Mycobacterium tuberculosis Rv3586 (DacA) Is a Diadenylate Cyclase That Converts ATP or ADP into c-di-AMP

Cyclic diguanosine monophosphate (c-di-GMP) and cyclic diadenosine monophosphate (c-di-AMP) are recently identified signaling molecules. c-di-GMP has been shown to play important roles in bacterial pathogenesis, whereas information about c-di-AMP remains very limited. Mycobacterium tuberculosis Rv3586 (DacA), which is an ortholog of Bacillus subtilis DisA, is a putative diadenylate cyclase. In this study, we determined the enzymatic activity of DacA in vitro using high-performance liquid chromatography (HPLC), mass spectrometry (MS) and thin layer chromatography (TLC). Our results showed that DacA was mainly a diadenylate cyclase, which resembles DisA. In addition, DacA also exhibited residual ATPase and ADPase in vitro. Among the potential substrates tested, DacA was able to utilize both ATP and ADP, but not AMP, pApA, c-di-AMP or GTP. By using gel filtration and analytical ultracentrifugation, we further demonstrated that DacA existed as an octamer, with the N-terminal domain contributing to tetramerization and the C-terminal domain providing additional dimerization. Both the N-terminal and the C-terminal domains were essential for the DacA's enzymatically active conformation. The diadenylate cyclase activity of DacA was dependent on divalent metal ions such as Mg2+, Mn2+ or Co2+. DacA was more active at a basic pH rather than at an acidic pH. The conserved RHR motif in DacA was essential for interacting with ATP, and mutation of this motif to AAA completely abolished DacA's diadenylate cyclase activity. These results provide the molecular basis for designating DacA as a diadenylate cyclase. Our future studies will explore the biological function of this enzyme in M. tuberculosis

Biological Characteristics of Severe Combined Immunodeficient Mice Produced by CRISPR/Cas9-Mediated Rag2 and IL2rg Mutation

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 is a novel and convenient gene editing system that can be used to construct genetically modified animals. Recombination activating gene 2 (Rag2) is a core component that is involved in the initiation of V(D)J recombination during T- and B-cells maturation. Separately, the interleukin-2 receptor gamma chain gene (IL2rg) encoded the protein-regulated activity of natural killer (NK) cells and shared common receptors of some cytokines. Rag2 and IL2rg mutations cause immune system disorders associated with T-, B-, and NK cell function and some cytokine activities. In the present study, 2 single-guide RNAs (sgRNAs) targeted on Rag2 and IL2rg genes were microinjected into the zygotes of BALB/c mice with Cas9 messenger RNA (mRNA) to create Rag2/IL2rg-/- double knockout mice, and the biological characteristics of the mutated mice were subsequently analyzed. The results showed that CRISPR/Cas9-induced indel mutation displaced the frameshift of Rag2 and IL2rg genes, resulting in a decrease in the number of T-, B-, and NK cells and the destruction of immune-related tissues like the thymus and spleen. Mycobacterium tuberculosis 85B antigen could not induce cellular and humoral immune response in mice. However, this aberrant immune activity compromised the growth of several tumor heterogenous grafts in the mutated mice, including orthotopic and subcutaneous transplantation tumors. Thus, Rag2/IL2rg-/- knockout mice possessed features of severe combined immunodeficiency (SCID), which is an ideal model for human xenograft

Bioinformatics and systems-biology approach to identify common pathogenic mechanisms for COVID-19 and systemic lupus erythematosus

The Coronavirus disease 2019 (COVID-19) pandemic has brought a heavy burden to the world, interestingly, it shares many clinical symptoms with systemic lupus erythematosus (SLE). It is unclear whether there is a similar pathological process between COVID-9 and SLE. In addition, we don’t know how to treat SLE patients with COVID-19. In this study, we analyse the potential similar pathogenesis between SLE and COVID-19 and explore their possible drug regimens using bioinformatics and systems biology approaches. The common differentially expressed genes (DEGs) were extracted from the COVID-19 datasets and the SLE datasets for functional enrichment, pathway analysis and candidate drug analysis. Based on the two transcriptome datasets between COVID-19 and SLE, 325 common DEGs were selected. Hub genes were identified by protein-protein interaction (PPI) analysis. few found a variety of similar functional changes between COVID-19 and SLE, which may be related to the pathogenesis of COVID-19. Besides, we explored the related regulatory networks. Then, through drug target matching, we found many candidate drugs for patients with COVID-19 only or COVID-19 combined with SLE. COVID-19 and SLE patients share many common hub genes, related pathways and regulatory networks. Based on these common targets, we found many potential drugs that could be used in treating patient with COVID-19 or COVID-19 combined with SLE.</p

