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PubMed2006

Neutrophil signaling pathways activated by bacterial DNA stimulation.

We have previously shown that bacterial DNA activates human neutrophils in a CpG-independent manner. In this study, we have characterized the signaling pathways involved in the activation mechanism. We found that p38 MAPK, ERK1/2, and JNK pathways, as well as the PI3K/Akt pathway, are activated by bacterial DNA. We also determined that bacterial DNA induces NF-kappaB and AP-1 activation. When analyzing the role of these pathways on neutrophil functions, we observed that up-regulation of CD11b triggered by bacterial DNA was decreased by pharmacological inhibitors of the p38 MAPK, ERK1/2, and JNK, whereas stimulation of IL-8 release was dependent on p38, ERK1/2, and NF-kappaB. Moreover, we found that IL-8 production was markedly enhanced by inhibition of JNK, suggesting that this pathway negatively modulates NF-kappaB-dependent transcription. We also observed that bacterial DNA stimulated IL-1R-associated kinase-1 kinase activity and its partial degradation. Finally, we determined that bacterial DNA stimulated CD11b up-regulation in TLR9(-/-) but not in MyD88(-/-) mouse neutrophils, supporting that bacterial DNA induces neutrophil activation through a TLR9-independent and MyD88-dependent pathway.

Animals↗
PubMed1997

Molecular properties of anti-DNA induced in preautoimmune NZB/W mice by immunization with bacterial DNA.

To elucidate the mechanism of Ag drive in the anti-DNA response, the Ab response to bacterial DNA has been analyzed in normal and autoimmune mice. Preautoimmune NZB/W mice immunized with Escherichia coli dsDNA produce Abs that resemble spontaneous autoantibodies and bind mammalian dsDNA. In contrast, normal mice, when immunized similarly, produce Abs that bind only bacterial dsDNA. To characterize further the responsiveness of NZB/W mice to bacterial DNA, we determined the molecular properties of mAbs from preautoimmune NZB/W mice immunized with E. coli DNA. Of nine Abs studied, all were IgM and all bound mammalian ssDNA, while four had appreciable reactivity with mammalian dsDNA. The induced anti-dsDNA resembled spontaneous anti-DNA from autoimmune mice in V gene utilization and V(H) CDR3 arginine content. These Abs lacked evidence of somatic mutation, however, indicating that affinity maturation via somatic mutation is not essential for dsDNA reactivity. The findings suggest that preautoimmune NZB/W mice have immunoregulatory defects that allow activation of mammalian dsDNA reactive B cells by bacterial DNA.

Amino Acid Sequence↗
PubMed1991

Specificity of anti-DNA antibodies induced in normal mice by immunization with bacterial DNA.

To determine the specificity of anti-DNA antibodies induced in normal mice by immunization with bacterial DNA, sera from BALB/c mice immunized with single-stranded DNA from Escherichia coli (EC) were tested for binding to a panel of synthetic DNA and RNA homopolymers as well as duplexes. Results of these studies indicate that sera from EC DNA immunized mice preferentially bind certain DNA and RNA homopolymers as well as DNA duplexes. Furthermore, the specificity of the antibodies from immunized mice resembled those of sera from autoimmune MRL-lpr/lpr mice in terms of the synthetic antigens recognized, although some differences were noted in the magnitude of the response to individual duplexes. These results suggest that anti-DNA antibodies induced by bacterial DNA bind to DNA structures dependent on both the base and the sugar phosphate moieties of the nucleic acid antigen and may resemble some anti-DNA antibodies expressed in spontaneous autoimmune disease in these binding properties.

Animals↗
PubMed2026

DNA polymerase I: structure, activity, and function in bacterial DNA replication and repair.

