Journal: mBio

Article Title: Cyclic di-GMP Signaling in Bacillus subtilis Is Governed by Direct Interactions of Diguanylate Cyclases and Cognate Receptors

doi: 10.1128/mBio.03122-19

Figure Lengend Snippet: Known components of the c-di-GMP signaling network in B. subtilis . The color code for protein domains is as follows: gray for the TM and putative soluble signaling domains, purple for the GGDEF domains, orange for the EAL domains, and blue for the PilZ domain. Inactive protein domains are illustrated with dashed lines. Note that the EAL domain activity of DgcW has not been tested so far in vitro .

Article Snippet: The amount of c-di-GMP contained in the protein preparations was quantified using a c-di-GMP standard (Jena Bioscience) with known concentrations as a reference.

Techniques: Activity Assay, In Vitro

Journal: mBio

Article Title: Cyclic di-GMP Signaling in Bacillus subtilis Is Governed by Direct Interactions of Diguanylate Cyclases and Cognate Receptors

doi: 10.1128/mBio.03122-19

Figure Lengend Snippet: Single-molecule tracking of exponentially growing DgcK-mVenus in B. subtilis 3610 wild-type cells. (A) Representative static molecule of DgcK-mVenus in the wild type localized at the cell pole (D), and dynamic track of DgcK-mVenus moving along the cell pole. Scale bars correspond to 2 μm. (B, E) Corresponding projections of all tracks in these cells showing the static (B) or mobile (E) track in a color-coded manner. The origin is highlighted in red and the end in yellow. All other tracks in the same cell are displayed in gray. (C, F) Normalized intensity profile of the track shaded in gray. (G to I) Average dwell times of DgcK-mVenus and DgcP-mVenus in different B. subtilis NCIB 3610 strains. (G) The bar plots depict the change in the average dwell times (calculated with “SMM Track”) of DgcK-mVenus in the NCIB 3610 wild type (3610 WT), in a ydaK deletion strain (3610 Δ ydaK ), and in two strains overexpressing ydaK (3610 4% EtOH, cells stressed with 4% ethanol for 30 min; 3610 P xyl - ydaKLMN , NCIB 3610 with P xyl - ydaKLMN ). The absence of YdaK leads to a decrease of the dwell time, whereas overexpression leads to an increase. (H, I) Dwell times of DgcK-mVenus (H) or DgcP-mVenus (I) in a stain lacking the c-di-GMP receptor dgrA (3610 Δ dgrA ) and in the NCIB 3610 wild type. All data are from three biological replicates.

Article Snippet: The amount of c-di-GMP contained in the protein preparations was quantified using a c-di-GMP standard (Jena Bioscience) with known concentrations as a reference.

Techniques: Over Expression, Staining

Journal: mBio

Article Title: Cyclic di-GMP Signaling in Bacillus subtilis Is Governed by Direct Interactions of Diguanylate Cyclases and Cognate Receptors

doi: 10.1128/mBio.03122-19

Figure Lengend Snippet: Analyses of the mobility of DGC DgcK and DgcP by the Gaussian mixture model (GMM) in different B. subtilis NCIB 3610 strains. For the determination of fraction sizes and of diffusion coefficients, the GMM was used. (A to C) GMM of DgcK-mVenus in NCIB 3610 (wild-type) (A), NCIB 3610 Δ ydaK (B), and NCIB 3610 Δ dgrA (C) cells. The histogram of the step-size distribution was fitted with a single fit and a double fit, which consists of a static/slow population and a mobile population, as indicated in the key. Because the double fit corresponds better to the data than the single fit, the double fit was taken for all determinations. (D, E) The bar plots depict the changes in the distributions of the two (static and mobile) subpopulations of DgcK-mVenus. (D) In the absence of the c-di-GMP receptor YdaK (NCIB 3610 Δ ydaK ), the size of the static DgcK-mVenus population decreases, whereas this population increased upon overexpression of YdaK (NCBI 3610 cells stressed with 4% ethanol for 30 min, or NCIB3610 Pxyl-ydaKLMN cells overexpressing YdaK via addition of xylose). (E) Deletion of the gene encoding the PilZ domain protein DgrA (NCIB 3610 Δ dgrA ) leads to an increase of the mobile population of DgcK-mVenus compared to that of the wild type. Data are analyzed from three biological replicates. (F, G) GMM of DgcP-mVenus in NCIB 3610 (F) and NCIB 3610 Δ dgrA (G). (H) The bar plots depict the changes in the two subpopulations of DgcP-mVenus. Deletion of the dgrA gene encoding the c-di-GMP receptor DgrA (strain 3610 Δ dgrA ) leads to an increase in the size of the mobile population of DgcK-mVenus compared to that of the wild type.

