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PubMed1989

Induction of anti-double stranded DNA antibodies in normal mice by immunization with bacterial DNA.

Because of recent observations suggesting that bacterial DNA is immunogenic, the induction in normal mice of antibodies to Escherichia coli (EC) dsDNA was investigated. BALB/c and C57BL/6 mice were immunized with dsEC or ds calf thymus (CT) DNA complexed to methylated BSA in adjuvant; antibody responses were measured by ELISA. In both strains, dsEC DNA immunization induced a much higher anti-dsDNA response to dsEC DNA than did dsCT DNA immunization. Neither immunized group showed an appreciable antibody response when tested on dsCT DNA. Anti-dsDNA antibodies were also demonstrated by ELISA using synthetic DNA duplexes as well as a filter binding assay using 3H-labeled dsEC DNA as Ag. These results suggest that bacterial dsDNA is immunogenic and that at least some anti-dsDNA specificities can arise by immunization.

Animals↗
PubMed2000

The features of arthritis induced by CpG motifs in bacterial DNA.

OBJECTIVE: To investigate the features of arthritis induced by bacterial DNA that contain CpG motifs. METHODS: Bacterial DNA originating from Escherichia coli and Staphylococcus aureus or synthetic oligonucleotides containing CpG motifs were injected directly into knee joints of mice. Histopathologic joint damage, antibody levels, cytokine levels, and synovial messenger RNA (mRNA) expression of cytokines and chemokines were assessed. RESULTS: Histopathologic signs of arthritis were evident within 2 hours and lasted for at least 3 weeks. Nonmethylated CpG motifs were responsible for the induction of arthritis since oligonucleotides containing these motifs triggered arthritis, whereas methylation of these nucleotides abrogated the inflammatory response. Arthritis was characterized by an influx of monocytic, Mac-1+ cells and by a scarcity of T lymphocytes. The disease was characterized locally by mRNA expression of macrophage-derived cytokines (tumor necrosis factor alpha, interleukin-12 [IL-12], IL-1beta) and chemokines (monocyte chemoattractant protein 1, RANTES) in arthritic joints. Systemically, the arthritis was characterized by increased levels of circulating IL-6 and immunoglobulins. CONCLUSION: These findings demonstrate that bacterial DNA that contain nonmethylated CpG motifs induces arthritis, suggesting an important pathogenic role for bacterial DNA in septic arthritis.

Animals↗
PubMed2003

Bacterial DNA activates human neutrophils by a CpG-independent pathway.

Bacterial DNA stimulates macrophages, monocytes, B lymphocytes, NK cells, and dendritic cells in a CpG-dependent manner. In this work we demonstrate that bacterial DNA, but not mammalian DNA, induces human neutrophil activation as assessed by L-selectin shedding, CD11b upregulation, and stimulation of cellular shape change, IL-8 secretion, and cell migration. Induction of these responses is not dependent on the presence of unmethylated CpG motifs, as neutrophil stimulatory properties were neither modified by CpG-methylation of bacterial DNA nor reproduced by oligonucleotides bearing CpG motifs. We found that human neutrophils express Toll-like receptor (TLR) 9 mRNA. However, as expected for a CpG-independent mechanism, activation does not involve a TLR9-dependent signaling pathway; neutrophil stimulation was not prevented by immobilization of bacterial DNA or by wortmannin or chloroquine, two agents that inhibit TLR9 signaling. Of note, both single-stranded and double-stranded DNA were able to induce activation, suggesting that neutrophils might be activated by bacterial DNA at inflammatory foci even in the absence of conditions required to induce DNA denaturation. Our findings provide the first evidence that neutrophils might be alerted to the presence of invading bacteria through recognition of its DNA via a novel mechanism not involving CpG motifs.

Bacteria↗
PubMed1997

CpG motifs in bacterial DNA cause inflammation in the lower respiratory tract.