Faithful replication and repair of the genome are essential processes for all life. Genome maintenance is coordinated by a complex suite of proteins, with bacteria evolving intricate systems despite their relatively simplistic genomes. DNA polymerases are a key class of proteins that mediate genome maintenance. DNA polymerases are all capable of extending nascent strands of DNA but contribute to DNA replication and repair in distinct ways depending on their active site and substrate specificity. The first discovered polymerase, bacterial DNA polymerase I (Pol I), has long been considered the primary enzyme responsible for Okazaki fragment maturation and resynthesis in many DNA repair pathways. These conclusions derive primarily from studies using the gram-negative bacterium, Escherichia coli. Given that some bacterial lineages diverged from E. coli over a billion years ago, these assumptions may not account for evolution in functional diversity. In this review, we examine the structural features of bacterial Pol I and discuss how each of its distinct enzymatic activities contribute to genome maintenance. Throughout, we introduce differences that have been discovered between gram-negative and gram-positive species and explore how activity differences may translate to functional adaptations in replication or repair. We focus on evidence from gram-positive bacteria, particularly Bacillus subtilis and Geobacillus stearothermophilus, that challenges the universality of Pol I's functions and reveals lineage-specific adaptations in replication and repair mechanisms. By synthesizing historical perspectives with recent discoveries, this review underscores both the importance of Pol I and the evolutionary diversification of Pol I in bacterial DNA metabolism.

Bacterial DNA replication↗
PubMed2005

Differences in macrophage activation by bacterial DNA and CpG-containing oligonucleotides.

Bacterial DNA activates mouse macrophages, B cells, and dendritic cells in a TLR9-dependent manner. Although short ssCpG-containing phosphodiester oligonucleotides (PO-ODN) can mimic the action of bacterial DNA on macrophages, they are much less immunostimulatory than Escherichia coli DNA. In this study we have assessed the structural differences between E. coli DNA and PO-ODN, which may explain the high activity of bacterial DNA on macrophages. DNA length was found to be the most important variable. Double-strandedness was not responsible for the increased activity of long DNA. DNA adenine methyltransferase (Dam) and DNA cytosine methyltransferase (Dcm) methylation of E. coli DNA did not enhance macrophage NO production. The presence of two CpG motifs on one molecule only marginally improved activity at low concentration, suggesting that ligand-mediated TLR9 cross-linking was not involved. The major contribution was from DNA length. Synthetic ODN >44 nt attained the same levels of activity as bacterial DNA. The response of macrophages to CpG DNA requires endocytic uptake. The length dependence of the CpG ODN response was found to correlate with the presence in macrophages of a length-dependent uptake process for DNA. This transport system was absent from B cells and fibroblasts.

Animals↗
PubMed1999

Specificity of antibodies to bacterial DNA in the sera of healthy human subjects and patients with systemic lupus erythematosus.

OBJECTIVE: To elucidate the epitope structure to DNA by identifying antigenic determinants on bacterial DNA bound by anti-DNA antibodies from normal human subjects (NHS) and patients with systemic lupus erythematosus (SLE). METHODS: Sera from NHS and patients with SLE were tested by ELISA for the presence of antibodies to single stranded DNA from calf thymus, Micrococcus lysodeikticus, Staphylococcus epidermidis, Clostridium perfringens, and Klebsiella pneumoniae. To assess binding to conserved and nonconserved determinants, sera were absorbed on DNA-cellulose affinity columns bearing each of the bacterial DNA and then tested for binding to the other DNA antigens. RESULTS: Absorption of SLE sera with any of the bacterial DNA caused a loss of binding to all other bacterial DNA as well as calf thymus DNA. In contrast, absorption of NHS sera with bacterial DNA caused a loss of binding to the DNA on the affinity column with much less effect on binding to the other DNA antigens. CONCLUSION: These results indicate a marked difference in the specificity of antibodies to bacterial DNA in NHS and patients with SLE. The binding of SLE anti-DNA to predominantly conserved determinants suggests that a shift in patterns of anti-DNA specificity may be associated with the autoimmune state.

Antibody Specificity↗
PubMed1996

Macrophages ingest and are activated by bacterial DNA.