Article Snippet: The amount of c-di-GMP contained in the protein preparations was quantified using a c-di-GMP standard (Jena Bioscience) with known concentrations as a reference.

Techniques: Diffusion-based Assay, Over Expression

Journal: mBio

Article Title: Cyclic di-GMP Signaling in Bacillus subtilis Is Governed by Direct Interactions of Diguanylate Cyclases and Cognate Receptors

doi: 10.1128/mBio.03122-19

Figure Lengend Snippet: Interaction of the DGC DgcK with its cognate receptor YdaK depends on an intact I-site within the GGDEF domain of YdaK. (A) In vitro interaction analysis of His 6 -DgcK-∆N175 and GST-YdaK-∆N111. Coomassie-stained SDS-PAGE of a pull-down assay employing His 6 -DgcK-∆N175 (bait), GST-YdaK-∆N111 (prey) and GST (control 2). 2 nmol His 6 -DgcK-∆N175 was immobilized on nickel-sepharose beads, followed by incubation with 20 nmol GST-YdaK-∆N111 with or without addition of c-di-GMP (cdG, second and fourth lane, respectively). First lane: His 6 -DgcK-∆N175 binding to nickel-sepharose beads. Third lane, Control 1: GST-YdaK-∆N111 does not bind to nickel-sepharose beads. Fifth lane, Control 2: His 6 -DgcK-∆N175 does not bind the affinity tag GST. (B) In vitro interaction analysis of GST-His 6 -YdaK-∆N111 and His 6 -DgcK-∆N175. Coomassie-stained SDS-PAGE of a pull-down assay employing GST-His 6 -YdaK-∆N111 (bait), His 6 -DgcK-∆N175 (prey). 2 nmol GST-His 6 -YdaK-∆N111 was immobilized on GST-sepharose beads, followed by incubation with 20 nmol His 6 -DgcK-∆N175 with or without addition of c-di-GMP (cdG, second and third lanes, respectively). First lane: GST-His 6 -YdaK-∆N111 binding to GST beads. Fourth lane: Control 1, His 6 -DgcK-∆N175 does not bind to GST-sepharose beads. Fifth lane: Control 2, His 6 -DgcK-∆N175 does not bind the affinity tag GST. (C) Coomassie-stained SDS-PAGE of a pull-down assay employing GST-His 6 -DgcK-∆N175 (bait), His 6 -YdaK-∆N111 (prey) and the I-site mutant His 6 -YdaK-∆N111_R202A. 2 nmol GST-His 6 -DgcK-∆N175 was immobilized on GST-sepharose beads, followed by incubation with 20 nmol His 6 -YdaK-∆N111 (first lane). Second and third lanes: GST-His 6 -DgcK-∆N175 does not bind His 6 -YdaK-∆N111_R202A with or without additional addition of c-di-GMP (cdG). Fourth lane: Control 1, His 6 -YdaK-∆N111_R202A does not bind to GST-sepharose beads. Fifth lane: Control 2, GST-His 6 -DgcK-∆N175 binding to GST beads.

Article Snippet: The amount of c-di-GMP contained in the protein preparations was quantified using a c-di-GMP standard (Jena Bioscience) with known concentrations as a reference.

