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Results for “DNA, BACTERIAL”

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PubMed1987

Homology between bacterial DNA and bovine mitochondrial DNA encoding cytochrome c oxidase subunit III.

A segment of mitochondrial DNA encoding the bovine cytochrome c oxidase subunit III gene was isolated and inserted into an Escherichia coli plasmid vector. A 556 base pair fragment of the insert DNA representing about 70% of the 3'-end of the subunit III gene was used to search for homology with bacterial DNA from strains that contain heme aa3-type cytochrome c oxidases. Bacillus subtilis, Thermus thermophilus, and PS3 DNAs all showed strong hybridization to the probe, whereas Paracoccus denitrificans and Rhodopseudomonas sphaeroides DNAs showed only weak hybridization to the probe, even under low stringency conditions.

Animals↗

PubMed2001

Bacterial DNA does not increase serum corticosterone concentration or prevent increases induced by other stimuli.

Bacterial DNA containing unmethylated CpG motifs (CpG DNA) and other microbial molecules such as lipopolysaccharide (LPS) have a broad range of immune stimulatory effects, which may include many shared cell signaling pathways leading to enhanced cytokine production. Some cytokines activate the hypothalamic-pituitary-adrenal (HPA) axis, and their production is downregulated by products of the HPA axis (glucocorticoids). Because such interactions have practical implications in the clinical use of CpG DNA, the present study was done to examine the effects of CpG DNA and LPS on serum corticosterone concentrations. In contrast to LPS, administration of CpG DNA (DNA from Escherichia coli) (30-300 microg) alone did not significantly increase serum corticosterone concentrations 1 or 4 h after administration. Administration of CpG DNA to mice prior to LPS caused a synergistic increase in serum tumor necrosis factor-alpha (TNF-alpha), indicative of an immune stimulatory effect. LPS and TNF-alpha, however, induced similar levels of corticosterone with or without concomitant CpG DNA. Increasing doses of LPS caused peak corticosterone levels similar to those induced by LPS in combination with CpG DNA. Exogenous TNF-alpha administered in vivo induced comparable concentrations of corticosterone with or without CpG DNA. An alternative stressor (restraint) yielded similar levels of corticosterone with or without CpG DNA. These results indicate that CpG DNA does not induce corticosterone release or alter its release by other stimuli, indicating biologically important differences in its immune effect compared to those of LPS, and possibly reduced toxicity.

Animals↗

PubMed2003

Pathogen DNA as target for host-generated oxidative stress: role for repair of bacterial DNA damage in Helicobacter pylori colonization.

Helicobacter pylori elicits an oxidative stress during host colonization. This oxidative stress is known to cause lesions in the host DNA. Here we addressed the question as to whether the pathogen DNA is subject to lethal or mutational damage by the host-generated oxidative response. H. pylori Hpnth mutants unable to repair oxidized pyrimidines from the bacterial DNA were generated. H. pylori strains lacking a functional endonuclease III (HpNth) showed elevated spontaneous and induced mutation rates and were more sensitive than the parental strain to killing by exposure to oxidative agents or activated macrophages. Although under laboratory conditions the Hpnth mutant strain grows as well as the wild-type strain, in a mouse infection the stomach bacterial load gradually decreases while the population in the wild-type strain remains stable, showing that endonuclease III deficiency reduces the colonization capacity of the pathogen. In coinfection experiments with a wild-type strain, Hpnth cells are eradicated 15 days postinfection (p.i.) even when inoculated in a 1:9 wild-type:mutant strain ratio, revealing mutagenic lesions that are counterselected under competition conditions. These results show that the host effectively induces lethal and premutagenic oxidative DNA adducts on the H. pylori genome. The possible consequences of these DNA lesions on the adaptability of H. pylori strains to new hosts are discussed.

Animals↗

PubMed1996

Bacterial DNA induces murine interferon-gamma production by stimulation of interleukin-12 and tumor necrosis factor-alpha.

