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) |
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| MBS6507358-005mL | MyBiosource | 0.05mL | 835 EUR |
cGMP (cyclic GMP) |
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| MBS6507358-5x005mL | MyBiosource | 5x0.05mL | 3600 EUR |
Cyclic GMP (TBAOH) |
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| HY-113469B | MedChemExpress | 10 mg | 70.35 EUR |
Cyclic GMP (sodium) |
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| HY-113469A | MedChemExpress | 10 mg | 70.35 EUR |
Cyclic GMP Antibody |
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| abx022640-02ml | Abbexa | 0.2 ml | 1128 EUR |
Cyclic GMP Antibody |
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| GWB-BA5CC2 | GenWay Biotech | 0.2 ml | Ask for price |
Cyclic GMP Antibody |
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| GWB-DFA25F | GenWay Biotech | 2 ml | Ask for price |
Cyclic GMP EIA Kit |
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| SKT-211-96 | Stressmarq | 1 plate of 96 wells | 568.8 EUR |
SHEEP ANTI CYCLIC GMP |
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| MBS222513-2mL | MyBiosource | 2mL | 455 EUR |
SHEEP ANTI CYCLIC GMP |
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| MBS222513-5x2mL | MyBiosource | 5x2mL | 1870 EUR |
Sheep anti Cyclic GMP |
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| MBS316214-02mL | MyBiosource | 0.2mL | 1235 EUR |
Sheep anti Cyclic GMP |
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| MBS316214-5x02mL | MyBiosource | 5x0.2mL | 5380 EUR |
Cyclic GMP (Direct) ELISA |
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| MBS494508-5x96Tests | MyBiosource | 5x96Tests | 3410 EUR |
Cyclic GMP (Direct) ELISA |
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| MBS494508-96Tests | MyBiosource | 96Tests | 740 EUR |
Cyclic GMP Standard, 125UL |
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| C080-125UL | Arbor Assays | 125UL | 85 EUR |
Cyclic GMP Standard, 350UL |
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| C080-350UL | Arbor Assays | 350UL | 207 EUR |
Cyclic GMP Standard, 625UL |
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| C080-625UL | Arbor Assays | 625UL | 207 EUR |
Cyclic GMP Standard, 70UL |
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| C080-70UL | Arbor Assays | 70UL | 85 EUR |
Anti- Cyclic GMP Antibody |
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| GWB-FDE020 | GenWay Biotech | 1000 TESTS | Ask for price |
Cyclic GMP Polyclonal Antibody |
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| MBS194212-02mL | MyBiosource | 0.2mL | 560 EUR |
Cyclic GMP Polyclonal Antibody |
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| MBS194212-5x02mL | MyBiosource | 5x0.2mL | 2510 EUR |
cGMP(Cyclic GMP)ELISA Kit |
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| MBS2516175-10x96Tests | MyBiosource | 10x96Tests | 3265 EUR |
cGMP(Cyclic GMP)ELISA Kit |
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| MBS2516175-24Tests | MyBiosource | 24Tests | 225 EUR |
cGMP(Cyclic GMP)ELISA Kit |
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| MBS2516175-48Test | MyBiosource | 48Test | 350 EUR |
cGMP(Cyclic GMP)ELISA Kit |
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| MBS2516175-5x96Test | MyBiosource | 5x96Test | 1655 EUR |
cGMP(Cyclic GMP)ELISA Kit |
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| MBS2516175-96Tests | MyBiosource | 96Tests | 395 EUR |
cGMP(Cyclic GMP) ELISA Kit |
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| YPJ1147-48wellsplate | UpingBio | 48 wells plate | 180 EUR |
cGMP(Cyclic GMP) ELISA Kit |
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| YPJ1147-96wellsplate | UpingBio | 96 wells plate | 220 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.