Techniques: In Vitro, Staining, SDS Page, Pull Down Assay, Incubation, Binding Assay, Mutagenesis

Journal: mBio

Article Title: Cyclic di-GMP Signaling in Bacillus subtilis Is Governed by Direct Interactions of Diguanylate Cyclases and Cognate Receptors

doi: 10.1128/mBio.03122-19

Figure Lengend Snippet: c-di-GMP alters the conformation of YdaK. (A) Amino acid residues of YdaK-ΔN111 are colored according to their differences in HDX profiles between c-di-GMP-bound YdaK-ΔN111 and apo-YdaK-ΔN111. The secondary structure of YdaK-ΔN111 based on a generated model is indicated. (B, left) Locations of regions with less HDX in the presence of c-di-GMP in a structural model of YdaK-ΔN111. The I-site and degenerated GGDEF motifs are in red and green, respectively. The I-site arginine 202 is shown as sticks. The position of c-di-GMP bound to the I-site is inferred from a superimposition of the YdaK-ΔN111 structural model upon the crystal structure of the GGDEF domain of Dcsbis from Pseudomonas aeruginosa (PDB accession number 4ZMM ). (Right) Location of representative peptides in the structural model of YdaK-ΔN111. (C) Hydrogen/deuterium exchange profiles of representative YdaK peptides in the c-di-GMP-bound (red) and unliganded (blue) states. Data represent the means ± standard deviations (SD) of results from three technical replicates. t, time.

Article Snippet: The amount of c-di-GMP contained in the protein preparations was quantified using a c-di-GMP standard (Jena Bioscience) with known concentrations as a reference.

Techniques: Generated

Journal: mBio

Article Title: Cyclic di-GMP Signaling in Bacillus subtilis Is Governed by Direct Interactions of Diguanylate Cyclases and Cognate Receptors

doi: 10.1128/mBio.03122-19

Figure Lengend Snippet: Interaction of the PilZ protein DgrA with DgcK and DgcP. (A, left) In vitro interaction analysis of GST-His 6 -DgrA and His 6 -DgcK-ΔN175. Coomassie blue-stained SDS-PAGE gel from a pulldown assay employing GST-His 6 -DgrA (bait), His 6 -DgcK-ΔN175 (prey), and GST (control [Ctrl]). Two nmol of GST-His 6 -DgrA was immobilized on glutathione-Sepharose beads (first lane), followed by incubation with 20 nmol His 6 -DgcK-ΔN175 (second lane) and 2 mM c-di-GMP (cdG) (third lane). Control 1 (fourth lane) is His 6 -DgcK-ΔN175, which does not bind to glutathione-Sepharose beads. Control 2 (fifth lane) is His 6 -DgcK-ΔN175, which does not bind the affinity tag GST. (A, right) Input controls for: GST-His-DgrA (first lane), His-DgcK-ΔN175 (second lane), GST-His 6 (third lane). (B, left) In vitro interaction analysis of DgcP-Strep and GST-His 6 -DgrA. Coomassie blue-stained SDS-PAGE gel from a pulldown assay employing DgcP-StrepII (bait) and GST-His 6 -DgrA (prey). Two nmol of DgcP-StrepII was immobilized on Strep-Sepharose beads (first lane), followed by incubation with 20 nmol GST-His 6 -DgrA (second lane), which shows that DgcP-StrepII can immobilize GST-His 6 -DgrA on Strep-Sepharose. In the third lane, 2 nmol DgcP-Strep was immobilized on Strep-Sepharose beads, followed by incubation with 20 nmol GST-His 6 -DgrA and of 2 mM c-di-GMP. Control 1 (fourth lane) was GST-His6-DgrA, which does not bind to Strep-Sepharose beads. (B, right) Input controls for DgcP-StrepII (first lane), GST-His 6 -DgrA (second lane).

Article Snippet: The amount of c-di-GMP contained in the protein preparations was quantified using a c-di-GMP standard (Jena Bioscience) with known concentrations as a reference.

Techniques: In Vitro, Staining, SDS Page, Incubation

Journal: PLOS Pathogens

Article Title: Protein kinase PfPK2 mediated signalling is critical for host erythrocyte invasion by malaria parasite