Bacterial, but not mammalian DNA, can induce interferon-gamma (IFN-gamma) in murine splenocytes. To elucidate the basis of this activity, we have assessed in vitro cytokine production by C3H/HeJ splenocytes stimulated with either DNA from Escherichia coli or a synthetic oligonucleotide containing an active palindromic sequence identified from DNA. Both DNAs induced IFN-gamma production, with the requirement for intact DNA shown by sensitivity to DNase digestion. Fractionated cell populations were evaluated to determine direct or indirect cellular effects of the DNA. Although bacterial DNA failed to induce IFN-gamma in the nonadherent cell population, supernatants from adherent cells stimulated by DNA induced IFN-gamma production by these cells. Interleukin-12 (IL-12) was detectable in supernatants from DNA-stimulated splenocytes before IFN-gamma, and neutralizing antibodies directed against IL-12 markedly inhibited the induction of IFN-gamma. Anti-tumor necrosis factor-alpha (TNF-alpha) antibodies also inhibited IFN-gamma production, and the combination of both anti-IL-12 and anti-TNF-alpha could totally inhibit production of IFN-gamma. Taken together, these results indicate that the stimulation of IFN-gamma production by bacterial DNA is mediated by IL-12 and TNF-alpha and point to macrophages/monocytes as targets of action of this macromolecule.

Animals↗

PubMed2006

Selective inhibitors of bacterial DNA adenine methyltransferases.

The authors describe the discovery and characterization of several structural classes of small-molecule inhibitors of bacterial DNA adenine methyltransferases. These enzymes are essential for bacterial virulence (DNA adenine methyltransferase [DAM]) and cell viability (cell cycle-regulated methyltransferase [CcrM]). Using a novel high-throughput fluorescence-based assay and recombinant DAM and CcrM, the authors screened a diverse chemical library. They identified 5 major structural classes of inhibitors composed of more than 350 compounds: cyclopentaquinolines, phenyl vinyl furans, pyrimidine-diones, thiazolidine-4-ones, and phenyl-pyrroles. DNA binding assays were used to identify compounds that interact directly with DNA. Potent compounds selective for the bacterial target were identified, whereas other compounds showed greater selectivity for the mammalian DNA cytosine methyltransferase, Dnmt1. Enzyme inhibition analysis identified mechanistically distinct compounds that interfered with DNA or cofactor binding. Selected compounds demonstrated cell-based efficacy. These small-molecule DNA methyltransferase inhibitors provide useful reagents to probe the role of DNA methylation and may form the basis of developing novel antibiotics.

DNA (Cytosine-5-)-Methyltransferase 1↗

PubMed1987

Defective degradation of bacterial DNA by phagocytes from patients with systemic and discoid lupus erythematosus.

The digestion of bacterial DNA by peripheral blood monocytes was impaired both in patients with systemic lupus erythematosus (SLE) and discoid lupus erythematosus (DLE). The monocytes of these patients had both a small quantitative defect in the solubilization of DNA and a marked qualitative defect in the extent to which this DNA was degraded. In addition, neutrophils from patients with SLE released significantly less high molecular-weight DNA than control cells. Digestion of bacterial RNA and protein by phagocytes was not defective in either disease. The reduced digestion of DNA by phagocytes resulted in concomitantly larger amounts of high molecular-weight DNA remaining in these cells. Such sequestration of DNA may contribute to the persistence of fairly large DNA fragments in the tissue of patients with lupus erythematosus.

Adult↗

PubMed1997

Specificity and immunochemical properties of antibodies to bacterial DNA in sera of normal human subjects and patients with systemic lupus erythematosus (SLE).

To elucidate the mechanisms of anti-DNA production, we assessed the binding of sera of normal human subjects (NHS) and patients with SLE to a panel of bacterial and mammalian DNA. Using single-stranded DNA as antigens in an ELISA, NHS showed significant binding to some but not all bacterial DNA, while lacking reactivity to calf thymus DNA. Among bacterial DNA, the highest levels of binding were observed with DNA from Micrococcus lysodeikticus and Staphylococcus aureus. In contrast, SLE sera showed high levels of binding to all DNA tested. To evaluate further immunochemical properties of the anti-DNA antibodies, the subclass distribution of these responses was evaluated by subclass-specific reagents. While NHS showed a predominance of IgG2 antibodies to bacterial DNA, SLE sera had a predominance of IgG1 antibodies to these antigens. Together, these results provide further evidence for the antigenicity of bacterial DNA and suggest that NHS and SLE anti-DNA differ in the patterns of epitope recognition as well as mechanisms of induction.

Animals↗

PubMed1995

Induction of cross-reactive anti-dsDNA antibodies in preautoimmune NZB/NZW mice by immunization with bacterial DNA.

