Nitrate Is an Environmental Cue in the Gut for Salmonella enterica Serovar Typhimurium Biofilm Dispersal through Curli Repression and Flagellum Activation via Cyclic-di-GMP Signaling

Curli, a major component of the bacterial biofilms in the intestinal tract, activates pattern recognition receptors and triggers joint inflammation after infection with Salmonella enterica serovar Typhimurium. The factors that allow S. Typhimurium to disperse from biofilms and invade the epithelium to establish a successful infection during acute inflammation remain unknown. Here, we studied S. Typhimurium biofilms in vitro and in vivo to understand how the inflammatory environment regulates the switch between multicellular and motile S. Typhimurium in the gut. We discovered that nitrate generated by the host is an environmental cue that induces S. Typhimurium to disperse from the biofilm. Nitrate represses production of an important biofilm component, curli, and activates flagella via the modulation of intracellular cyclic-di-GMP levels. We conclude that nitrate plays a central role in pathogen fitness by regulating the sessile-to-motile lifestyle switch during infection.
IMPORTANCE Recent studies provided important insight into our understanding of the role of c-di-GMP signaling and the regulation of enteric biofilms. Despite an improved understanding of how c-di-GMP signaling regulates S. Typhimurium biofilms, the processes that affect the intracellular c-di-GMP levels and the formation of multicellular communities in vivo during infections remain unknown. Here, we show that nitrate generated in the intestinal lumen during infection with S. Typhimurium is an important regulator of biofilm formation in vivo.

Second Messenger 2’3′-cyclic GMP-AMP (2’3′-cGAMP):Synthesis, transmission, and degradation

Cyclic GMP-AMP synthase (cGAS) senses foreign DNA to produce 2’3′-cyclic GMP-AMP (2’3′-cGAMP). 2’3′-cGAMP is a second messenger that binds and activates the adaptor protein STING, which triggers the innate immune response. As a STING agonist, the small molecule 2’3′-cGAMP plays pivotal roles in antiviral defense and has adjuvant applications, and anti-tumor effects. 2’3′-cGAMP and its analogs are thus putative targets for immunotherapy and are currently being testedin clinical trials to treat solid tumors.
However, several barriers to further development have emerged from these studies, such as evidence of immune and inflammatory side-effects, poor pharmacokinetics, and undesirable biodistribution. Here, we review the status of 2’3′-cGAMP research and outline the role of 2’3′-cGAMP in immune signaling, adjuvant applications, and cancer immunotherapy, as well as various 2’3′-cGAMP detection methods.

A pGpG-specific phosphodiesterase regulates cyclic di-GMP signaling in Vibrio cholerae

The bacterial second messenger cyclic diguanylate monophosphate (c-di-GMP) controls various cellular processes, including motility, toxin production and biofilm formation. c-di-GMP is enzymatically synthesized by GGDEF domain-containing diguanylate cyclases and degraded by HD-GYP domain-containing phosphodiesterases (PDEs) to 2 GMP or by EAL domain-containing PDE-As to 5′-phosphoguanylyl-(3′,5′)-guanosine (pGpG). Since excess pGpG feedback inhibits PDE-A activity and thereby can lead to the uncontrolled accumulation of c-di-GMP, a PDE that degrades pGpG to 2 GMP (PDE-B) has been presumed to exist.
To date, the only enzyme known to hydrolyze pGpG is Oligoribonuclease Orn, which degrades all kinds of oligoribonucleotides. Here, we identified a pGpG-specific PDE, which we named PggH, using biochemical approaches in the gram-negative bacteria Vibrio cholerae. Biochemical experiments revealed that PggH exhibited specific PDE activity only toward pGpG, thus differing from the previously reported Orn. Furthermore, the high-resolution structure of PggH revealed the basis for its PDE activity and narrow substrate specificity. Finally, we propose that PggH could modulate the activities of PDE-As and the intracellular concentration of c-di-GMP, resulting in phenotypic changes including in biofilm formation.

The Campylobacter jejuni Response Regulator and Cyclic-Di-GMP Binding CbrR Is a Novel Regulator of Flagellar Motility

A leading cause of bacterial gastroenteritis, Campylobacter jejuni is also associated with broad sequelae, including extragastrointestinal conditions such as reactive arthritis and Guillain-Barré Syndrome (GBS). CbrR is a C. jejuni response regulator that is annotated as a diguanylate cyclase (DGC), an enzyme that catalyzes the synthesis of c-di-GMP, a universal bacterial second messenger, from GTP. In C. jejuni DRH212, we constructed an unmarked deletion mutant, cbrR, and complemented mutant, cbrR+. Motility assays indicated a hyper-motile phenotype associated with cbrR, whereas motility was deficient in cbrR+.
The overexpression of CbrR in cbrR+ was accompanied by a reduction in expression of FlaA, the major flagellin. Biofilm assays and scanning electron microscopy demonstrated similarities between DRH212 and cbrR; however, cbrR+ was unable to form significant biofilms. Transmission electron microscopy showed similar cell morphology between the three strains; however, cbrR+ cells lacked flagella. Differential radial capillary action of ligand assays (DRaCALA) showed that CbrR binds GTP and c-di-GMP. Liquid chromatography tandem mass spectrometry detected low levels of c-di-GMP in C. jejuni and in E. coli expressing CbrR. CbrR is therefore a negative regulator of FlaA expression and motility, a critical virulence factor in C. jejuni pathogenesis.

