C di amp

Cells were grown to an optical density at 750 nm (OD₇₅₀) of 1 and transferred to new 250 ml flasks with BG-11/HEPES medium containing 0.2 M sorbitol for the osmotic stress, or 0.2 M NaCl for the ionic stress, except for halophile Synechocystis for which the NaCl concentration was 0.6 M. As a control, BG-11/HEPES medium without sorbitol or NaCl added was utilized. Cells were maintained under osmotic or salt stress for 24 h. After 24 h, c-di-AMP and c-di-GMP were quantified as described (Massie et al., 2012 (link); Agostoni et al., 2013 (link); Barker et al., 2013 (link)). In brief, c-di-AMP and c-di-GMP were quantified by UPLC-MS/MS. Prior to analysis, an aliquot of each sample was dried under vacuum to remove extraction buffer and the pellet was resuspended in an equal volume of water. A 10-μl volume of the resuspended sample was analyzed together with an eight-point standard curve of purified c-di-AMP or c-di-GMP (Biolog). c-di-AMP and c-di-GMP concentrations determined for samples were normalized to total soluble protein content from an equal volume of cells from which second messengers were extracted as previously described (Agostoni et al., 2013 (link); Zhu et al., 2016 (link)). Growth over time of WT, OE DAC and OE PDE Synechocystis stains in the presence of sorbitol (0.5 M) or NaCl (0.6 M) was measured using OD₇₅₀ as described above.

Separation and quantification of intracellular nucleotides by HPLC-UV were performed essentially by the method of Strauch et al. (48 (link)) using an Agilent HP1100 HPLC. Briefly, aliquots of nucleotide extracts were separated at 20°C by anion-exchange chromatography using a Partisil 10SAX column (10-μm particle size; 25 cm by 4.6 mm internal diameter). Gradient elution was performed by the following gradient elution program using mobile phase A (MPA) (7 mM potassium hydrogen phosphate [pH 4.0]) and mobile phase B (MPB) (0.5 M potassium dihydrogen phosphate–0.5 M sodium sulfate pH 5.4) and a flow rate of 1.5 ml/min: 0 to 5 min, 100% MPA; 5 to 10 min, 100% to 85% MPA; 10 to 15 min, 85% to 81% MPA; 15 to 20 min, 81% to 50% MPA; 20 to 25 min, 50% to 30% MPA; 25 to 30 min, 30% to 25% MPA; 30 to 40 min, 30% to 0% MPA (postrun equilibration, 100% MPA for 15 min). Eluting nucleotides were detected using a UV diode-array detector and quantified by their absorbance at 254 nm in comparison to known standards. Quantified values in picomoles were normalized to the cells present in 1 ml of a culture with an OD₆₀₀ value of 1. All nucleotide standards were purchased from Sigma, with the exception of cdiAMP, cdiGMP, pApA, and pGpG which came from Biolog.

c-di-GMP, c-di-AMP and cGAMP used for bacterial growth inhibition assays were purchased from BIOLOG Life Science Institute (Germany). Fe-CMBs and Fe-Ent were purchased from Biophore Research Products (Germany), thrombin was sourced from Biosharp (China) and glutathione-sepharose 4B column was purchased from GE Healthcare. Both radioactive-labelled [α-³²P]GTP and [α-³²P]ATP were purchased from Perkin Elmer (USA). Middlebrook 7H9 medium and Oleic acid-Albumin-Dextrose-Catalase (OADC) enrichment were sourced from Becton-Dickinson Company (USA) and were used for growing M. tuberculosis. GTP, dNTP and DNA polymerase for PCR assays were obtained from TaKaRa Co. (Japan). Both pET28a and pGEX-4 T-1vectors, which were used for expressing rLCN2 proteins. All restriction enzymes for cloning were obtained from TaKaRa Co.

Animals (n = 10) were immunized 3 times at day 0, 14, and 28 by intramuscular route. Each animal received a dose of 50 μl containing 15 μg of β-Gal protein (Sigma-Aldrich, Germany) as antigen. ß-Gal was either adsorbed to alum [1:1 v/v, aluminum hydroxyphosphate (Adju-Phos^®), Brenntag Biosector, Denmark] at pH 7.4 and 25°C or co-administered with c-di-AMP (Biolog, Germany) at a concentration of 5 μg per dose. Fourteen days after the third immunization, spleens of vaccinated mice were collected, immune cells were extracted, pooled and restimulated with β-Gal. The cytokine concentration was measured by cytometric bead array (CBA). Results from one representative out of two independent experiments are shown.

Single cell suspensions of splenic cells were prepared. In some experiments red blood cells were lysed using ammonium chloride TRIS. In most experiments, live cells were isolated using Lympholyte M kit (Cedarlane, Burlington, Ontario, Canada) and B cells were purified by CD43 negative selection using MACS CD43-microbeads (Miltenyi Biotec Inc., San Diego, CA.). Resultant populations were routinely >97% B cells based on B220 staining and FACS analysis. Ex vivo B cells and splenocytes were cultured in IMDM supplemented with 10% FBS, sodium pyruvate (1 mM), L-glutamine (2 mM), 1% penicillin/ streptomycin, 2-ME (50 μM), HEPES buffer (10 mM), and 1% non-essential amino acids.
Ex vivo B cells or splenocytes were seeded (3 × 10⁵ cells/100 μL/well) in 96 well flat-bottom or U-bottom plates and stimulated with various concentrations of CDNs or IL-4 for the indicated time. The following stimuli were used: IL-4 (Supernatant from a J558L culture, 1:200 dilution); c-diGMP (C 057, Biolog life science institute, Federal Republic of Germany); 2′, 3′ cGAMP (c[G(2′,5′)pA(3′,5′)p], C 161, Biolog life science institute, Federal Republic of Germany); 3′, 3′ cGAMP (c-(ApGp), C 117, Biolog life science institute, Federal Republic of Germany); c-diAMP (C 088, Biolog life science institute, Federal Republic of Germany); Rp, Rp-c-diAMPSS (C118, Biolog life science institute, Federal Republic of Germany).