To investigate the role of antigen drive in anti-double-stranded (ds) DNA production, the antibody response induced in lupus-prone NZB/NZW mice by E. coli (EC) dsDNA was evaluated. Preautoimmune NZB/NZW female mice were immunized with complexes of EC dsDNA with methylated bovine serum albumin (mBSA) in complete Freund's adjuvant; control mice received either mBSA complexes with calf thymus (CT) dsDNA or mBSA alone in adjuvant. IgG antibody responses were assessed by ELISA. Similar to normal mice, immunized NZB/NZW mice produced significant levels of anti-dsDNA when measured with EC dsDNA as antigen. Whereas normal mice produce antibodies which are specific for the immunizing bacterial DNA, NZB/NZW mice produced antibodies that bound crossreactively to CT dsDNA by ELISA. Furthermore, the induced antibodies resembled lupus anti-DNA in their fine specificity for polynucleotide antigens and reactivity with Crithidia luciliae DNA. Despite their response to EC dsDNA, NZB/NZW mice immunized with CT dsDNA failed to generate significant anti-dsDNA responses. These results provide further evidence for the enhanced immunogenicity of bacterial DNA and suggest that immune cell abnormalities in NZB/NZW mice promote the generation of crossreactive autoantibody responses when confronted with a foreign DNA.

Aging↗

PubMed1999

Bacterial DNA or oligonucleotides containing unmethylated CpG motifs can minimize lipopolysaccharide-induced inflammation in the lower respiratory tract through an IL-12-dependent pathway.

To determine whether the systemic immune activation by CpG DNA could alter airway inflammation, we pretreated mice with either i.v. bacterial DNA (bDNA) or oligonucleotides with or without CpG motifs, exposed these mice to LPS by inhalation, and measured the inflammatory response systemically and in the lung immediately following LPS inhalation. Compared with non-CpG oligonucleotides, i. v. treatment with CpG oligonucleotides resulted in higher systemic concentrations of polymorphonuclear leukocytes, IL-10, and IL-12, but significantly reduced the concentration of total cells, polymorphonuclear leukocytes, TNF-alpha, and macrophage inflammatory protein-2 in the lavage fluid following LPS inhalation. The immunoprotective effect of CpG-containing oligonucleotides was dose-dependent and was most pronounced in mice pretreated between 2 and 4 h before the inhalation challenge, corresponding to the peak levels of serum cytokines. bDNA resulted in a similar immunoprotective effect, and methylation of the CpG motifs abolished the protective effect of CpG oligonucleotides. The protective effect of CpG oligonucleotides was observed in mice with either a disrupted IL-10 or IFN-gamma gene, but release of cytokines in the lung was increased, especially in the mice lacking IFN-gamma. In contrast, CpG DNA did not protect mice with a disrupted IL-12 gene against the LPS-induced cellular influx, even though CpG DNA reduced the release of TNF-alpha and macrophage inflammatory protein-2 in the lung. These findings indicate that CpG-containing oligonucleotides or bDNA are protected against LPS-induced cellular airway inflammation through an IL-12-dependent pathway, and that the pulmonary cytokine and cellular changes appear to be regulated independently.

Administration, Inhalation↗

PubMed2000

Bacterial dna sequences in prostate tissue from patients with prostate cancer and chronic prostatitis.

PURPOSE: Although bacterial genetic material has been detected in prostate tissue from patients with various disorders, the prevalence of these organisms is unknown. We tested the hypothesis that bacterial detection rates differ between patients with prostate cancer and those with the chronic prostatitis/pelvic pain syndrome. MATERIALS AND METHODS: Sterile prostate biopsies were obtained during radical retropubic prostatectomy from 107 patients with prostate cancer and using a perineal approach from 170 with the chronic prostatitis/pelvic pain syndrome. Numerous controls were also evaluated. Bacterial ribosomal encoding DNA (165 rDNA) sequences were detected using a polymerase chain reaction assay. Selected positives were cloned, sequenced and compared with DNA databases. RESULTS: Bacterial DNA sequences were detected in 21 (19. 6%) of 107 patients with prostate cancer compared to 79 (46.4%) of 170 with chronic prostatitis (p <0.0001). These bacteria included urogenital pathogens, other described microorganisms and bacteria not reported previously. CONCLUSIONS: Bacterial DNA sequences may be identified in prostate tissue from many patients. Bacterial detection rates in prostate tissue appear to differ among populations, with higher rates among patients with the chronic prostatitis/pelvic pain syndrome than among those with prostate cancer. Future studies of the role of various bacteria in the prostate may provide insight into the pathophysiology of prostate disease.

Adolescent↗

PubMed1999

Improved method for purification of bacterial DNA from bovine milk for detection of Brucella spp. by PCR.