Since unmethylated CpG motifs are more frequent in DNA from bacteria than vertebrates, and the unmethylated CpG motif has recently been reported to have stimulatory effects on lymphocytes, we speculated that bacterial DNA may induce inflammation in the lower respiratory tract through its content of unmethylated CpG motifs. To determine the role of bacterial DNA in lower airway inflammation, we intratracheally instilled prokaryotic and eukaryotic DNA in C3H/HeBFEJ mice and performed whole lung lavage 4 h after the exposure. Heat denatured, single stranded Escherichia coli genomic DNA (0.06 ng endotoxin/microg DNA) was compared to heat denatured, single stranded calf thymus DNA (0.007 endotoxin/microg DNA). 10 microg of bacterial DNA, in comparison to 10 microg of calf thymus DNA, resulted in a fourfold increase in the concentration of cells (P = 0.0002), a fivefold increase in the concentration of neutrophils (P = 0.0002), a 50-fold increase in the concentration of TNF-alpha (P = 0.001), and a fourfold increase in the concentration of both IL-6 (P = 0.0003) and macrophage inflammatory protein-2 (P = 0.0001) in the lavage fluid. Importantly, instillation of 0.60 ng of E. coli LPS resulted in a negligible inflammatory response. To test whether the stimulatory effects of bacterial DNA are due to its unmethylated CpG dinucleotides, we methylated the bacterial DNA and also prepared 20 base pair oligonucleotides with and without CpG motifs. In comparison to instillation of untreated bacterial DNA, methylation of the bacterial DNA resulted in a significant reduction in the concentration of cells and cytokines in the lower respiratory tract. Moreover, oligonucleotides containing embedded unmethylated CpG motifs resulted in inflammation in the lower respiratory tract that was indistinguishable from that observed with untreated bacterial DNA. In contrast, oligonucleotides without the embedded CpG motifs or with embedded but methylated CpG motifs resulted in significantly less inflammation in the lower respiratory tract. The possible relevance of these data to human disease was shown by extracting and analyzing DNA in sputum from patients with cystic fibrosis (CF). Approximately 0.1 to 1% of this sputum DNA was bacterial. Intratracheal instillation of highly purified CF sputum DNA caused acute inflammation similar to that induced by bacterial DNA. These findings suggest that bacterial DNA, and unmethylated CpG motifs in particular, may play an important pathogenic role in inflammatory lung disease.

Animals↗
PubMed1999

Intra-articularly localized bacterial DNA containing CpG motifs induces arthritis.

Unmethylated CpG motifs are often found in bacterial DNA, and exert immunostimulatory effects on hematopoietic cells. Bacteria produce severe joint inflammation in septic and reactive arthritides; bacterial DNA may be involved in this process. We injected bacterial DNA originating from Escherichia coli and Staphylococcus aureus and oligonucleotides containing CpG directly into the knee joints of mice of different strains. Arthritis was seen by histopathology within 2 hours and lasted for at least 14 days. Unmethylated CpG motifs were responsible for this induction of arthritis, as oligonucleotides containing these motifs produced the arthritis. The arthritis was characterized by an influx of monocytic, Mac-1+ cells and by a lack of T lymphocytes. Depletion of monocytes resulted in abrogation of the synovial inflammation. Tumor necrosis factor (TNF)-alpha, a cytokine produced by cells of the monocyte/macrophage lineage, is an important mediator of this disease, as expression of mRNA for TNF-alpha was evident in the inflamed joints, and the CpG-mediated inflammation was abrogated in mice genetically unable to produce this cytokine. These findings demonstrate that bacterial DNA containing unmethylated CpG motifs induces arthritis, and indicate an important pathogenic role for bacterial DNA in septic arthritis.

Animals↗
PubMed2005

STAT3 activation and c-FOS expression in the brain following peripheral administration of bacterial DNA.