Recent evidence suggests that bacterial DNA activates immune responses. Here we showed that TNF-alpha mRNA was induced in bone marrow-derived macrophages and the macrophage cell line RAW 264 by plasmid DNA, but not by DNaseI-digested plasmid, plasmid methylated on CpG dinucleotides, or by vertebrate genomic DNA, which is naturally largely methylated on these sequences. Synthetic polynucleotides poly d(I-C) and poly I x poly C also induced TNF-alpha. IL-1 beta and plasminogen activator inhibitor-2 mRNAs were induced by plasmid DNA, and IFN-gamma-pretreated macrophages responded to DNA with induction of inducible nitric oxide synthase. The HIV-1 long terminal repeat was activated by exogenous DNA in a manner similar to TNF-alpha, and was also activated by a CpG-containing oligonucleotide. Transcription factor nuclear factor-kappa B (NF-kappa B) is involved in regulation of the HIV-1 long terminal repeat and many inflammatory response genes. NF-kappa B binding activity was increased by plasmid DNA. An important question is whether these effects involve DNA binding to a cell surface receptor that signals to the interior, or whether internalization is necessary. Here we found that plasmid was taken up by RAW 264 cells and remained sufficiently intact to code for luciferase protein. Results suggest that DNA is taken up by macrophages and characteristic bacterial DNA sequences, which include an unmethylated CpG sequence, activate a signaling cascade leading to activation of NF-kappa B and inflammatory gene induction. Relevance to DNA vaccination, gene therapy, antisense, and transfection studies is discussed.

Animals↗
PubMed1993

Molecular characterization of anti-DNA antibodies induced in normal mice by immunization with bacterial DNA. Differences from spontaneous anti-DNA in the content and location of VH CDR3 arginines.

Immunization of normal mice with bacterial DNA induces a significant anti-DNA response that includes antibodies resembling some lupus anti-DNA in their binding properties, although lacking specificity for mammalian dsDNA. To determine the structure of these induced antibodies and their relationship to anti-DNA from lupus mice, we have characterized the clonality and selected V-region sequences of a panel of 20 anti-DNA antibodies from 3 BALB/c mice immunized with ssDNA from Escherichia coli. Southern blot analysis of H and L chain rearrangements indicated that two of the animals expressed pairs of clonally related antibodies. Amino acid sequences of 10 of the induced antibodies demonstrated predominant utilization of J558 family VH genes and JH4 in association with various DH, J kappa and V kappa genes. Among the VH CDR3 of these 10 antibodies, 4 displayed arginine residues as a result of N region additions. None of these antibodies, however, had more than one arginine residue in VH CDR3 nor arginines at positions 100 or 100a, characteristic features of lupus antibodies to dsDNA. These results suggest that normal mice immunized with bacterial DNA display certain facets of DNA Ag drive, although lacking the mechanisms for the production of antibodies to mammalian dsDNA.

Amino Acid Sequence↗
PubMed2003

Bacterial DNA in house and farm barn dust.

BACKGROUND: Early in life, natural exposure to microbial components (eg, endotoxin) may mitigate allergy and asthma development in childhood. Bacterial DNA is a potent stimulus for the innate immune system; its immune stimulatory potential in dust is unknown. OBJECTIVES: We sought to quantify bacterial DNA and endotoxin content in dust from urban homes, rural homes, farm homes, and farm barns and to determine if dust DNA is immune-stimulatory. METHODS: Total DNA, bacterial DNA, and endotoxin were measured in 32 dust samples. To measure bacterial DNA content, a quantitative polymerase chain reaction assay specific for bacterial ribosomal DNA was developed. Peripheral blood mononuclear cells from 5 adults were stimulated with endotoxin-free dust DNA with/without lipopolysaccharide (LPS) from selected dust samples. IL-12p40, IL-10, and tumor necrosis factor-alpha were measured in cell supernatants by enzyme-linked immunosorbent assay. RESULTS: Bacterial DNA in dust correlated with endotoxin (r = 0.56, P <.001) and total DNA content (r = 0.51, P =.003). The highest bacterial DNA levels were measured in farm barns (mean, 22.1 microg/g dust; range, 1.3 to 56.2), followed by rural homes (6.3 microg/g; 0.2 to 20), farm homes (2.2 microg/g; 0.1 to 9.1), and urban homes (0.6 microg/g; 0.1 to 1.2). Farm barn DNA significantly potentiated (P < or =.05) LPS-induced IL-10 and IL-12 p40 but not tumor necrosis factor-alpha release (13-fold, 3-fold, and 1.5-fold increases, respectively). DNA from 6 urban homes did not demonstrate this LPS-potentiating effect. CONCLUSIONS: Endotoxin is a marker for bacterial DNA, which is also higher in locales of lower asthma and allergy prevalence. DNA from farm barn dust augments the immune modulatory effects of endotoxin and may combine with exposure to other such naturally occurring microbial components to mitigate allergy and asthma development.