Different methods of extraction of bacterial DNA from bovine milk to improve the direct detection of Brucella by PCR were evaluated. We found that the use of a lysis buffer with high concentrations of Tris, EDTA, and NaCl, high concentrations of sodium dodecyl sulfate and proteinase K, and high temperatures of incubation was necessary for the efficient extraction of Brucella DNA. The limit of detection by PCR was 5 to 50 Brucella CFU/ml of milk.

Animals↗

PubMed2001

From bugs to drugs: therapeutic immunomodulation with oligodeoxynucleotides containing CpG sequences from bacterial DNA.

Several types of immune cells possess pattern recognition receptors (PRR) that can distinguish prokaryotic DNA from vertebrate DNA by detecting unmethylated CpG dinucleotides in particular base contexts (CpG motifs). Bacterial DNA or synthetic oligodeoxynucleotides containing these CpG motifs activate both innate and acquired immune responses that have evolved to protect against intracellular infections. These T helper 1 (Th1)-like immune responses include activation of B cells, dendritic cells, macrophages, and natural killer (NK) cells. CpG DNA-induced immune activation can protect against infection either alone or in combination with a vaccine and is effective in the immunotherapy of allergic diseases and cancer. Human clinical trials using such CpG DNA are currently underway.

Adjuvants, Immunologic↗

PubMed2000

Immunostimulatory bacterial DNA sequences activate dendritic cells and promote priming and differentiation of CD8+ T cells.

CD8+ T lymphocytes producing high levels of interferon-gamma (IFN-gamma) and expressing antigen specific cytotoxic activity are effectively induced after plasmid DNA vaccination and mediate protection against several intracellular micro-organisms. Recent evidence suggests that the priming of CD8+ T-cell responses following DNA injection involves antigen presentation mediated by dendritic cells. Here, we show that bacterial DNA and synthetic oligonucleotides containing dinucleotide (CpG) motifs activate cytokine expression in dendritic cells and modulate in vivo CD8+ T-cell priming and differentiation.

Animals↗

PubMed2006

Breast milk lactoferrin regulates gene expression by binding bacterial DNA CpG motifs but not genomic DNA promoters in model intestinal cells.

High-affinity binding of DNA by lactoferrin (LF) is an established phenomenon, but the biologic function of this interaction remains unclear. LF is an abundant breast milk protein (12.5-87.5 micromol/L) and is resistant to digestion in the infant gut. Regulation of gene expression by LF appears to be a major activity, particularly in modulating immune responses. We hypothesized that LF binding to DNA is a mechanism of gene regulation and aimed to identify the mechanism and physiologic sites of this activity. Our studies focused on two major biologic compartments of DNA: LF binding to proinflammatory bacterial DNA sequences (CpG motifs) in extracellular compartments and LF binding to genomic DNA promoters in the nucleus. LF 0.5 mmol/L inhibited CpG motif-induced nuclear factor-kappaB (NF-kappaB) activation and interleukin (IL)-8 and IL-12 cytokine gene transcription in B cells. Intestinal epithelial cells were unresponsive to CpG motifs. However, significant LF transferred across M cell-like monolayers, specialized epithelial cells that transcytose intact macromolecules to underlying B-cell follicles in the intestine. LF did not activate gene expression by binding to putative response elements in epithelial and lymphoid cells. Nor did LF bind to putative response elements specifically in gel-shift assays. No nuclear localization of LF was detected in green fluorescent protein (GFP) tagging experiments. We conclude that breast milk LF regulates gene expression by binding CpG motifs extracellularly, with follicular B cells in the infant intestine a likely target.

B-Lymphocytes↗

PubMed2007

Identification of bacterial DNA markers for the detection of human fecal pollution in water.

We used genome fragment enrichment and bioinformatics to identify several microbial DNA sequences with high potential for use as markers in PCR assays for detection of human fecal contamination in water. Following competitive solution-phase hybridization of total DNA from human and pig fecal samples, 351 plasmid clones were sequenced and were determined to define 289 different genomic DNA regions. These putative human-specific fecal bacterial DNA sequences were then analyzed by dot blot hybridization, which confirmed that 98% were present in the source human fecal microbial community and absent from the original pig fecal DNA extract. Comparative sequence analyses of these sequences suggested that a large number (43.5%) were predicted to encode bacterial secreted or surface-associated proteins. Deoxyoligonucleotide primers capable of annealing to a subset of 26 of the candidate sequences predicted to encode factors involved in interactions with host cells were then used in the PCR and did not amplify markers in DNA from any additional pig fecal specimens. These 26 PCR assays exhibited a range of specificity in tests with 11 other animal sources, with more than half amplifying markers only in specimens from dogs or cats. Four assays were more specific, detecting markers only in specimens from humans, including those from 18 different human populations examined. We then demonstrated the potential utility of these assays by using them to detect human fecal contamination in several impacted watersheds.

