Levels Of Cyclic Diguanylate Research Articles

Design and Synthesis of Novel c-di-GMP G-Quadruplex Inducers as Bacterial Biofilm Inhibitors

The formation of biofilms by clinical pathogens typically leads to chronic and recurring antibiotic-resistant infections. High cellular levels of cyclic diguanylate (c-di-GMP), a ubiquitous secondary messenger of bacteria, have been proven to be associated with a sessile biofilm lifestyle of pathogens. A promising antibiofilm strategy involving the induction of c-di-GMP to form dysfunctional G-quadruplexes, thereby blocking the c-di-GMP-mediated biofilm regulatory pathway, was proposed in this study. In this new strategy, a series of novel c-di-GMP G-quadruplex inducers were designed and synthesized for development of therapeutic biofilm inhibitors. Compound 5h exhibited favorable c-di-GMP G-quadruplex-inducing activity and 62.18 ± 6.76% biofilm inhibitory activity at 1.25 μM without any DNA intercalation effect. Moreover, the favorable performance of 5h in interfering with c-di-GMP-related biological functions, including bacterial motility and bacterial extracellular polysaccharide secretion, combined with the reporter strain and transcriptome analysis results confirmed the c-di-GMP signaling-related action mechanism of 5h.

A partner-switching system controls activation of mixed-linkage β-glucan synthesis by c-di-GMP in Sinorhizobium meliloti.

Sinorhizobium meliloti synthesizes a linear mixed-linkage (1 → 3)(1 → 4)-β-d-glucan (ML β-glucan, MLG) in response to high levels of cyclic diguanylate (c-di-GMP). Two proteins BgsA and BgsB are required for MLG synthesis, BgsA being the glucan synthase which is activated upon c-di-GMP binding to its C-terminal domain. Here we report that the product of bgrR (SMb20447) is a diguanylate cyclase (DGC) that provides c-di-GMP for the synthesis of MLG by BgsA. bgrR is the first gene of a hexacistronic bgrRSTUWV operon, likely encoding a partner-switching regulatory network where BgrR is the final target. Using different approaches, we have determined that the products of genes bgrU (containing a putative PP2C serine phosphatase domain) and bgrW (with predicted kinase effector domain), modulate the phosphorylation status and the activity of the STAS domain protein BgrV. We propose that unphosphorylated BgrV inhibits BgrR DGC activity, perhaps through direct protein-protein interactions as established for other partner switchers. A bgrRSTUWV operon coexists with MLG structural bgsBA genes in many rhizobial genomes but is also present in some MLG non-producers, suggesting a role of this partner-switching system in other processes besides MLG biosynthesis.

Complex Interplay between FleQ, Cyclic Diguanylate and Multiple σ Factors Coordinately Regulates Flagellar Motility and Biofilm Development in Pseudomonas putida.

Most bacteria alternate between a free living planktonic lifestyle and the formation of structured surface-associated communities named biofilms. The transition between these two lifestyles requires a precise and timely regulation of the factors involved in each of the stages that has been likened to a developmental process. Here we characterize the involvement of the transcriptional regulator FleQ and the second messenger cyclic diguanylate in the coordinate regulation of multiple functions related to motility and surface colonization in Pseudomonas putida. Disruption of fleQ caused strong defects in flagellar motility, biofilm formation and surface attachment, and the ability of this mutation to suppress multiple biofilm-related phenotypes associated to cyclic diguanylate overproduction suggests that FleQ mediates cyclic diguanylate signaling critical to biofilm growth. We have constructed a library containing 94 promoters potentially involved in motility and biofilm development fused to gfp and lacZ, screened this library for FleQ and cyclic diguanylate regulation, and assessed the involvement of alternative σ factors σN and FliA in the transcription of FleQ-regulated promoters. Our results suggest a dual mode of action for FleQ. Low cyclic diguanylate levels favor FleQ interaction with σN-dependent promoters to activate the flagellar cascade, encompassing the flagellar cluster and additional genes involved in cyclic diguanylate metabolism, signal transduction and gene regulation. On the other hand, characterization of the FleQ-regulated σN- and FliA-independent PlapA and PbcsD promoters revealed two disparate regulatory mechanisms leading to a similar outcome: the synthesis of biofilm matrix components in response to increased cyclic diguanylate levels.

Raffinose, a plant galactoside, inhibits Pseudomonas aeruginosa biofilm formation via binding to LecA and decreasing cellular cyclic diguanylate levels.

Biofilm formation on biotic or abiotic surfaces has unwanted consequences in medical, clinical, and industrial settings. Treatments with antibiotics or biocides are often ineffective in eradicating biofilms. Promising alternatives to conventional agents are biofilm-inhibiting compounds regulating biofilm development without toxicity to growth. Here, we screened a biofilm inhibitor, raffinose, derived from ginger. Raffinose, a galactotrisaccharide, showed efficient biofilm inhibition of Pseudomonas aeruginosa without impairing its growth. Raffinose also affected various phenotypes such as colony morphology, matrix formation, and swarming motility. Binding of raffinose to a carbohydrate-binding protein called LecA was the cause of biofilm inhibition and altered phenotypes. Furthermore, raffinose reduced the concentration of the second messenger, cyclic diguanylate (c-di-GMP), by increased activity of a c-di-GMP specific phosphodiesterase. The ability of raffinose to inhibit P. aeruginosa biofilm formation and its molecular mechanism opens new possibilities for pharmacological and industrial applications.

The hmsT 3' untranslated region mediates c-di-GMP metabolism and biofilm formation in Yersinia pestis.

