Cloning, sequence analysis and heterologous expression of the DNA adenine-(N6) methyltransferase from the human pathogen Actinobacillus actinomycetemcomitans

Eberhard, Jörg

doi:10.1016/s0378-1097(01)00014-3

Cited by 6 publications

(11 citation statements)

References 18 publications

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“…Dam was required for virulence in Pasteurella multocida, known to cause bovine respiratory disease (14). Recently, the gene encoding Dam was cloned and sequenced from Actinobacillus actinomycetemcomitans, which is implicated in causing human periodontal diseases (21). Therefore, Dam presents the exciting possibility that it may play a role in the virulence of a broad range of pathogens and, thus, further investigation is merited.…”

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confidence: 99%

DNA Adenine Methyltransferase Influences the Virulence of Aeromonas hydrophila

et al. 2006

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Among the various virulence factors produced by Aeromonas hydrophila, a type II secretion system (T2SS)-secreted cytotoxic enterotoxin (Act) and the T3SS are crucial in the pathogenesis of Aeromonas-associated infections. Our laboratory molecularly characterized both Act and the T3SS from a diarrheal isolate, SSU of A. hydrophila, and defined the role of some regulatory genes in modulating the biological effects of Act. In this study, we cloned, sequenced, and expressed the DNA adenine methyltransferase gene of A. hydrophila SSU (dam AhSSU ) in a T7 promoter-based vector system using Escherichia coli ER2566 as a host strain, which could alter the virulence potential of A. hydrophila. Recombinant Dam, designated as M.AhySSUDam, was produced as a histidine-tagged fusion protein and purified from an E. coli cell lysate using nickel affinity chromatography. The purified Dam had methyltransferase activity, based on its ability to transfer a methyl group from S-adenosyl-L-methionine to N 6 -methyladenine-free lambda DNA and to protect methylated lambda DNA from digestion with DpnII but not against the DpnI restriction enzyme. The dam gene was essential for the viability of the bacterium, and overproduction of Dam in A. hydrophila SSU, using an arabinose-inducible, P BAD promoter-based system, reduced the virulence of this pathogen. Specifically, overproduction of M.AhySSUDam decreased the motility of the bacterium by 58%. Likewise, the T3SS-associated cytotoxicity, as measured by the release of lactate dehydrogenase enzyme in murine macrophages infected with the Dam-overproducing strain, was diminished by 55% compared to that of a control A. hydrophila SSU strain harboring the pBAD vector alone. On the contrary, cytotoxic and hemolytic activities associated with Act as well as the protease activity in the culture supernatant of a Dam-overproducing strain were increased by 10-, 3-, and 2.4-fold, respectively, compared to those of the control A. hydrophila SSU strain. The Dam-overproducing strain was not lethal to mice (100% survival) when given by the intraperitoneal route at a dose twice that of the 50% lethal dose, which within 2 to 3 days killed 100% of the animals inoculated with the A. hydrophila control strain. Taken together, our data indicated alteration of A. hydrophila virulence by overproduction of Dam.Aeromonas hydrophila is both a human and an animal pathogen, and the diseases associated with this bacterium in humans include gastroenteritis, wound infections, and septicemia (36, 88). In immuno-compromised individuals, some of the more serious complications associated with A. hydrophila infections are cellulitis, myonecrosis, and ecthyma gangrenosum, which often are fatal (2, 36). A variety of virulence factors/mechanisms, such as the surface layer, capsule, different forms of pili (e.g., type IV [Tap] and bundle-forming pili), toxins, quorum sensing, biofilm formation and, more recently, the type III secretion system (T3SS), were identified in various Aeromonas species that are involved in the pathogenesis...