Animals↗

PubMed2004

Detection of bacterial DNA in synovial fluid from horses with infectious synovitis.

Standard culturing techniques are often unrewarding in confirming diagnosis of synovial infection in the equine patient. Several human studies report the use of sensitive polymerase chain reaction (PCR) techniques for the detection of bacterial involvement in acute synovitis. However, successful extraction of bacterial DNA directly from clinical samples from horses without prior culture has not been reported yet. The goal of this study was to develop a sensitive and reliable method for molecular detection and identification of bacterial species in synovial fluid from horses with infectious synovitis. Synovial fluid samples from 6 horses with culture confirmed synovial infection were used for broad range 16S rRNA gene PCR. Synovial aspirates of 2 healthy horses were used as negative controls. Following extraction and purification of synovial fluid DNA, all samples were processed by touchdown PCR. Amplicons were detected by reverse line blot hybridisation and visualised with chemiluminescence. Pathogen-specific detection of 16S rRNA gene sequences was successful in all 6 synovial fluid samples. No bacterial DNA was detected in the aspirates from the negative control horses using touchdown PCR followed by 25 additional cycles of amplification. The identity of the pathogens was confirmed by DNA sequencing of the amplicons. It can be concluded that broad range 16S rRNA gene PCR followed by reverse line blot hybridisation is a promising technique for detection of bacterial DNA in synovial fluid samples. Further research should aim at the detection of bacterial DNA in synovial fluid samples suspected of infection but having negative culture results. When the 16S PCR proves to be reliable and more sensitive than standard culturing techniques, it may become a powerful tool in the diagnosis of synovial infection.

Animals↗

PubMed1999

Bacterial DNA in mixed cholesterol gallstones.

OBJECTIVE: Numerous investigators have proposed a role for bacteria in biliary lithogenesis. We hypothesized that bacterial DNA is present in gallstones, and that categorical differences exist between gallstone type and the frequency of bacterial sequences. METHODS: Polymerase chain reaction (PCR) was used to amplify bacterial 16S rRNA and uidA (encoding Escherichia coli [E. coli] beta-glucuronidase) genes in different types of gallstones. PCR products were sequenced. RESULTS: Bacterial 16S rRNA and uidA DNA sequences in E. coli were detected in all brown pigment, common bile duct, and mixed cholesterol gallstones (n = 14). In contrast, only one (14%) of seven pure cholesterol gallstones yielded a PCR product. Most (88%) mixed cholesterol gallstones yielded PCR amplification products from their central, as well as their outer, portions. Sequenced products possessed 88-98% identity to 16S rRNA genes of E. coli and Pseudomonas species. CONCLUSIONS: Bacterial DNA sequences are usually present in mixed cholesterol (to 95% cholesterol content), brown pigment, and common bile duct, but rarely in pure cholesterol gallstones. The presence of bacterial beta-glucuronidase is also suggested. The role of bacteria and their products in the formation of mixed cholesterol gallstones, which comprise the majority of cholesterol gallstones, warrants further study.

Adult↗

PubMed2000

DNA-PKcs is required for activation of innate immunity by immunostimulatory DNA.

Bacterial DNA and related synthetic immunostimulatory oligodeoxyribonucleotides (ISS-ODN) stimulate innate immunity. However, the molecular recognition mechanism that initiates signaling in response to bacterial DNA and ISS-ODN has not been identified. Herein, we demonstrate that administration of bacterial DNA and ISS-ODN to mice lacking the catalytic subunit of DNA-PK (DNA-PKcs) and in vitro stimulation of BMDM from these mice result in defective induction of IL-6 and IL-12. Further analysis using BMDM of IKKbeta(-/-) revealed that both DNA-PKcs and IKKbeta are essential for normal cytokine production in response to ISS-ODN or bacterial DNA. ISS-ODN and bacterial DNA activate DNA-PK, which in turn contributes to activation of IKK and NF-kappaB. These results reveal a novel role of DNA-PKcs in innate immune responses and a link between DNA repair and innate immunity.

Androstadienes↗