scholarly journals Brucella Melitensis invA Gene (BME_RS01060) Transcription Is Promoted Under Acidic Stress Conditions.

2021 ◽  
Author(s):  
Raúl Sauceda-Becerra ◽  
Hugo Barrios-García ◽  
Julio Martínez-Burnes ◽  
Beatriz Arellano-Reynoso ◽  
Alejandro Benítez-Guzmán ◽  
...  
Keyword(s):  
Real Time ◽  
Gene Transcription ◽  
Real Time Pcr ◽  
Brucella Melitensis ◽  
Survival Rates ◽  
Time Dependent ◽  
Acidic Stress ◽  
Wild Type ◽  
Mutant Strains ◽  
Acidic Conditions

Abstract The invA gene of Brucella melitensis codes for a NUDIX (nucleoside diphosphate linked to moiety X) hydrolase related to invasiveness. The objective of this work was to evaluate invA transcription under acidic conditions. The invA gene transcription was up regulated at pH 3 and pH 5 observed with semiquantitative real-time PCR in B. melitensis 133 strain. Results indicated that invA gene transcription at pH 3 showed a basal and decreased transcription compared to that of pH 5 incubation. Transcription levels of the dnaK gene were similar to those obtained with invA gene. The survival rates of wild type and invA mutant strains at pH 5 were above 90% in all post-incubation times. In contrast, at pH 3 there was a time-dependent reduction on both strains at 15 min (P < 0.05). These results suggest that invA gene transcription is promoted under acidic conditions in Brucella melitensis.

scholarly journals 3,3′-Thiodipropionic acid (TDP), a possible precursor for the synthesis of polythioesters: identification of TDP transport proteins in Variovorax paradoxus TBEA6

2021 ◽  
Author(s):  
M. Venkateswar Reddy ◽  
Alexander Steinbüchel
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Significant Shift ◽  
Wild Type ◽  
The Real ◽  
E Coli ◽  
Variovorax Paradoxus ◽  
Protein Ligand Interactions ◽  
Mutant Strains ◽  
Ligand Interactions

Abstract3,3′-Thiodipropionic acid (TDP) is an antioxidant, which can be used as precursor carbon source to synthesize polythioesters. The bacterium Variovorax paradoxus TBEA6 strain can use TDP as a single source of carbon and energy. In the present study, experiments were carried out to identify proteins involved in the transport of TDP into the cells of strain TBEA6. Hence, eight putative tctC genes, which encode for the TctC proteins, were amplified from genomic DNA of TBEA6 strain using polymerase chain reaction and expressed in E. coli BL21 cells. Cells were grown in auto-induction medium, and protein purification was done using His Spin Trap affinity columns. Purity and molecular weight of each protein were confirmed by SDS-PAGE analysis. Protein-ligand interactions were monitored in thermoshift assays using the real-time PCR system. Two TctC proteins (locus tags VPARA-44430 and VPARA-01760) out of eight proteins showed a significant shift in their melting temperatures when they interact with the ligand (TDP or gluconate). The responsible genes were deleted in the genome of TBEA6 using suicide plasmid pJQ200mp18Tc, and single deletion mutants of the two candidate genes were subsequently generated. Finally, growth of the wild-type strain (TBEA6) and the two mutant strains (ΔVPARA-44430 and ΔVPARA-01760) were monitored and compared using TDP or gluconate as carbon sources. Wild type strains were successfully grown with TDP or gluconate. From the two mutant strains, one (ΔVPARA-44430) was unable to grow with TDP indicating that the tctC gene with locus tag VPARA-44430 is involved in the uptake of TDP.Key Points• Putative tctC genes from V. paradoxus TBEA6 were heterologously expressed in E. coli.• Protein-ligand interactions monitored in thermoshift assays using the real-time PCR.• tctC gene with locus tag VPARA-44430 is involved in the uptake of TDP.

