Bacterial Proteins ; chemistry ; genetics ; metabolism ; DNA, Bacterial ; chemistry ; genetics ; metabolism ; Gene Expression Regulation, Bacterial ; physiology ; Glutathione ; metabolism ; Listeria monocytogenes ; chemistry ; genetics ; metabolism ; Peptide Termination Factors ; chemistry ; genetics ; metabolism

Objective To investigate the effect of long intergenic non-coding RNA COX2 (lincRNA-COX2) on apoptosis and polarization of Listeria monocytogenes (Lm)-infected RAW264.7 cells. Methods RAW264.7 cells were cultured and divided into control group (uninfected cells), Lm infection group, negative control of small interfering RNA (si-NC) group, si-NC and Lm infection group, small interfering RNA of lincRNA-COX2 (si-lincRNA-COX2) group, si-lincRNA-COX2 and Lm infection group. RAW264.7 cells were infected with MOI=10 Lm for 6 hours, and then the inhibition efficiency of siRNA transfection was detected by fluorescence microscope and quantitative real-time PCR (qRT-PCR). The expression levels of cleaved-caspase-3(c-caspase-3), caspase-3, B-cell lymphoma-2 (Bcl2), Bcl2 associated X protein (BAX), arginase 1 (Arg1), inducible nitric oxide synthase (iNOS) were detected by Western blot analysis. Results c-caspase-3/caspase-3, BAX/Bcl2 and iNOS were significantly up-regulated, while the level of Arg1 was down-regulated in Lm-infected RAW264.7 cells compared with control group. LincRNA-COX2 knockdown inhibited the increase of protein levels for BAX/Bcl2, c-caspase-3/caspase-3 and iNOS in Lm-infected RAW264.7 cells, while the level of Arg1 in Lm-infected RAW264.7 cells was up-regulated. Conclusion Knockdown of lincRNA-COX2 can inhibit cell apoptosis and suppress the macrophage polarization into M1 type in Lm-infected RAW264.7 cells.
Apoptosis/genetics* ; bcl-2-Associated X Protein/metabolism* ; Caspase 3/metabolism* ; Cyclooxygenase 2/metabolism* ; Listeria monocytogenes/pathogenicity* ; Macrophages/microbiology* ; RNA, Long Noncoding/metabolism* ; RNA, Small Interfering/genetics* ; Animals ; Mice

Listeria monocytogenes is an important zoonotic foodborne pathogen that can tolerate a number of environmental stresses. RsbR, an upstream regulator of the sigma B (SigB) factor, is thought to sense environmental challenges and trigger the SigB pathway. In Bacillus subtilis, two phosphorylation sites in RsbR are involved in activating the SigB pathway and a feedback mechanism, respectively. In this study, the role of RsbR in L. monocytogenes under mild and severe stresses was investigated. Strains with genetic deletion (ΔrsbR), complementation (C-ΔrsbR), and phosphorylation site mutations in the rsbR (RsbR-T175A, RsbR-T209A, and RsbR-T175A-T209A) were constructed to evaluate the roles of these RsbR sequences in listerial growth and survival. SigB was examined at the transcriptional and translational levels. Deletion of rsbR reduced listerial growxth and survival in response to acidic stress. Substitution of the phosphorylation residue RsbR-T175A disabled RsbR complementation, while RsbR-T209A significantly upregulated SigB expression and listerial survival. Our results provide clear evidence that two phosphorylation sites of RsbR are functional in L. monocytogenes under acidic conditions, similar to the situation in B. subtilis.
Alanine/genetics* ; Bacillus subtilis ; Bacterial Proteins/metabolism* ; Binding Sites ; Gene Deletion ; Gene Expression Regulation, Bacterial ; Genetic Complementation Test ; Homeostasis ; Hydrogen-Ion Concentration ; Listeria monocytogenes/metabolism* ; Listeriosis/microbiology* ; Mutation ; Phenotype ; Phosphoproteins/metabolism* ; Phosphorylation ; Sigma Factor/metabolism* ; Stress, Physiological

NB-Cl gene is a potential class IIa bacteriocin gene. To obtain its soluble expression, NB-C1 was fused with the green fluorescent protein (GFP) gene and a recombinant expression vector plVEX 2.4d-GFP-NB-C1 was constructed, which was transformed into Escherichia coli BL21(DE3) pLysS. The expressed fusion protein GFP-NB-CI was purified by Ni-NTA affinity chromatography and the bioactivity was examined using Listeria monocytogenes as the indicator bacteria. The results showed that the expressed fusion protein GFP-NB-C1 was soluble and the final concentration of the purified fusion protein was 36.1 mg/L E. coli culture and had the purity above 95%. The antimicrobial assay of GFP-NB-C1 was analyzed and showed its high activity against Listeria monocytogenes.
Amino Acid Sequence ; Anti-Bacterial Agents ; pharmacology ; Bacteriocins ; biosynthesis ; genetics ; Chromatography, Affinity ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Listeria monocytogenes ; drug effects ; Molecular Sequence Data ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