To study the role of bacterial DNA in the brain function, we investigated signal transducer and activator of transcription 3 (STAT3) activation and c-FOS expression in the brain by immunohistochemistry in response to peripheral administration of CpG-DNA. CpG-DNA induced phospho-STAT3-immunoreactive cells and c-FOS-positive cells in several brain regions in a different manner. Phospho-STAT3-immunoreactive cells were observed in the circumventricular organs where the blood-brain barrier is weak. On the other hand, CpG-DNA increased c-FOS-positive cells in the paraventricular nucleus of the hypothalamus, and the nucleus of the tractus solitarius (NTS) and the area postrema. Unilateral cervical vagotomy did not modify CpG-DNA-induced c-FOS expression in the NTS, indicating that CpG-DNA-induced activation of the NTS is independent of the afferent vagus nerve input originating from the subdiaphragmatic organs. On the other hand, Toll-like receptor 9 mRNA was expressed in the nodose ganglion. Therefore, it is possible that CpG-DNA activates afferent vagus nerve through the nodose ganglion which subsequently activates the NTS. Present observations represented that peripheral CpG-DNA induced immune event in the brain, and that not only c-FOS but also phosphorylation of STAT3 can be a useful indicator for evaluation of neuro-immune interaction.

Animals↗
PubMed1997

Deficient expression of antibodies specific for bacterial DNA by patients with systemic lupus erythematosus.

Antibodies to DNA occur commonly in the sera of normal human subjects and bind to nonconserved sites exclusive to DNA from certain bacterial species. These antibodies are primarily IgG2 and differ from anti-DNA antibodies in systemic lupus erythematosus (SLE) sera, which are primarily IgG1 and bind conserved sites on all DNA. To investigate the immune response to bacterial DNA in SLE, antibodies to single-stranded DNA from Micrococcus lysodeikticus (MC) were measured in normal human subjects and in SLE sera by enzyme-linked immunosorbent assay either directly or following absorption by cellulose bearing calf thymus (CT) DNA; isotype-specific reagents were used to assess the IgG2 response. In these assays, SLE sera had a marked deficiency in antibodies specific for MC single-stranded DNA as demonstrated by the reduction of antibody levels by CT DNA cellulose absorption. Furthermore, IgG2 anti-DNA in SLE sera were cross-reactive with MC and CT single-stranded DNA, whereas IgG2 anti-DNA in normal human subjects' sera were specific for the bacterial DNA antigen. Together, these findings indicate that SLE patients have a marked deficiency in the production of antibodies specific for bacterial DNA antigen. This deficiency could predispose to anti-DNA autoantibody production and result from tolerance defects that distort the array of specificities in the B-cell repertoire.

Adolescent↗
PubMed1997

Specificity and immunochemical properties of antibodies to bacterial DNA.

DNA is a structurally heterogeneous molecule that elicits antibody production in both normal and aberrant immunity. In the prototypic autoimmune disease systemic lupus erythematosus, anti-DNA antibodies occur prominently and are serological markers for diagnosis and prognosis. These antibodies target conserved sites on both single-stranded (ss) and double-stranded (ds) DNA from essentially all species. In contrast, sera of normal human subjects (NHS) contain antibodies that selectively bind to DNA from certain bacteria. The NHS antibodies differ from lupus anti-DNA in their exclusive binding to foreign DNA as well as in their isotype, light chain distribution, and mode of DNA interaction. The properties of these antibodies suggest that they arise as a specific response to bacterial DNA introduced during infection or colonization. This conclusion is supported by studies demonstrating that bacterial DNA is a potent immunogen in normal mice and can induce antibodies specific for determinants on bacterial ss and dsDNA. Furthermore, immunization of normal animals with bacterial DNA elicits antibodies that bind mammalian as well as bacterial ssDNA; this immunization can also provoke glomerulonephritis. In addition to its immunogenicity, bacterial DNA is mitogenic, stimulating polyclonal activation of murine B cells; bacterial DNA can also induce cytokine production in the mouse. The range of immunological activities of bacterial DNA suggests an important role of this molecule in stimulating host defense in normal individuals and provoking autoimmunity in individuals predisposed to SLE.

Animals↗
PubMed2000

Sequence analysis of bacterial DNA in the colon and stomach of the Tyrolean Iceman.