Prevotella Induces the Production of Th17 Cells in the Colon of Mice

Th17-mediated mucosal inflammation is related to increased Prevotella bacterial abundance. The actual involvement of Prevotella in the development and accumulation of intestinal Th17 cells at a steady state, however, remains undefined. Herein, we investigated the role of Prevotella in inducing intestinal Th17 cells in mice. Mice were treated with a combination of broad-spectrum antibiotics (including ampicillin, neomycin sulfate, vancomycin hydrochloride, and metronidazole) in their drinking water for 4 weeks and then gavaged with Prevotella for 4 weeks. After inoculation, 16S rDNA sequencing was used to verify the colonization of Prevotella in the colon of mice. The IL-17A as well as IL-17A-expressing T cells was localized and quantified by an immunofluorescence assay (IFA) of colon sections. Th17 cells in the mesenteric lymph nodes of mice were counted by flow cytometry. Systemic immune response to Prevotella colonization was evaluated based on the serum levels of IL-6, TNF-α, IL-1β, IL-17A, IL-10, IL-4, IFN-γ, and IL-2. Th17-polarizing cytokines (IL-6, TNF-α, IL-1β, and IL-2) induced by Prevotella were evaluated by stimulation of bone marrow-derived dendritic cells (BMDCs). Results revealed that after inoculation, Prevotella successfully colonized the intestine of mice and induced the production and accumulation of colonic Th17 cells in the colon. Moreover, Prevotella elevated some of the Th17-related cytokines in the serum of mice. And Th17-polarizing cytokines (IL-6 and IL-1β) produced by BMDCs were mediated mainly through the interaction between Prevotella and Toll-like receptor 2 (TLR2). In conclusion, our data suggest that Prevotella induces the production of Th17 cells in the colon of mice, thus highlighting the potential role of Prevotella in training the intestinal immune system

Function of the DGA and RHR motifs in DacA.

<p>(A) Partial sequence alignment of <i>M. tuberculosis</i> DacA and <i>T maritima</i> DisA showing the conserved DGA and RHR motifs. Identical residues between the two proteins are highlighted in yellow blocks. (B) Potential contact of DGA and RHR motifs with ATP generated from <i>T. maritima</i> DisA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035206#pone.0035206-Witte1" target="_blank">[22]</a> using the Cn3D software. The three amino acids highlighted in yellow represent either DGA or RHR, as indicated. (C) SDS-PAGE of purified DacA, DacA_RHR and DacA_G73A. Lane M, MW marker; lanes 1–3 are purified DacA, DacA_RHR and DacA_G73A, respectively. (D) ATP binding by DacA_RHR and DacA_G73A. Proteins either in the presence (+) or absence (−) of ATP were separated by electrophoresis with a native gel and stained with Coomassie Brilliant Blue. (E) Analysis of diadenylate cyclase activity of 10 µM DacA_G73A and DacA_RHR in the presence of ATP using HPLC. (F) Structural modeling of DacA and its derivative polypeptides. Three domains of DacA from the N-terminus to the C-terminus are colored in green, blue and orange, respectively. A red star represents one molecule of ATP. A yellow line in DacA_RHR indicates the mutation of RHR motif.</p

Effect of divalent metal ions and pH on DacA's activities.

<p>(A and B) Effect of metal ions on c-di-AMP production catalyzed by DacA in the presence of 2 mM ATP (A) or 0.5 mM ADP (B). (C and D) Effect of pH on c-di-AMP production catalyzed by DacA in the presence of 2 mM ATP (C) or 0.5 mM ADP (D). Note that less ADP was used in the reactions compared with ATP, and thus the arbitrary units between reactions with ATP and ADP are not directly comparable.</p

Primers used for protein expression in this study^a.

a<p>, Primer JY078 was used in combination with KM2948, and primer JY077 was used in combination with KM2949. The mutations for the respective amino acids in primers JY178 to JY219 are indicated in bold.</p

Determination of DacA's activities using HPLC and LC-MS.

<p>(A) Analysis of the products from reaction of ATP with DacA using HPLC. Reaction of ATP with DisA was included as a positive control. The reactions were carried out as described in the Methods. ATP, c-di-AMP, ADP, AMP and pApA standards were also analyzed under the same conditions. (B and C) LC/UV/MS profiles of the products formed by DacA with ATP. The products were detected by monitoring EMS at mass range from 100 to 1000 amu (B) or monitored by UV absorption at 254 nm (C).</p

Determination of DacA's activities using TLC.

<p>(A) Separation of nucleotides generated from [α-³³P]ATP by DacA and DisA. The positions of ATP, ADP, AMP and c-di-AMP are indicated based on the <i>R_f</i> of each standard analyzed in panel B under the same conditions. (B) Separation of nucleotide standards using TLC. Spots 1–5 are ATP, ADP, AMP, c-di-AMP and pApA, respectively. (C) Quantitation of c-di-AMP production. The relative intensity of c-di-AMP generated by DacA or DisA at various time points as in panel A was analyzed using the ImageQuant software. Data shown are representative of two repeat experiments. (D) Production of c-di-AMP with various concentrations of DacA at 30 min of incubation. Reactions contain 2-fold serial diluted DacA protein as indicated on the top of the TLC graph (in log₂ µg). “N” indicates a control with no protein, and “Ctl” contains 1 µg DisA as a positive control. “0” equals 1 µg of protein. (E) Quantitation of ATP depletion and c-di-AMP production by DacA from panel D. Data shown are representative of two repeat experiments.</p

Catalytic activities of DacA with different nucleotides.

<p>(A) Reaction of DacA with ADP, AMP, c-di-AMP or pApA. Samples were separated by HPLC. The peaks in “DacA+ADP” are labeled according to the retention time of each standard as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035206#pone-0035206-g002" target="_blank">Fig. 2A</a>. (B) Reactions catalyzed by DacA using ATP as a substrate, based on the results shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035206#pone-0035206-g002" target="_blank">Fig. 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035206#pone-0035206-g004" target="_blank">Fig. 4A</a>. “A” stands for adenosine, and “P” stands for phosphate. The thickness of arrows denotes priority of reaction, and the thickest arrow shows the major catalytic reaction.</p