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。     

Cyclic di-GMP signalling in the virulence and environmental adaptation of Xanthomonas campestris

Cyclic di-GMP is a second messenger with a role in regulation of a range of cellular functions in diverse bacteria including the virulence of pathogens. Cellular levels of cyclic di-GMP are controlled through synthesis, catalysed by the GGDEF protein domain, and degradation by EAL or HD-GYP domains. Here we report a comprehensive study of cyclic di-GMP signalling in bacterial disease in which we examine the contribution of all proteins with GGDEF, EAL or HD-GYP domains to virulence and virulence factor production in the phytopathogen Xanthomonas campestris pathovar campestris (Xcc). Genes with significant roles in virulence to plants included those encoding proteins whose probable function is in cyclic-di-GMP synthesis as well as others (including the HD-GYP domain regulator RpfG) implicated in cyclic di-GMP degradation. Furthermore, RpfG controlled expression of a subset of these genes. A partially overlapping set of elements controlled the production of virulence factors in vitro. Other GGDEF-EAL domain proteins had no effect on virulence factor synthesis but did influence motility. These findings indicate the existence of a regulatory network that may allow Xcc to integrate information from diverse environmental inputs to modulate virulence factor synthesis as well as of cyclic di-GMP signalling systems dedicated to other specific tasks.     

Bacterial Signal Transduction by Cyclic Di-GMP and Other Nucleotide Second Messengers

The first International Symposium on c-Di-GMP Signaling in Bacteria (22 to 25 March 2015, Harnack-Haus, Berlin, Germany) brought together 131 molecular microbiologists from 17 countries to discuss recent progress in our knowledge of bacterial nucleotide second messenger signaling. While the focus was on signal input, synthesis, degradation, and the striking diversity of the modes of action of the current second messenger paradigm, i.e., cyclic di-GMP (c-di-GMP), “classics” like cAMP and (p)ppGpp were also presented, in novel facets, and more recent “newcomers,” such as c-di-AMP and c-AMP-GMP, made an impressive appearance. A number of clear trends emerged during the 30 talks, on the 71 posters, and in the lively discussions, including (i) c-di-GMP control of the activities of various ATPases and phosphorylation cascades, (ii) extensive cross talk between c-di-GMP and other nucleotide second messenger signaling pathways, and (iii) a stunning number of novel effectors for nucleotide second messengers that surprisingly include some long-known master regulators of developmental pathways. Overall, the conference made it amply clear that second messenger signaling is currently one of the most dynamic fields within molecular microbiology, with major impacts in research fields ranging from human health to microbial ecology.     

The HD-GYP domain, cyclic di-GMP signaling, and bacterial virulence to plants

Cyclic di-GMP is an almost ubiquitous second messenger in bacteria that was first described as an allosteric activator of cellulose synthase but is now known to regulate a range of functions, including virulence in human and animal pathogens. Two protein domains, GGDEF and EAL, are implicated in the synthesis and degradation, respectively, of cyclic di-GMP. These domains are widely distributed in bacteria, including plant pathogens. The majority of proteins with GGDEF and EAL domains contain additional signal input domains, suggesting that their activities are responsive to environmental cues. Recent studies have demonstrated that a third domain, HD-GYP, is also active in cyclic di-GMP degradation. In the plant pathogen Xanthomonas campestris pv. campestris, a two-component signal transduction system comprising the HD-GYP domain regulatory protein RpfG and cognate sensor RpfC positively controls virulence. The signals recognized by RpfC may include the cell-cell signal DSF, which also acts to regulate virulence in X. campestris pv. campestris. Here, we review these recent advances in our understanding of cyclic di-GMP signaling with particular reference to one or more roles in the bacterial pathogenesis of plants.     

Capturing cyclic nucleotides in action: snapshots from crystallographic studies

Fifty years ago, cyclic AMP was discovered as a second messenger of hormone action, heralding the age of signal transduction. Many cellular processes were found to be regulated by cAMP and the related cyclic GMP. Cyclic nucleotides function by binding to and activating their effectors - protein kinase A, protein kinase G, cyclic-nucleotide-regulated ion channels and the guanine nucleotide-exchange factor Epac. Recent structural insights have now made it possible to propose a general structural mechanism for how cyclic nucleotides regulate these proteins.     