The male human body found in an Alpine glacier on September 19, 1991 ("Tyrolean Iceman") has, for the first time in history, given scientists a chance to perform detailed anatomical, histological, and molecular investigations on the organs of a person from the Neolithic Age (5350-5100 B.P.). In the present study, tissue samples aseptically taken from the stomach and the colon of the mummy were utilized for DNA extraction, and the DNA was PCR-amplified, using primer pairs designed to bind to fragments of the 16s ribosomal RNA gene (16s rDNA) of a broad range of bacteria. The PCR products were cloned in plasmid vectors, and the recombinant clones (amplicons) were sequenced. The sequence data were finally used for scanning data libraries containing the corresponding sequences of present-day bacteria, to infer the putative ecophysiology of the ancient ones. The same procedure was repeated on some fragments of grass from the clothing found near the corpse. These fragments were taken as a control of the microbiological situation of the glacier. The results show that the flora of the Iceman's stomach is entirely composed of Burkholderia pickettii, an organism commonly found in aquatic habitats. The colon, on the other hand, contains several members of the fecal flora of humans, such as Clostridium perfringens, C. ghonii, C. sordellii, Eubacterium tenue, and Bacteroides sp. The Iceman's colon, however, was found to contain, rather unexpectedly, also some members of the genus Vibrio. The results are discussed in light of what is known about the preservation of microbial DNA at the Iceman's site and of previous parasitological studies performed on the Iceman himself and on human coprolites.

Animals↗
PubMed1992

Potassium ions and changes in bacterial DNA supercoiling under osmotic stress.

Escherichia coli transiently increases both the [ATP]/[ADP] ratio and the negative supercoiling of plasmid DNA when it is shifted to high osmolarity. Here we report that a mutant lacking all saturable K+ transport systems increases the negative supercoiling of the plasmid DNA under upshock but cannot further relax DNA. The mutant dnaK756 behaves like the K+ transport mutant.

Adenosine Diphosphate↗
PubMed2005

Real-time polymerase chain reaction for detecting bacterial DNA directly from blood of neonates being evaluated for sepsis.

Speed is of the essence when evaluating an infant with symptoms consistent with sepsis. Because of the high morbidity and mortality associated with neonatal sepsis, infants receive multiple, broad-spectrum antibiotics before receiving finalized blood culture results. Incorporating an additional, reliable, yet rapid assay to detect bacteria directly from blood would facilitate timely diagnosis and appropriate care. To this end, we designed a real-time polymerase chain reaction (PCR) assay targeting the highly conserved 380 bases of 16S rDNA. DNA was extracted from whole-blood samples using a Qiagen column. The limit of detection for the TaqMan-based assay, using a Smartcycler instrument, was 40, 50, or 2000 colony-forming units per milliliter of blood (CFU/ml) of Escherichia coli, group B Streptococcus, and Listeria monocytogenes, respectively, when white blood cell counts were below 39,000/microl. Implementing this approach requires less than 4 hours for both sample preparation and real-time PCR compared with 1 to 2 days to detect growth in culture or 5 days to finalize no-growth culture results. There was an overall agreement between the results of culture and real-time PCR of 94.1% (80 of 85) in this study. These results suggest that molecular techniques can augment culture-based methods for diagnosing neonatal sepsis, especially in infants whose mothers have received intrapartum antibiotic prophylaxis.

DNA, Bacterial↗
PubMed1982

Design and characterization of N2-arylaminopurines which selectively inhibit replicative DNA synthesis and replication-specific DNA polymerases: guanine derivatives active on mammalian DNA polymerase alpha and bacterial DNA polymerase III.

The 2-amino substituted derivatives of guanine, N2-(p-n-butylphenyl)guanine (BuPG) and N2-(3',4'-trimethylenephenyl) guanine (TMPG), were synthesized and found to selectively inhibit, respectively, HeLa cell DNA polymerase alpha (po1 alpha) and B. subtilis DNA polymerase III (po1 III). Both purines, like their corresponding uracil analogs, BuAu and TMAU (2,9), were specifically competitive with dGTP in their inhibitory action on their target polymerases. BuPG, the pol alpha-specific purine, was also toxic for HeLa cells in vivo, selectively inhibiting DNA synthesis. These N2-substituted purines, in contrast to the 6-substituted uracils, provide a structural basis for the synthesis of nucleosides and nucleotides with considerable potential as probes for the analysis of the structure of specific replicative DNA polymerases and their function in cellular DNA metabolism.

Bacillus subtilis↗