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DNA Adenine Methyltransferase Influences the Virulence of Aeromonas hydrophila

et al. 2006

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“…We identified and characterized the A. actinomycetemcomitans DAM (M.AacDAM) of strain Y4. 17 A DAM-deficient mutant of A. actinomycetemcomitans showed increased levels of extracellular leukotoxin and the reduced capacity to invade epithelial cells. 19 On various epithelial surfaces as well as in the oral cavity structured bacterial biofilms can be pathogenic and induce chemical defense mechanisms or inflammatory responses in epithelial cells.…”

Section: Dammentioning

confidence: 99%

“…have been identified in the oral bacteria Porphyromonas gingivalis and A. actinomycetemcomitans 17,18 and both microorganisms are associated with periodontal diseases. The pattern of DNA methylation is crucial to the regulation of cellular processes, including the expression of various virulence genes.…”

Section: Dammentioning

confidence: 99%

Differential epithelial cell response upon stimulation with theAggregatibacter actinomycetemcomitansstrains VT 1169, VT 1560 DAM^-and ATCC 4318

Eberhard

Banasch²,

Jepsen³

et al. 2010

Epigenetics

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“…DAM (DNA adenine methyltransferase) is one such orphan enzyme, and is prevalent among enteric bacteria, such as E. coli , Salmonella species, Yersinia species and Vibrio cholerae (2). DAM enzymes have also been identified in the oral bacteria Porphyromonas gingivalis (2) and A. actinomycetemcomitans (8). Patterns of DNA methylation are crucial to the regulation of important cellular processes (5, 20, 22, 28), including the expression of various virulence genes.…”

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Inactivation of DNA adenine methyltransferase alters virulence factors in Actinobacillus actinomycetemcomitans

Lippmann

Oza

et al. 2006

Oral Microbiology and Immunology

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DNA adenine methyltransferase (DAM) plays critical roles in diverse biological pathways in gram-negative bacteria, and specifically in regulating the expression of virulence genes in several organisms. Actinobacillus actinomycetemcomitans plays an important role in the pathogenesis of juvenile and adult periodontal disease, yet little is known about its mechanisms of gene regulation. DAM is shown here to directly or indirectly affect well-known A. actinomycetemcomitans virulence factors. A mutant A. actinomycetemcomitans strain lacking the dam gene was created by homologous recombination and shows normal growth phenotypes when grown exponentially. This mutant strain has four sixfold increased levels of extracellular leukotoxin, altered cellular levels of leukotoxin, and significant changes in bacterial invasion of KB oral epithelial cells. These results provide a basis for further characterization of regulatory mechanisms that control A. actinomycetemcomitans virulence.

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Cloning, sequence analysis and heterologous expression of the DNA adenine-(N6) methyltransferase from the human pathogen Actinobacillus actinomycetemcomitans

Cited by 6 publications

References 18 publications

DNA Adenine Methyltransferase Influences the Virulence of Aeromonas hydrophila

DNA Adenine Methyltransferase Influences the Virulence of Aeromonas hydrophila

Differential epithelial cell response upon stimulation with theAggregatibacter actinomycetemcomitansstrains VT 1169, VT 1560 DAM^-and ATCC 4318

Inactivation of DNA adenine methyltransferase alters virulence factors in Actinobacillus actinomycetemcomitans

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Cloning, sequence analysis and heterologous expression of the DNA adenine-(N6) methyltransferase from the human pathogen Actinobacillus actinomycetemcomitans

Cited by 6 publications

References 18 publications

DNA Adenine Methyltransferase Influences the Virulence of Aeromonas hydrophila

DNA Adenine Methyltransferase Influences the Virulence of Aeromonas hydrophila

Differential epithelial cell response upon stimulation with theAggregatibacter actinomycetemcomitansstrains VT 1169, VT 1560 DAM-and ATCC 4318

Inactivation of DNA adenine methyltransferase alters virulence factors in Actinobacillus actinomycetemcomitans

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Differential epithelial cell response upon stimulation with theAggregatibacter actinomycetemcomitansstrains VT 1169, VT 1560 DAM^-and ATCC 4318