Characteristics of Biofilms from Urinary Tract Catheters and Presence of Biofilm-Related Components in Escherichia coli

Long term catheterization of the urinary tract leads to bacterial colonization of the urine, whereby adherence to the catheter surface is a major determinative factor for colonization. Collection of bacterial isolates from urine and urinary catheters of 45 patients showed multi-species catheter-colonization, while Escherichia coli isolates were frequently found in the urine in high numbers. Biofilm formation of catheter and urine-derived E. coli isolates was associated with the presence of the fluA gene, loss of O-antigen, and expression of type 1 fimbriae. The second messenger cyclic di-GMP (cdiGMP), a major regulator of biofilm formation, regulated adherence to the catheter surface in a selected clinical isolate suggesting that the cdiGMP second messenger pathway may be a target for anti-biofilm therapeutic approaches.     

Second messengers and divergent HD-GYP phosphodiesterases regulate 3′,3′-cGAMP signaling

3′,3′-cyclic GMP-AMP (cGAMP) is the third cyclic dinucleotide (CDN) to be discovered in bacteria. No activators of cGAMP signaling have yet been identified, and the signaling pathways for cGAMP have been inferred to display a narrow distribution based upon the characterized synthases, DncV and Hypr GGDEFs. Here, we report that the ubiquitous second messenger cyclic AMP (cAMP) is an activator of the Hypr GGDEF enzyme GacB from Myxococcus xanthus. Furthermore, we show that GacB is inhibited directly by cyclic di-GMP, which provides evidence for cross-regulation between different CDN pathways. Finally, we reveal that the HD-GYP enzyme PmxA is a cGAMP-specific phosphodiesterase (GAP) that promotes resistance to osmotic stress in M. xanthus. A signature amino acid change in PmxA was found to reprogram substrate specificity and was applied to predict the presence of non-canonical HD-GYP phosphodiesterases in many bacterial species, including phyla previously not known to utilize cGAMP signaling.     

Synthesis and Characterization of a Fluorescent Analogue of Cyclic di-GMP

Cyclic di-GMP (c-di-GMP), a ubiquitous bacterial second messenger, has emerged as a key controller of several biological processes. Numbers of reports that deal with the mechanistic aspects of this second messenger have appeared in the literature. However, the lack of a reporter tag attached to the c-di-GMP at times limits the understanding of further details. In this study, we have chemically coupled N-methylisatoic anhydride (MANT) with c-di-GMP, giving rise to Mant-(c-di-GMP) or MANT-CDG. We have characterized the chemical and physical properties and spectral behavior of MANT-CDG. The fluorescence of MANT-CDG is sensitive to changes in the microenvironment, which helped us study its interaction with three different c-di-GMP binding proteins (a diguanylate cyclase, a phosphodiesterase, and a PilZ domain-containing protein). In addition, we have shown here that MANT-CDG can inhibit diguanylate cyclase activity; however, it is hydrolyzed by c-di-GMP specific phosphodiesterase. Taken together, our data suggest that MANT-CDG behaves like native c-di-GMP, and this study raises the possibility that MANT-CDG will be a valuable research tool for the in vitro characterization of c-di-GMP signaling factors.     

Phenotypic convergence mediated by GGDEF-domain-containing proteins

GGDEF domain-containing proteins have been implicated in bacterial signal transduction and synthesis of the second messenger molecule cyclic-di-GMP. A number of GGDEF proteins are involved in controlling the formation of extracellular matrices. AdrA (Salmonella enterica serovar Typhimurium) and HmsT (Yersinia pestis) contain GGDEF domains and are required for extracellular cellulose production and biofilm formation, respectively. Here we show that hmsT is able to restore cellulose synthesis to a Salmonella serovar Typhimurium adrA mutant and that adrA can replace hmsT in Y. pestis Hms-dependent biofilm formation. Like Y. pestis HmsT overproducers, Y. pestis cells carrying adrA under the control of an arabinose-inducible promoter produced substantial biofilms in the presence of arabinose. Finally, we demonstrate that HmsT is involved in the synthesis of cyclic di-GMP.     

