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PubMed2001

Downregulation of bactericidal peptides in enteric infections: a novel immune escape mechanism with bacterial DNA as a potential regulator.

Antibacterial peptides are active defense components of innate immunity. Several studies confirm their importance at epithelial surfaces as immediate barrier effectors in preventing infection. Here we report that early in Shigella spp. infections, expression of the antibacterial peptides LL-37 and human beta-defensin-1 is reduced or turned off. The downregulation is detected in biopsies from patients with bacillary dysenteries and in Shigella- infected cell cultures of epithelial and monocyte origin. This downregulation of immediate defense effectors might promote bacterial adherence and invasion into host epithelium and could be an important virulence parameter. Analyses of bacterial molecules causing the downregulation indicate Shigella plasmid DNA as one mediator.

Adult↗
PubMed1993

Testing potential gyrase inhibitors of bacterial DNA gyrase: a comparison of the supercoiling inhibition assay and "cleavable complex" assay.

Inhibitory activity of test compounds against Escherichia coli DNA gyrase in a "cleavable complex" assay, readily observed in vitro at the enzyme level by the artificial addition of a denaturing agent, is found to be an excellent indicator of 4-quinolone inhibition of DNA gyrase, and as accurate a predictor of target enzyme inhibitory activity as the measurement of the inhibition of DNA gyrase supercoiling. This study was designed to examine the specificity of DNA gyrase inhibitors of various chemical classes in these two DNA gyrase assays, and define the use of these two assays in understanding the nature of inhibition by experimental compounds. Supercoiling inhibition was detected by determination of the 50% inhibition level, and cleavable complex inhibition measured by the determination of the drug concentration at which 50% of the maximal (of control) formation of linear, cleaved DNA was obtained. Results indicate that these two assays can serve several different functions in microbiological research, among them: (1) quantitation of enzyme inhibitory activity at the target level; and (2) distinguishing between nonspecific inhibition or artifactual inhibition of DNA gyrase and true, mechanism-based inhibition of the catalytic activity of DNA gyrase.

Anti-Bacterial Agents↗
PubMed2005

Diagnosis and genotyping of Helicobacter pylori by polymerase chain reaction of bacterial DNA from gastric juice.

BACKGROUND: Efficient and accurate detection of Helicobacter pylori infection as well as identification of virulence-associated alleles are important for the treatment of gastroduodenal diseases caused by this gastric pathogen. The present study was performed to test the efficiency of gastric juice polymerase chain reaction (PCR) method for the rapid detection of H. pylori infection and to determine the bacterial genotypes without the need for culture, which is often not feasible especially in developing countries. METHODS: DNA was extracted from gastric juice samples collected from 45 subjects and was used to amplify urease B gene (ureB) for H. pylori. Results obtained from this method were further confirmed by rapid urease test (RUT), histology and culture. Genotypes of the infected strains predicted from gastric juice PCR were compared to the genotype data obtained from the isolated strains. RESULTS: Among 45 cases, 32 were positive by RUT, 37 by histological examination, 25 by gastric juice PCR method, while culture yielded positive results for 19 samples. Except for one case, all the 19 culture-positive strains gave the same genotype with the gastric juice PCR result. It was found that the gastric juice PCR is more efficient for detection of multiple-strain infection as compared to genotype data obtained from strains isolated as pooled culture. CONCLUSIONS: This moderately sensitive technique could be employed with good efficiency, particularly in cases where it is difficult to obtain biopsy. Moreover, with this method bacterial genotype could be obtained.

Bangladesh↗
PubMed1998

TIP49, homologous to the bacterial DNA helicase RuvB, acts as an autoantigen in human.

TATA-binding protein (TBP), a central component for transcriptional regulation, forms complexes with various transcription regulators. We have isolated a novel human cDNA for a 49-kD TBP-interacting protein (TIP49). The human TIP49 was highly homologous to bacterial RuvB proteins that function as a DNA helicase to promote branch migration of the Holliday junction. Immunofluorescence analysis using anti-TIP49 antibody showed a typical dot-shaped nuclear staining pattern, suggesting that TIP49 is included in a macromolecular structure in the nucleus and may participate in nuclear events such as transcription and recombination. Moreover, glycerol gradient analysis demonstrated that TIP49 is present in a macromolecular complex in nuclear extracts. Interestingly, we detected a high level of autoantibodies against TIP49 in sera of patients with autoimmune diseases such as polymyositis/dermatomyositis and autoimmune hepatitis. This indicates that the autoantibody against this protein is a new marker for particular connective tissue diseases. These findings provide further evidence that the macromolecular structures described above are targeted by an autoimmune mechanism. The anti-TIP49 antibodies can be useful probes for clinical diagnosis and for investigation of intranuclear structure.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2004

Structural rearrangement accompanying NAD+ synthesis within a bacterial DNA ligase crystal.