The Regulatory Network of Cyclic GMP-AMP Synthase-Stimulator of Interferon Genes Pathway in Viral Evasion

Virus infection has been consistently threatening public health. The cyclic GMP-AMP synthase (cGAS)-Stimulator of Interferon Genes (STING) pathway is a critical defender to sense various pathogens and trigger innate immunity of mammalian cells. cGAS recognizes the pathogenic DNA in the cytosol and then synthesizes 2’3′-cyclic GMP-AMP (2’3’cGAMP). As the second messenger, cGAMP activates STING and induces the following cascade to produce type I interferon (IFN-I) to protect against infections. However, viruses have evolved numerous strategies to hinder the cGAS-STING signal transduction, promoting their immune evasion.
Here we outline the current status of the viral evasion mechanism underlying the regulation of the cGAS-STING pathway, focusing on how post-transcriptional modifications, viral proteins, and non-coding RNAs involve innate immunity during viral infection, attempting to inspire new targets discovery and uncover potential clinical antiviral treatments.

cGMP (cyclic GMP)

MBS6507358-005mL MyBiosource 0.05mL 835 EUR

cGMP (cyclic GMP)

MBS6507358-5x005mL MyBiosource 5x0.05mL 3600 EUR

Cyclic GMP (TBAOH)

HY-113469B MedChemExpress 10 mg 70.35 EUR

Cyclic GMP (sodium)

HY-113469A MedChemExpress 10 mg 70.35 EUR

Cyclic GMP Antibody

abx022640-02ml Abbexa 0.2 ml 1128 EUR

Cyclic GMP Antibody

GWB-BA5CC2 GenWay Biotech 0.2 ml Ask for price

Cyclic GMP Antibody

GWB-DFA25F GenWay Biotech 2 ml Ask for price

Cyclic GMP EIA Kit

SKT-211-96 Stressmarq 1 plate of 96 wells 568.8 EUR


MBS222513-2mL MyBiosource 2mL 455 EUR


MBS222513-5x2mL MyBiosource 5x2mL 1870 EUR

Sheep anti Cyclic GMP

MBS316214-02mL MyBiosource 0.2mL 1235 EUR

Sheep anti Cyclic GMP

MBS316214-5x02mL MyBiosource 5x0.2mL 5380 EUR

Cyclic GMP (Direct) ELISA

MBS494508-5x96Tests MyBiosource 5x96Tests 3410 EUR

Cyclic GMP (Direct) ELISA

MBS494508-96Tests MyBiosource 96Tests 740 EUR

Cyclic GMP Standard, 125UL

C080-125UL Arbor Assays 125UL 85 EUR

Cyclic GMP Standard, 350UL

C080-350UL Arbor Assays 350UL 207 EUR

Cyclic GMP Standard, 625UL

C080-625UL Arbor Assays 625UL 207 EUR

Cyclic GMP Standard, 70UL

C080-70UL Arbor Assays 70UL 85 EUR

Anti- Cyclic GMP Antibody

GWB-FDE020 GenWay Biotech 1000 TESTS Ask for price

Cyclic GMP Polyclonal Antibody

MBS194212-02mL MyBiosource 0.2mL 560 EUR

Cyclic GMP Polyclonal Antibody

MBS194212-5x02mL MyBiosource 5x0.2mL 2510 EUR


MBS2516175-10x96Tests MyBiosource 10x96Tests 3265 EUR


MBS2516175-24Tests MyBiosource 24Tests 225 EUR


MBS2516175-48Test MyBiosource 48Test 350 EUR


MBS2516175-5x96Test MyBiosource 5x96Test 1655 EUR


MBS2516175-96Tests MyBiosource 96Tests 395 EUR

ELISA kit for cGMP (Cyclic GMP)

E-EL-0083 Elabscience Biotech 1 plate of 96 wells 452.4 EUR

DetectX® Cyclic GMP Antibody, 3ML

C078-3ML Arbor Assays 3ML 218 EUR

BldD-based bimolecular fluorescence complementation for in vivo detection of the second messenger cyclic di-GMP

The widespread bacterial second messenger bis-(3′-5′)-cyclic diguanosine monophosphate (c-di-GMP) is an important regulator of biofilm formation, virulence and cell differentiation. C-di-GMP-specific biosensors that allow detection and visualization of c-di-GMP levels in living cells are key to our understanding of how c-di-GMP fluctuations drive cellular responses. Here, we describe a novel c-di-GMP biosensor, CensYBL, that is based on c-di-GMP-induced dimerization of the effector protein BldD from Streptomyces resulting in bimolecular fluorescence complementation of split-YPet fusion proteins.
As a proof-of-principle, we demonstrate that CensYBL is functional in detecting fluctuations in intracellular c-di-GMP levels in the Gram-negative model bacteria Escherichia coli and Salmonella enterica serovar Typhimurium. Using deletion mutants of c-di-GMP diguanylate cyclases and phosphodiesterases, we show that c-di-GMP dependent dimerization of CBldD-YPet results in fluorescence complementation reflecting intracellular c-di-GMP levels. Overall, we demonstrate that the CensYBL biosensor is a user-friendly and versatile tool that allows to investigate c-di-GMP variations using single-cell and population-wide experimental set-ups.