Abstract
The cytolethal distending toxin (CDT) of the oral pathogen Aggregatibacter actinomycetemcomitans induces cell cycle arrest and apoptosis in various cell types. Western analysis, pharmacological inhibition and siRNA silencing were performed in human immortalized gingival keratinocytes (HIGK) to dissect the functional role of the ataxia telangiectasia mutated (ATM) pathway in the signal transduction steps triggered by the CDT. Infection of HIGK was associated with a time-dependent induction of cytoplasmic histone-associated DNA fragmentation. However, in the absence of CDT, infected HIGK underwent reversible DNA strand breaks but not apoptosis, while caspase 3 activity, p21 levels, and HIGK viability were unaffected. Caspase 9 activity was attenuated in the CDT mutant-infected HIGK compared to wild-type infected cells. Pharmacological inhibition and siRNA-silencing of the ATM downstream effector, the protein kinase checkpoint kinase 2 (Chk2), significantly impacted CDT-mediated apoptosis. Together, these findings provide insight on the specificity of the ATM-Chk2 pathway in response to the CDT of A. actinomycetemcomitans in oral epithelial cells, which ultimately leads to apoptosis. We further propose the existence of an unidentified factor that is distinct from the CDT, and involved with a reversible DNA fragmentation that does not trigger terminal apoptosis in oral epithelial cells. This model potentially explains conflicting reports on the biological activity of the A. actinomycetemcomitans CDT.
Highlights
Aggregatibacter actinomycetemcomitans is the etiologic agent for localized aggressive periodontitis [1] and can cause severe infections outside of the oral cavity, including endocarditis and brain abcesses [2] and systemic conditions such as cardiovascular disease and pregnancy complications [3,4]
human immortalized gingival keratinocytes (HIGK) pulsed with two serotypes of A. actinomycetemcomitans showed a similar and timedependent induction pattern of DNA fragmentation (Fig. 1B), which provided direct evidence of apoptosis induced by two different wild-type organisms
We investigated the signal transduction steps involved in the CDTmediated apoptosis induced by A. actinomycetemcomitans
Summary
Aggregatibacter actinomycetemcomitans is the etiologic agent for localized aggressive periodontitis [1] and can cause severe infections outside of the oral cavity, including endocarditis and brain abcesses [2] and systemic conditions such as cardiovascular disease and pregnancy complications [3,4]. The pathogenicity of A. actinomycetemcomitans is influenced by both microbial and host determinants. A. actinomycetemcomitans produces a cytolethal distending toxin (CDT) that is part of a family of cytotoxins found in other pathogenic bacterial species such as Campylobacter jejuni, enteropathogenic Escherichia coli, Haemophilus ducreyi and Shigella species [5,6,7]. Human immune cells and diverse non-lymphoid cell types exhibit variable levels of sensitivity to the CDT of A. actinomycetemcomitans [8,9]. The effects of CDT on lymphocytes are apparently different and the molecular basis for an increased lymphocyte sensitivity to CDT remains moot [10,11]
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