Adult↗
PubMed2004

A sequential study of serum bacterial DNA in patients with advanced cirrhosis and ascites.

Bacterial translocation is currently considered the main pathogenic mechanism leading to spontaneous bacterial peritonitis in patients with advanced cirrhosis and ascites. However, to the authors' knowledge there is no information regarding the characteristics of this process in humans. The goals of the current study were to pursue partially identified bacterial DNA in blood (what the authors consider molecular evidence of bacterial translocation) through its relative quantification in a 72-hour study period by using real-time polymerase chain reaction (PCR). A consecutive series of 17 patients with advanced cirrhosis and culture-negative, nonneutrocytic ascites were studied. Therapeutic paracentesis was performed at the time of admission, and blood samples were obtained at baseline and every 8 hours in a 3-day period. Bacterial DNA was detected by a PCR-based method, relatively quantified by real-time PCR, and identified by automated nucleotide sequencing. Seven of 17 patients demonstrated the simultaneous presence of bacterial DNA in blood and ascitic fluid at the time of admission. After therapeutic paracentesis was performed, bacterial DNA persisted in the blood for a minimum of 24 hours, and was reported to last as long as 72 hours in some patients. In addition, different patterns of bacterial DNA appearance and clearance from the blood were identified. The nucleotide sequencing process demonstrated that bacteria detected in the first sample were identical to those noted in subsequent detections over time. In conclusion, bacterial translocation is a single-species, dynamic process that appears to develop in a subgroup of patients with advanced cirrhosis.

Adult↗
PubMed1993

Induction of immune-mediated glomerulonephritis in normal mice immunized with bacterial DNA.

Normal mice immunized with bacterial DNA produce high titers of anti-DNA antibodies and represent a new model for autoantibody production in systemic lupus erythematosus. To determine whether DNA immunization can also provoke clinical manifestations of lupus, the occurrence of nephritis in immunized mice was assessed and correlated with levels of anti-DNA as well as antibodies to glomerular antigens. BALB/c mice immunized with Escherichia coli single-stranded DNA in complexes with methylated bovine serum albumin in adjuvant showed increased proteinuria compared to control mice immunized with mBSA alone. Furthermore, DNA immunized mice had significantly greater glomerular proliferative changes and immunoglobulin deposition than control mice. In an in vitro assay, sera from DNA immunized mice exhibited greater binding to glomerular antigens than sera from control mice. Compared to sera, renal eluates from DNA-immunized mice were enriched for anti-DNA and glomerular binding activity. These data indicate that immunization of normal mice with E. coli DNA induces an immune-mediated proliferative glomerulonephritis that is likely secondary to the renal deposition of anti-DNA antibodies.

Animals↗
PubMed2005

Detection and identification of bacterial DNA in serum from patients with acute pancreatitis.

BACKGROUND AND AIMS: Bacterial infections are common complications in patients with acute pancreatitis, and translocation of bacteria from the intestinal lumen is probably the first step in the pathogenesis of these infections. As blood cultures in afebrile patients are usually negative, more sensitive methods to investigate this hypothesis in patients are needed. Our group has recently developed a method to detect the presence of bacterial DNA in biological fluids, and we aimed to detect bacterial DNA in patients with acute pancreatitis, as molecular evidences of bacterial translocation. METHODS: Samples of blood were obtained on three consecutive days within the first six days after admission. Bacterial DNA was detected using a polymerase chain reaction based method, and an automated DNA nucleotide sequencing process allowed identification of bacteria species. RESULTS: Thirty one consecutively admitted patients with acute pancreatitis were studied. Bacterial DNA was detected in six patients (19.3%), and the sequencing process allowed identification of Citrobacter freundii and Pseudomonas aeruginosa. In two patients the same bacteria detected at admission was detected 24 hours later (above 99.9% homology of nucleotide sequence). Basic clinical and biochemical characteristics were similar among patients with or without the presence of bacterial DNA. CONCLUSION: Detection of gram negative bacteria derived bacterial DNA in our series supports the contention that bacterial translocation is a systemic process in approximately 20% of patients with acute pancreatitis that does not seem to be related to the severity of the episode or immediate development of infection.