A novel capture compound for the identification and analysis of cyclic di-GMP binding proteins

The second messenger cyclic di-GMP is a near-ubiquitous signaling molecule that globally alters bacterial cell physiology to promote biofilm formation and community behavior. Much progress was made in recent years towards the identification and characterization of diguanylate cyclases and phosphodiersterases, enzymes involved in the synthesis and degradation of this signaling compound. In contrast, our knowledge of the nature and mechanistic details of c-di-GMP effector proteins lags behind, primarily because effective tools for their specific enrichment and rapid analysis are missing. In this report we demonstrate that a novel tri-functional c-di-GMP-specific Capture Compound (cdG-CC) can be effectively used to identify and validate c-di-GMP binding proteins. The cdG-CC was able to specifically and efficiently pull down bona fide c-di-GMP effector proteins. Furthermore, in combination with mass spectrometry (CCMS), this technology robustly identified a substantial fraction of the known c-di-GMP signaling components directly from cell extracts of different model organisms. Finally, we applied the CCMS technique to profile c-di-GMP binding proteins of Pseudomonas aeruginosa and Salmonella enterica serovar typhimurium. Our studies establish CCMS as a powerful and versatile tool to identify and analyze components of the cellular c-di-GMP pathway in a wide range of different organisms.     

Inherent Regulation of EAL Domain-catalyzed Hydrolysis of Second Messenger Cyclic di-GMP

The universal second messenger cyclic di-GMP (cdG) is involved in the regulation of a diverse range of cellular processes in bacteria. The intracellular concentration of the dinucleotide is determined by the opposing actions of diguanylate cyclases and cdG-specific phosphodiesterases (PDEs). Whereas most PDEs have accessory domains that are involved in the regulation of their activity, the regulatory mechanism of this class of enzymes has remained unclear. Here, we use biophysical and functional analyses to show that the isolated EAL domain of a PDE from Escherichia coli (YahA) is in a fast thermodynamic monomer-dimer equilibrium, and that the domain is active only in its dimeric state. Furthermore, our data indicate thermodynamic coupling between substrate binding and EAL dimerization with the dimerization affinity being increased about 100-fold upon substrate binding. Crystal structures of the YahA-EAL domain determined under various conditions (apo, Mg²⁺, cdG·Ca²⁺ complex) confirm structural coupling between the dimer interface and the catalytic center. The built-in regulatory properties of the EAL domain probably facilitate its modular, functional combination with the diverse repertoire of accessory domains.     

A-kinase anchoring proteins: scaffolding proteins in the heart

The pleiotropic cyclic nucleotide cAMP is the primary second messenger responsible for autonomic regulation of cardiac inotropy, chronotropy, and lusitropy. Under conditions of prolonged catecholaminergic stimulation, cAMP also contributes to the induction of both cardiac myocyte hypertrophy and apoptosis. The formation of localized, multiprotein complexes that contain different combinations of cAMP effectors and regulatory enzymes provides the architectural infrastructure for the specialization of the cAMP signaling network. Scaffolds that bind protein kinase A are called "A-kinase anchoring proteins" (AKAPs). In this review, we discuss recent advances in our understanding of how PKA is compartmentalized within the cardiac myocyte by AKAPs and how AKAP complexes modulate cardiac function in both health and disease.     

Binding of bacterial secondary messenger molecule c di-GMP is a STING operation

Initial skirmishes between the host and pathogen result in spillage of the contents of the bacterial cell. Amongst the spillage, the secondary messenger molecule, cyclic dimeric guanosine monophosphate (c di-GMP), was recently shown to be bound by stimulator of interferon genes (STING). Binding of c di-GMP by STING activates the Tank Binding Kinase (TBK1) mediated signaling cascades that galvanize the body' defenses for elimination of the pathogen. In addition to c di-GMP, STING has also been shown to function in innate immune responses against pathogen associated molecular patterns (PAMPs) originating from the DNA or RNA of pathogens. The pivotal role of STING in host defense is exemplified by the fact that STING^-/- mice die upon infection by HSV-1. Thus, STING plays an essential role in innate immune responses against pathogens. This opens up an exciting possibility of targeting STING for development of adjuvant therapies to boost the immune defenses against invading microbes. Similarly, STING could be targeted for mitigating the inflammatory responses augmented by the innate immune system. This review summarizes and updates our current understanding of the role of STING in innate immune responses and discusses the future challenges in delineating the mechanism of STING-mediated responses.