Yersinia pestis, the cause of plague, forms a biofilm in the proventriculus of its flea vector to enhance transmission. Biofilm formation in Y. pestis is regulated by the intracellular levels of cyclic diguanylate (c-di-GMP). In this study, we investigated the role of the 3' untranslated region (3'UTR) in hmsT mRNA, a transcript that encodes a diguanylate cyclase that stimulates biofilm formation in Y. pestis by synthesizing the second messenger c-di-GMP. Deletion of the 3'UTR increased the half-life of hmsT mRNA, thereby upregulating c-di-GMP levels and biofilm formation. Our findings indicate that multiple regulatory sequences might be present in the hmsT 3'UTR that function together to mediate mRNA turnover. We also found that polynucleotide phosphorylase is partially responsible for hmsT 3'UTR-mediated mRNA decay. In addition, the hmsT 3'UTR strongly repressed gene expression at 37°C and 26°C, but affected gene expression only slightly at 21°C. Our findings suggest that the 3'UTR might be involved in precise and rapid regulation of hmsT expression, allowing Y. pestis to fine-tune c-di-GMP synthesis and consequently regulate biofilm production to adapt to the changing host environment.

The LonA Protease Regulates Biofilm Formation, Motility, Virulence, and the Type VI Secretion System in Vibrio cholerae.

The presence of the Lon protease in all three domains of life hints at its biological importance. The prokaryotic Lon protease is responsible not only for degrading abnormal proteins but also for carrying out the proteolytic regulation of specific protein targets. Posttranslational regulation by Lon is known to affect a variety of physiological traits in many bacteria, including biofilm formation, motility, and virulence. Here, we identify the regulatory roles of LonA in the human pathogen Vibrio cholerae. We determined that the absence of LonA adversely affects biofilm formation, increases swimming motility, and influences intracellular levels of cyclic diguanylate. Whole-genome expression analysis revealed that the message abundance of genes involved in biofilm formation was decreased but that the message abundances of those involved in virulence and the type VI secretion system were increased in a lonA mutant compared to the wild type. We further demonstrated that a lonA mutant displays an increase in type VI secretion system activity and is markedly defective in colonization of the infant mouse. These findings suggest that LonA plays a critical role in the environmental survival and virulence of V. cholerae. Bacteria utilize intracellular proteases to degrade damaged proteins and adapt to changing environments. The Lon protease has been shown to be important for environmental adaptation and plays a crucial role in regulating the motility, biofilm formation, and virulence of numerous plant and animal pathogens. We find that LonA of the human pathogen V. cholerae is in line with this trend, as the deletion of LonA leads to hypermotility and defects in both biofilm formation and colonization of the infant mouse. In addition, we show that LonA regulates levels of cyclic diguanylate and the type VI secretion system. Our observations add to the known regulatory repertoire of the Lon protease and the current understanding of V. cholerae physiology.

The surface charge of anti-bacterial coatings alters motility and biofilm architecture.

Bacterial biofilms affect many areas of human activity including food processing, transportation, public infrastructure, and most importantly healthcare. This study addresses the prevention of biofilms and shows that the surface charge of an abiotic substrate influences bacterial motility as well as the morphology and physiology of the biofilm. Grafting-from polymerisation was used to create polymer brush surfaces with different characteristics, and the development of Pseudomonas aeruginosa biofilms was followed using confocal microscopy. Interestingly, two types of biofilms developed on these surfaces: mushroom structures with high levels of cyclic diguanylate (c-di-GMP) were found on negatively charged poly (3-sulphopropylmethacrylate) (SPM) and zwitterionic poly (2-(methacryloyloxy)ethyl)dimethyl-3-sulphoproyl) ammonium hydroxide) (MEDSAH), while flat biofilms developed on glass, positively charged poly (2-(methacryloyloxy)-ethyl trimethyl ammonium chloride) (METAC), protein-repellent poly oligo(ethylene glycol methyl ether methacrylate) (POEGMA) and hydrophobic polymethylmethacrylate (PMMA). The results show that of all the surfaces studied, overall the negatively charged polymer brushes were most efficient in reducing bacterial adhesion and biofilm formation. However, the increased level of regulatory c-di-GMP in mushroom structures suggests that bacteria are capable of a quick physiological response when exposed to surfaces with varying physicochemical characteristics enabling some bacterial colonization also on negatively charged surfaces.

A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels

Pseudomonas aeruginosa causes chronic biofilm infections, and its ability to attach to surfaces and other cells is important for biofilm formation and maintenance. Mutations in a gene called wspF, part of a putative chemosensory signal-transduction operon, have been shown to result in cell aggregation and altered colony morphology. The WspF phenotypes depend on the presence of WspR, which is a member of a family of signal transduction proteins known as response regulators. It is likely that the effect of the wspF mutation is to cause constitutive activation of WspR by phosphorylation. WspR contains a GGDEF domain known to catalyze formation of a cytoplasmic signaling molecule cyclic diguanylate (c-diGMP). We determined that purified WspR catalyzed the formation of c-diGMP in vitro and phosphorylation stimulated this activity. We observed increased cellular levels of c-diGMP and increased biofilm formation in a wspF mutant. Expression of a protein predicted to catalyze degradation of c-diGMP reversed the phenotypes of a wspF mutant and inhibited biofilm initiation by wild-type cells, indicating that the presence of c-diGMP is necessary for biofilm formation. A transcriptome analysis showed that expression levels of at least 560 genes were affected by a wspF deletion. The psl and pel operons, which are involved in exopolysaccharide production and biofilm formation, were expressed at high levels in a wspF mutant. Together, the data suggest that the wsp signal transduction pathway regulates biofilm formation through modulation of cyclic diguanylate levels.