DNA ligase is an enzyme important for DNA repair and replication. Eukaryotic genomes encode ligases requiring ATP as the cofactor; bacterial genomes encode NAD(+)-dependent ligase. This difference in substrate specificities and the essentiality of NAD(+)-dependent ligase for bacterial survival make NAD(+)-dependent ligase a good target for designing highly specific anti-infectives. Any such structure-guided effort would require the knowledge of the precise mechanism of NAD+ recognition by the enzyme. We report the principles of NAD+ recognition by presenting the synthesis of NAD+ from nicotinamide mononucleotide (NMN) and AMP, catalyzed by Enterococcus faecalis ligase within the crystal lattice. Unprecedented conformational change, required to reorient the two subdomains of the protein for the condensation to occur and to recognize NAD+, is captured in two structures obtained using the same protein crystal. Structural data and sequence analysis presented here confirms and extends prior functional studies of the ligase adenylation reaction.

Cloning, Molecular↗
PubMed2002

[Amplification reaction to bacterial DNA for diagnosing infective endocarditis].

The aim of this study was to evaluate the usefulness of broad-range bacterial PCR in infective endocarditis of bacterial etiology, and to determine its specificity and sensitivity. Twenty five blood samples were taken for analysis from patients with infective endocarditis and acquired valvular heart disease. Infective endocarditis was diagnosed according to Duke criteria. There were two control groups consisting of patients with acquired valvular heart disease: 10 patients with urinary tract infection and 15 patients without. Three different primer pairs for the region of the gene coding for 16S rRNA were tested, to find the most specific one. The highest specificity was found for F/R primers, as the relevant amplified PCR product was present in every blood sample with infective endocarditis, and also in 4 out of 10 patients with urinary tract infection. Broad-range PCR in bacterial endocarditis is a fast, sensitive and inexpensive tool for the detection of bacteria, but it is far more prone to contamination than species specific-PCR. However, in controlled conditions it may be valuable in the identification of non-specific infection allowing for a more rapid clinical diagnosis of endocarditis.

Bacteria↗
PubMed2001

Bacterial DNA ligases.

DNA ligases join breaks in the phosphodiester backbone of DNA molecules and are used in many essential reactions within the cell. All DNA ligases follow the same reaction mechanism, but they may use either ATP or NAD+ as a cofactor. All Bacteria (eubacteria) contain NAD+-dependent DNA ligases, and the uniqueness of these enzymes to Bacteria makes them an attractive target for novel antibiotics. In addition to their NAD+-dependent enzymes, some Bacteria contain genes for putative ATP-dependent DNA ligases. The requirement for these different isozymes in Bacteria is unknown, but may be related to their utilization in different aspects of DNA metabolism. The putative ATP-dependent DNA ligases found in Bacteria are most closely related to proteins from Archaea and viruses. Phylogenetic analysis suggests that all NAD+-dependent DNA ligases are closely related, but the ATP-dependent enzymes have been acquired by Bacterial genomes on a number of separate occasions.

Adenosine Triphosphate↗
PubMed1988

Coumarins as inhibitors of bacterial DNA gyrase.

Chlorobiocic acid and 3-(carbobenzoxyamino)-4,7-dihydroxy-8-methylcoumarin were identified as two new inhibitors of Micrococcus luteus DNA gyrase. Both compounds possess weak antibacterial activity against whole M. luteus cells which indicates that they probably lack efficient transport functions to penetrate the cell envelope.

Anti-Bacterial Agents↗
PubMed1983

The effect of bacterial DNA gyrase inhibitors on DNA synthesis in mammalian mitochondria.

Using isolated rat liver mitochondria, which have previously been shown to carry out true replicative DNA synthesis, we have obtained results which are in accord with the presence and functioning of a DNA gyrase in this organelle. The effects of the Escherichia coli DNA gyrase inhibitors, novobiocin, coumermycin, nalidixic acid and oxolinic acid, upon mtDNA replication suggest the involvement of the putative mitochondrial enzyme in various aspects of this process. First, the preferential inhibition of [3H]dATP incorporation into highly supercoiled DNA together with the appearance of labeled, relaxed DNA are consistent with the involvement of a gyrase in the process of generating negative supercoils in mature mtDNA. Second, the overall depression of incorporation of labeled dATP into mtDNA, including the reduction of radioactivity incorporated into replicative intermediates, suggests a 'swivelase' role for the putative gyrase, and this hypothesis is further supported by results obtained on sucrose gradient centrifugation of heat-denatured, D-loop mtDNA. Here, the synthesis of the completed clean circles is inhibited while 9 S initiator strand synthesis is not, suggesting that chain elongation is blocked by the gyrase inhibitors.

Aminocoumarins↗
PubMed2000

Does the bacterial DNA found in middle ear effusions come from viable bacteria?