EvaGreen-based Multiplex Real-time PCR Assay for Rapid Differentiation of Wild-Type and Glycoprotein E-Deleted Bovine Herpesvirus-1 Strains

2017 ◽  
Vol 28 (4) ◽  
pp. 248-252 ◽  
Author(s):  
Sachin S. Pawar ◽  
Chetan D. Meshram ◽  
Niraj K. Singh ◽  
Mohini Saini ◽  
B. P. Mishra ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Bovine Herpesvirus ◽  
Wild Type ◽  
Pcr Assay ◽  
Bovine Herpesvirus 1 ◽  
Glycoprotein E ◽  
Herpesvirus 1

Identification of T-DNA Inserted Mutant Gene Transcription by Using Real-time PCR in Arabidopsis

2011 ◽  
Vol 18 (2) ◽  
pp. 41-47
Author(s):  
Man YUAN ◽  
Xi BAI ◽  
Hua CAI ◽  
Yong LI ◽  
Wei JI ◽  
...  
Keyword(s):  
Real Time ◽  
Gene Transcription ◽  
Real Time Pcr ◽  
Mutant Gene

scholarly journals Proteomics Investigation of the Time Course Responses of RAW264.7 Macrophages to Infections With the Wild-Type and Twin-Arginine Translocation Mutant Strains of Brucella melitensis

2021 ◽  
Vol 11 ◽  
Author(s):  
Xin Yan ◽  
Sen Hu ◽  
Yan Yang ◽  
Da Xu ◽  
Wenxing Liu ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Time Course ◽  
Nitrosative Stress ◽  
Brucella Melitensis ◽  
No Production ◽  
Wild Type ◽  
Twin Arginine Translocation ◽  
Raw264.7 Macrophages ◽  
Mutant Strains ◽  
Related Proteins

Brucella, a notorious intracellular pathogen, causes chronic infections in many mammals, including humans. The twin-arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane; protein substrates translocated by Brucella include ABC transporters, oxidoreductases, and cell envelope biosynthesis proteins. Previously, we showed that a Tat mutant of Brucella melitensis M28 exhibits reduced survival within murine macrophages. In this study, we compared the host responses elicited by wild-type M28 and its Tat-mutant strains ex vivo. We utilized label-free quantitative proteomics to assess proteomic changes in RAW264.7 macrophages after infection with M28 and its Tat mutants. A total of 6085 macrophage proteins were identified with high confidence, and 79, 50, and 99 proteins were differentially produced upon infection with the Tat mutant at 4, 24, and 48 hpi, respectively, relative to the wild-type infection. Gene ontology and KEGG enrichment analysis indicated that immune response-related proteins were enriched among the upregulated proteins. Compared to the wild-type M28 infection, the most upregulated proteins upon Tat-mutant infection included the cytosolic nucleic acid signaling pathway-related proteins IFIH1, DHX58, IFI202, IFI204, and ISG15 and the NF-κB signaling pathway-related proteins PTGS2, CD40, and TRAF1, suggesting that the host increases the production of these proteins in response to Tat mutant infection. Upregulation of some proteins was further verified by a parallel reaction monitoring (PRM) assay. ELISA and qRT-PCR assays indicated that Tat mutant infection significantly induced proinflammatory cytokine (TNF-α and IL-6) and nitric oxide (NO) production. Finally, we showed that the Tat mutant displays higher sensitivity to nitrosative stress than the wild type and that treatment with the NO synthase inhibitor L-NMMA significantly increases the intracellular survival of the Tat mutant, indicating that NO production contributes to restricting Tat mutant survival within macrophages. Collectively, this work improves our understanding of host immune responses to Tat mutants and provides insights into the mechanisms underlying the attenuated virulence of Tat mutants.

scholarly journals Establishment of Systemic Brucella melitensis Infection through the Digestive Tract Requires Urease, the Type IV Secretion System, and Lipopolysaccharide O Antigen

2009 ◽  
Vol 77 (10) ◽  
pp. 4197-4208 ◽  
Author(s):  
Tatiane A. Paixão ◽  
Christelle M. Roux ◽  
Andreas B. den Hartigh ◽  
Sumathi Sankaran-Walters ◽  
Satya Dandekar ◽  
...  
Keyword(s):  
Digestive Tract ◽  
Cell Model ◽  
Brucella Melitensis ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Human Brucellosis ◽  
Type Iv Secretion System ◽  
Wild Type ◽  
Type Iv ◽  
Mutant Strains