Acute Disease↗
PubMed1998

IFN-gamma primes macrophage responses to bacterial DNA.

Macrophages recognize and are activated by unmethylated CpG motifs in bacterial DNA. Here we demonstrate that production of nitric oxide (NO) from murine RAW 264 macrophages and bone marrow-derived macrophages (BMM) in response to bacterial DNA is absolutely dependent on interferon-gamma (IFN-gamma) priming. Similarly, arginine uptake and expression of the inducible nitric oxide synthase (iNOS) gene in response to bacterial DNA in BMM occurred only after IFN-gamma priming. In contrast, mRNA for the cationic amino acid transporter, CAT2, was induced by plasmid DNA alone, and priming with IFN-gamma had no effect on this response. Tumor necrosis factor-alpha (TNF-alpha) release from RAW 264 and BMM in response to bacterial DNA was augmented by IFN-gamma pretreatment. In a stably transfected HIV-1 long terminal repeat (LTR) luciferase RAW 264 cell line, IFN-gamma and bacterial DNA synergized in activation of the HIV-1 LTR. Bacterial DNA has been shown to induce IFN-gamma production in vivo as an indirect consequence of interleukin-12 (IL-12) and TNF-alpha production from macrophages. The results herein suggest the existence of a self-amplifying loop that may have implications for therapeutic applications of bacterial DNA.

Animals↗
PubMed2000

The role of bacterial DNA in autoantibody induction.

Bacterial DNA has potent immunological properties that can stimulate the immune system in SLE in both specific and non-specific ways. As such, this molecule may play an important role in disease pathogenesis, because it can exert immunomodulatory activity and function as a molecular mimic. Future studies will hopefully both determine the role of foreign nucleic acids in the induction of autoantibodies and lead to strategies for their elimination.

Animals↗
PubMed2000

The role of bacterial DNA in septic arthritis.

Unmethylated CpG motifs are frequently found in bacterial DNA and have recently been shown to exert immunostimulatory effects on leukocytes. Bacteria produce severe septic arthritis; bacterial DNA may be involved in this process. We injected intraarticularly bacterial DNA and oligonucleotides containing unmethylated CpG motifs into knee joints of mice. Arthritis was seen by histopathology within 2 h and lasted for at least 14 days, and was characterized by an influx of monocytic, Mac-1+ cells and by a lack of T lymphocytes. Macrophages and their products such as tumor necrosis factor (TNF) alpha are essential for development of arthritis triggered by bacterial DNA containing CpG motifs. In contrast, neurophils, NK cells, and T/B cells were not instrumental in this condition. This review demonstrates that bacterial DNA containing unmethylated CpG motifs induces arthritis and indicates an important pathogenic role for bacterial DNA in septic arthritis.

Animals↗
PubMed1981

A plasmid DNA primase active in discontinuous bacterial DNA replication.

A DNA primase encoded by an IncI alpha plasmid promotes efficient DNA replication in a primase-defective mutant of Escherichia coli. This finding implies that the plasmid enzyme can prime discontinuous DNA synthesis of the bacterial chromosome. The plasmid gene encodes two large, antigenically related proteins which differ from E. coli primase.

Bacteriocin Plasmids↗
PubMed1995

DNA-mediated transformation of a fungus employing a vector devoid of bacterial DNA sequences.

Acremonium chrysogenum, a producer of cephalosporin C, was subjected to DNA-mediated transformations using a vector without bacterial DNA sequences. Recombinant fungal strains were generated with a gel-purified DNA fragment, carrying only the mutated beta-tubulin gene from A. chrysogenum. The lack of any bacterial DNA was verified by Southern hybridization analysis and polymerase chain reaction amplifications to detect even residual DNA sequences. This procedure can be referred to as a self-cloning experiment for which less restricted working regulations are needed. Finally, the transfer of a synthetic hirudin gene by cotransformation demonstrated that any DNA molecule can be introduced into the A. chrysogenum genome without bacterial marker genes. This seems to be highly relevant for biotechnical processes in which safe recombinant producer strains are required to satisfy governmental restrictions.

Acremonium↗