doi: 10.1371/journal.ppat.1011770

Figure Lengend Snippet: A. Schematic illustrating the domain architecture of PfGCα, which has an ATPase domain fused to the guanylyl cyclase, various subdomains are indicated . The Nucleotide binding domain (N), which splits the phosphorylation (P) domain, has a long insert and PfPK2-regulated phosphorylation site S1214 resides in an insert. Sequence alignment of GCα from P . falciparum , P . berghei and Toxoplasma gondii and human ATPase ATP8A1/2 indicates that this site is conserved only in Plasmodium spp . B. The phosphorylation of S1214 of PfGCα was reduced upon PfPK2 depletion. MS/MS spectra is provided for the corresponding phosphopeptide. TMT channel indicated in the inset represents independent biological replicates of PfPK2 DMSO (black) or RAP (cyan) parasites and wild type samples. C. DMSO or RAP-treated PfPK2-loxP schizonts were lysed and an ELISA-based assay kit was used to quantitate intracellular cGMP. The data provides fold change in cGMP upon RAP-treatment (mean ± SEM, n = 5, ***, P < 0.001, paired t test). D. Fluorimetry was used to assess calcium mobilization, which was determined by using Fluo-4-loaded mature DMSO- and RAP-treated PfPK2-loxP schizonts that were treated with zaprinast (left panel) or A23187 (right panel). The signals were normalised with respect to DMSO (0%) and A23187 (100%). The data presented are mean of four independent biological replicates that were done in duplicate (mean ± SEM, n = 4, *, P < 0.05, ns, P > 0.05, ns- not significant, paired t test).

Article Snippet: Intracellular cyclic nucleotide levels in mature segmented schizonts were measured using enzyme-linked immunosorbent assay (ELISA)-based high-sensitivity cGMP assay kit as suggested by the manufacturer (Cell Signalling Technology, Cat No. #4360).

Techniques: Binding Assay, Phospho-proteomics, Sequencing, Tandem Mass Spectroscopy, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Betrixaban Activates cGAS and ERVs to Promote Dual Nucleic-Sensing Antiviral Immunity

doi: 10.1101/2025.08.08.669242

Figure Lengend Snippet: (A) Predicted binding affinity scores of BT against pattern recognition receptors using the DeepAVC model. (B) SPR sensorgrams of BT binding to recombinant human cGAS at the indicated concentrations (0.196-50 µM). (C) Dose–response curve fitted from SPR data in (B). (D) RT-qPCR of IFNB1 mRNA in wild-type (WT), cGAS knockout and STING knockout HT1080 cells treated with BT (50, 75 or 100 µM) or DMSO for 12 h. (E) Secreted type I IFN measured by ELISA in the same cell lines and treatments as in (D). (F) Flow cytometry of VSV-GFP infection in WT, cGAS knockout and STING knockout HT1080 cells treated with BT (50, 75, 100 µM) and infected (MOI = 0.1) for 12 h. (G) Western blots of WT, cGAS knockout and STING knockout HT1080 cells treated as in (D). (H-I) In vivo VSV and HSV-1 challenges in cGAS knockout mice. Left panel is survival curves of mice, right panel is viruses RNA levels in liver measured by RT-qPCR. (J) Confocal micrographs of RAW 264.7 cells treated with DMSO or 50 µM BT for 12 h, stained for DAPI (blue) and cytosolic dsDNA (green). Scale bars, 5 µm. (K) Confocal images of RAW 264.7 cells treated as in (J), stained with DAPI (blue) and MitoTracker (magenta) to visualize mitochondrial morphology. Scale bars, 5 µm. (L) Comparison of mitochondrial structure by confocal versus STED super-resolution microscopy in RAW 264.7 cells treated with DMSO or BT and stained with PK Mito. Scale bars, 2 µm. (M) Transmission electron micrographs of mitochondria in RAW 264.7 cells treated with DMSO or BT. (N) In vitro cGAS enzymatic assays: LC-MS quantification of cGAMP production by recombinant cGAS incubated with dsDNA in the presence of BT (25 or 100 µM). (O) LC-MS analysis of cGAMP production by cGAS incubated with BT (25 or 100 µM) in the absence of exogenous DNA. Data are shown as mean ± SEM. N.S., not significant, p > 0.05; *p < 0.05; **p < 0.01; ****p < 0.0001.

Article Snippet: Recombinant human cGAS protein were purchased from MedChemExpress (catalog no. HY-P72337).

Techniques: Binding Assay, Recombinant, Quantitative RT-PCR, Knock-Out, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Infection, Western Blot, In Vivo, Staining, Comparison, Super-Resolution Microscopy, Transmission Assay, In Vitro, Liquid Chromatography with Mass Spectroscopy, Incubation