ABSTRACT Human brucellosis is caused mainly by Brucella melitensis, which is often acquired by ingesting contaminated goat or sheep milk and cheese. Bacterial factors required for food-borne infection of humans by B. melitensis are poorly understood. In this study, a mouse model of oral infection was characterized to assess the roles of urease, the VirB type IV secretion system, and lipopolysaccharide for establishing infection through the digestive tract. B. melitensis strain 16M was consistently recovered from the mesenteric lymph node (MLN), spleen, and liver beginning at 3 or 7 day postinfection (dpi). In the gut, persistence of the inoculum was observed up to 21 dpi. No inflammatory lesions were observed in the ileum or colon during infection. Mutant strains lacking the ureABC genes of the ure1 operon, virB2, or pmm encoding phosphomannomutase were constructed and compared to the wild-type strain for infectivity through the digestive tract. Mutants lacking the virB2 and pmm genes were attenuated in the spleen (P < 0.05) and MLN (P < 0.001), respectively. The wild-type and mutant strains had similar levels of resistance to low pH and 5 or 10% bile, suggesting that the reduced colonization of mutants was not the result of reduced resistance to acid pH or bile salts. In an in vitro lymphoepithelial cell (M-cell) model, B. melitensis transited rapidly through polarized enterocyte monolayers containing M-like cells; however, transit through monolayers containing only enterocytes was reduced or absent. These results indicate that B. melitensis is able to spread systemically from the digestive tract after infection, most likely through M cells of the mucosa-associated lymphoid tissue.

scholarly journals Sublytic C5b-9 Induces Glomerular Mesangial Cell Apoptosis Through miR-3546/SOX4/Survivin Axis in Rat Thy-1 Nephritis

2018 ◽  
Vol 49 (5) ◽  
pp. 1898-1917 ◽  
Author(s):  
Chunyan Yao ◽  
Fengxia He ◽  
Longfei Liu ◽  
Zhiwei Zhang ◽  
Chenhui Zhao ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Real Time ◽  
Gene Transcription ◽  
Real Time Pcr ◽  
Mesangial Cell ◽  
Survivin Expression ◽  
Luciferase Reporter ◽  
Glomerular Mesangial Cell

Background/Aims: The activation of complement system and the formation of C5b-9 complex have been confirmed in the glomeruli of patients with mesangioproliferative glomerulonephritis (MsPGN). However, the role and mechanism of C5b-9-induced injury in glomerular mesangial cell (GMC) are poorly understood. Rat Thy-1N is an animal model for studying MsPGN. It has been revealed that the attack of C5b-9 to the GMC in rat Thy-1N is sublytic, and sublytic C5b-9 can cause GMC apoptosis, but the underlying mechanism is not fully elucidated. To explore the role and regulatory mechanism of C5b-9 in MsPGN lesion, we used rat Thy-1N model and first detected the change of microRNA (miRNA) profiles both in Thy-1N rat renal tissues (in vivo) and in the cultured GMCs with sublytic C5b-9 stimulation (in vitro). Then we determined the effect of miR-3546, which increased both in vivo and in vitro, on GMC apoptosis upon sublytic C5b-9 as well as the involved mechanism. Methods: Rat Thy-1N model was established and GMCs were treated with sublytic C5b-9. The rat renal cortex and the stimulated GMCs were obtained for miRNA microarray detection. Subsequently, the increased miRNAs were verified by real-time PCR. Meanwhile, to ascertain the ability of some miRNAs to upregulate cleaved caspase 3 and induce GMC apoptosis, the corresponding miRNA mimics were transfected into GMCs, followed by western blotting (WB) and flow cytometry mesurement. Thereafter, the miR-3546-targeted gene (SOX4) was predicted using bioinformatics approaches, and SOX4 expression in Thy-1N tissues and in the GMCs upon sublytic C5b-9 stimulation or miR-3546 mimic/inhibitor transfection were detected using real-time PCR and WB. To prove that miR-3546 can affect SOX4 gene transcription and SOX4 can regulate survivin expression, dual luciferase reporter assay, real-time PCR, WB and chromatin immunoprecipitation (ChIP) assays were performed. Furthermore, the role of miR-3546/SOX4/survivin axis in the GMC apoptosis induced by sublytic C5b-9 was examined using WB and flow cytometry. Results: Compared with normal renal tissues and untreated GMCs, there were 43 and 62 upregulated miRNAs (> 2-fold) in Thy-1N tissues and sublytic C5b-9-stimulated GMCs respectively. A total of 17 miRNAs were increased both in vivo and in vitro, 11 of which were validated by real-time PCR. Among them, miR-3546 could markedly promote GMC apoptosis and inhibit SOX4 or survivin expression in response to sublytic C5b-9, and either SOX4 or survivin overexpression markedly rescued the GMC apoptosis mediated by miR-3546 mimic. Additionally, SOX4 overexpression could reverse the survivin suppression by miR-3546 mimic, and SOX4 could bind to survivin promoter (-1,278 to -853 nt) and activate survivin gene transcription. Conclusion: MiR-3546/ SOX4/survivin axis has a promoting role in the GMC apoptosis triggered by sublytic C5b-9, and our findings may provide a new insight into the pathogenesis of rat Thy-1N and human MsPGN.

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