INTRODUCTION: Between 20% and 50% of middle ear effusions in otitis media with effusion test positive for bacteria, i.e. H. influenzae, M. catarrhalis and S. pneumoniae. In one study 48% of effusions that tested negative by culture wre positive by polymerase chain reaction (PCR).1 This has been advanced as evidence for the presence of bacterial biofilms, as agents leading to the persistence of glue ear. There is, however, the possibility that the DNA detected is the fossilized remains of bacteria from previously cleared infections. If this is the case, then the effusioin must in some way be protecting the DNA from breakdown by DNases. METHODS: Here we demonstrate, using a viscosity assay, that middle ear effusions taken from children during myringotomy inhibit the breakdown of DNA in a concentration-dependent manner. Middle ear effusion homogenates 0.5-3.0 ml (1 : 10 Vol. vol/effusion: PBS) were incubated with 3 ml of DNA (0.5 mg/ml in PBS) plus 0.2 ml of DNase 1 (500 Kunitz) at 37 degrees C for 24 h. Viscosity measurements were taken at regular intervals and the changes in viscosity expressed as a percentage of time 0. DNase activity was inhibited by 1.5 ml and 3.0 ml of effusion 48% and 91%, respectively, after 30 min incubation. CONCLUSION: This study demonstrated that effusions have the ability to inhibit nuclease activity, and the reported presence of non-culturable bacteria based on DNA detection by PCR could still represent fossilized remains and not viable bacteria. The same could also be true of recent reports of bacterial mRNA.2

Journal Article↗
PubMed2006

Immunostimulatory oligodeoxynucleotide isolated from genome wide screening of Mycobacterium bovis chromosomal DNA.

Bacterial DNA has a variety of immunostimulatory activities, such as the activation of B cells and natural killer cells, the induction of interferon-gamma, and the induction of Th1-type immune responses. In contrast, mammalian DNA does not have these activities. To evaluate the genomic DNA sequences of Mycobacterium bovis that have immunostimulatory activity, we used a computer to analyze the M. bovis genome and we designed a series of synthetic, 20 base length, phosphodiester backbone oligodeoxynucleotides (ODNs) that contain CpG motifs (MB-ODNs). We screened the immunostimulatory MB-ODNs that induce the activation of the NF-kappaB-responsive IL-8 promoter in RAW 264.7 cells. Our experimental analyses demonstrate that the potent CpG DNA in the M. bovis genome has functional effects as a Th1-responsive adjuvant, and that it activates the transcription factor NF-kappaB. Moreover, we found that both the CpG motifs and the context of the sequence surrounding the CpG motif are important for the immunostimulatory activities. The identification of the potent immunostimulatory DNA sequence in a native bacterial genome may give insights to the optimal sequence for well-controlled immune responses.

Adjuvants, Immunologic↗
PubMed1980

A study of substrate specificity of mammalian and bacterial DNA polymerases with 5-alkyl-2'-deoxyuridine 5'-triphosphates.

DNA polymerases from procaryotic sources can utilize a variety of dTTP analogues as substrates. We studied here in vitro DNA syntheses catalyzed by DNA polymerase alpha and beta of calf thymus, and for comparison, by the Escherichia coli DNA polymerase I large fragment enzyme in the presence of 5-alkyl derivatives of dUTP as dTTP substrate analogues, using activated DNA as template-primer. The alkyl substituents were n-alkyl (from ethyl to hexyl) and iso-alkyl (isopropyl and tert-butyl) groups. All enzymes were active in the presence of each modified dTTP, incorporation rates of [3H]dAMP or [3H]dGMP were, however, much lower with the analogues than with dTTP. According to relative incorporation rates, alpha-polymerase in DNA synthesis was found to be less sensitive to changes in the length of the alkyl substituent of 5-n-alkyl-dUTPs than beta-polymerase or the E. coli enzyme. Evidence for the incorporation of the analogues was presented for 5-[2-14C]isopropyl-dUTP.

Animals↗
PubMed1994

Autonomous replication of bacterial DNA plasmid oligomers in Leishmania.

Extrachromosomal amplicons are frequently observed in drug-resistant Leishmania. A dominant selectable marker, the neomycin phosphotransferase gene, was introduced by gene targeting in a circular amplicon derived from the H locus of Leishmania in a mutant cell. This recombinant amplicon was isolated and transfected in a wild-type cell. The amplicon was kept in the wild-type cells, provided the selective pressure was maintained, suggesting that it was capable of autonomous replication. Novel Leishmania expression vectors suited for stable transfections were made to isolate, by a high transformation assay, the putative origin of replication in the amplicons. However, these plasmids, which did not contain a single Leishmania nucleotide, were found as extrachromosomal circular oligomers in Leishmania transfectants. Their relative stability, in addition to changes in their methylation pattern, indicated that these plasmids were most likely replicating. No specific sequences seem to be required for replication (and expression) in Leishmania, therefore precluding the isolation of origins of replication by genetic transformation.

Animals↗