The aim of this study was to develop and validate an oligonucleotide suspension array for rapid identification of 15 bacterial species responsible for bacteremia, particularly prevalent in Chinese hospitals. The multiplexed array, based on the QIAGEN LiquiChip Workstation, included 15 oligonucleotide probes which were covalently bound to different bead sets. PCR amplicons of a variable region of the bacterial 23S rRNA genes were hybridized to the bead-bound probes. Thirty-eight strains belonging to 15 species were correctly identified on the basis of their corresponding species-specific hybridization profiles. The results show that the suspension array, in a single assay, can differentiate isolates over a wide range of strains and species, and suggest the potential utility of suspension array system to clinical laboratory diagnosis.
Bacteremia ; diagnosis ; genetics ; Bacterial Typing Techniques ; Bacteriological Techniques ; DNA Probes ; Genetic Techniques ; Listeria monocytogenes ; metabolism ; Nucleic Acid Hybridization ; Oligonucleotide Array Sequence Analysis ; Oligonucleotides ; chemistry ; RNA, Ribosomal ; chemistry ; RNA, Ribosomal, 23S ; genetics ; Stem Cells

In order to study the pathogenesis of Listeria monocytogenes (LM), we cloned listeriolysin gene into prokaryotic expression vector PET21a. The expression vector was transformed into Escherichia coli BL21 for expression of listeriolysin O (LLO). LLO-His tag fusion protein was purified with a Ni-NTA affinity column and was used as an immunogen to vaccinate BALB/C mice. Hybridomas were developed by fusing mouse myeloma cells Sp2/0 and splenocytes from the immunized mice and screened with purified LLO. Three hybridomas secreting antibodies against listeriolysin O were obtained and named anti-LLO1, anti-LLO2 and anti-LLO3, respectively. Western blotting analysis showed that all of them could specifically bind to the LLO secreted by the LM. The titers of anti-LLO monoclonal antibodies in the supernatants of three hybridomas cultures were 1:3.6 x 10(4), 1:6.4 x 10(4) and 1:1.6 x 10(4), respectively, and the titers of ascites from the hybridoma-injected mice were 1:2 x 10(7), 1:2 x 10(7) and 1:1 x 10(7), respectively, based on ELISA test. The isotypes of the monoclonal antibodies were determined to be IgG1. The dissociation constants (Kd) of these three monoclonal antibodies were determined to be 6.18 x 10(-11), 7.50 x 10(-11) and 6.27 x 10(-11) respectively. These data and reagents will be of great assistance to elucidate the pathogenesis of Listeriosis.
Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Bacterial Toxins ; biosynthesis ; genetics ; immunology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Heat-Shock Proteins ; biosynthesis ; genetics ; immunology ; Hemolysin Proteins ; biosynthesis ; genetics ; immunology ; Listeria monocytogenes ; genetics ; pathogenicity ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

The incidence of diabetes mellitus is increasing among companion animals. This disease has similar characteristics in both humans and animals. Diabetes is frequently identified as an independent risk factor for infections associated with increased mortality. In the present study, homozygous diabetic (db/db) mice were infected with Listeria (L.) monocytogenes and then treated with the anti-diabetic drug exendin-4, a glucagon-like peptide 1 analogue. In aged db/db mice, decreased CD11b+ macrophage populations with higher lipid content and lower phagocytic activity were observed. Exendin-4 lowered high lipid levels and enhanced phagocytosis in macrophages from db/db mice infected with L. monocytogenes. Exendin-4 also ameliorated obesity and hyperglycemia, and improved ex vivo bacteria clearance by macrophages in the animals. Liver histology examined during L. monocytogenes infection indicated that abscess formation was much milder in exendin-4-treated db/db mice than in the control animals. Moreover, mechanistic studies demonstrated that expression of ATP binding cassette transporter 1, a sterol transporter, was higher in macrophages isolated from the exendin-4-treated db/db mice. Overall, our results suggest that exendin-4 decreases the risk of infection in diabetic animals by modifying the interaction between intracellular lipids and phagocytic macrophages.
ATP-Binding Cassette Transporters/metabolism ; Age Factors ; Animals ; Blood Chemical Analysis ; Cholesterol/metabolism ; Diabetes Mellitus, Type 2/*drug therapy/genetics ; Dyslipidemias/drug therapy/genetics ; Female ; Hyperglycemia/drug therapy/genetics ; Hypoglycemic Agents/*therapeutic use ; Injections, Intraperitoneal ; *Lipid Metabolism/drug effects ; Listeria monocytogenes/*drug effects/immunology ; Listeriosis/*drug therapy/immunology/microbiology ; Macrophages/drug effects/*metabolism ; Mice ; Obesity/drug therapy/genetics ; Peptides/*therapeutic use ; Phagocytosis/drug effects ; Venoms/*therapeutic use