively studied, how c-di-GMP signaling influences environmental bioprocess activities such as biodegradation remains unexplored. To elucidate the impacts of elevating c-di-GMP level on environmental bioprocesses, we constructed a Comamonas testosteroni strain constitutively expressing a c-di-GMP synthase YedQ from Escherichia coli and examined its capability in biofilm formation and biodegradation of 3-chloroaniline (3-CA). The high c-di-GMP strain exhibited an increased binding to Congo red dye, a decreased motility, and an enhanced biofilm formation capability. In planktonic cultures, the strain with an elevated c-di-GMP concentration and the wild type could degrade 3-CA comparably well. However, under batch growth conditions with a high surface to volume ratio, an elevated c-di-GMP concentration in C. testosteroni significantly increased the contribution of biofilms in 3-CA biodegradation. In continuous submerged biofilm reactors, C. testosteroni with an elevated c-di-GMP level exhibited an enhanced 3-CA biodegradation and a decreased cell detachment rate. Taken together, this study provides a novel strategy to enhance biofilm-based biodegradation of toxic xenobiotic compounds through manipulating bacterial c-di-GMP signaling.     

The second messenger bis-(3'-5')-cyclic-GMP and its PilZ domain-containing receptor Alg44 are required for alginate biosynthesis in Pseudomonas aeruginosa

The ubiquitous bacterial second messenger c-di-GMP regulates the expression of various virulence determinants in a wide range of bacterial pathogens. Several studies have suggested that proteins with a PilZ domain function as c-di-GMP receptors. We have identified in the Pseudomonas aeruginosa genome eight genes encoding for PilZ orhologues and demonstrated binding of c-di-GMP to all but one of these proteins in a direct ligand binding assay. One protein with the PilZ domain, Alg44, is involved in biosynthesis of the extracellular polysaccharide alginate. We have shown that increasing c-di-GMP levels by overexpression of highly active diguanylate cyclases, or hydrolysis of c-di-GMP by phosphodiesterases, enhanced or reduced formation of alginate in mucoid strains, respectively. We have engineered substitutions in several conserved residues of the PilZ domain of Alg44 determined that they resulted in simultaneous loss of c-di-GMP binding and the ability to support production of alginate in P. aeruginosa. A 6xHis-tagged Alg44 fusion was also shown to localize in the membrane fraction of P. aeruginosa independently from its ability to bind c-di-GMP. Alg44 appears to be an essential component of the alginate biosynthetic apparatus, where, following binding of c-di-GMP, it controls polymerization or transport of the polysaccharide.     

PilZ domain is part of the bacterial c-di-GMP binding protein

Recent studies identified c-di-GMP as a universal bacterial secondary messenger regulating biofilm formation, motility, production of extracellular polysaccharide and multicellular behavior in diverse bacteria. However, except for cellulose synthase, no protein has been shown to bind c-di-GMP and the targets for c-di-GMP action remain unknown. Here we report identification of the PilZ ("pills") domain (Pfam domain PF07238) in the sequences of bacterial cellulose synthases, alginate biosynthesis protein Alg44, proteins of enterobacterial YcgR and firmicute YpfA families, and other proteins encoded in bacterial genomes and present evidence indicating that this domain is (part of) the long-sought c-di-GMP-binding protein. Association of the PilZ domain with a variety of other domains, including likely components of bacterial multidrug secretion system, could provide clues to multiple functions of the c-di-GMP in bacterial pathogenesis and cell development.     

Sensing the messenger: the diverse ways that bacteria signal through c-di-GMP

An intracellular second messenger unique to bacteria, c-di-GMP, has gained appreciation as a key player in adaptation and virulence strategies, such as biofilm formation, persistence, and cytotoxicity. Diguanylate cyclases containing GGDEF domains and phosphodiesterases containing either EAL or HD-GYP domains have been identified as the enzymes controlling intracellular c-di-GMP levels, yet little is known regarding signal transmission and the sensory targets for this signaling molecule. Although limited in number, identified c-di-GMP receptors in bacteria are characterized by prominent diversity and multilevel impact. In addition, c-di-GMP has been shown to have immunomodulatory effects in mammals and several eukaryotic c-di-GMP sensors have been proposed. The structural biology of c-di-GMP receptors is a rapidly developing field of research, which holds promise for the development of novel therapeutics against bacterial infections. In this review, we highlight recent advances in identifying bacterial and eukaryotic c-di-GMP signaling mechanisms and emphasize the need for mechanistic structure-function studies on confirmed signaling targets.