Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin-Induces Cell Cycle Arrest in a Glycogen Synthase Kinase (GSK)-3-Dependent Manner in Oral Keratinocytes

Abstract

Cytolethal distending toxins (Cdt) are produced by a diverse group of pathogens. One Cdt-producing organism, Aggregatibacter actinomycetemcomitans, plays a critical role in the pathogenesis of a unique form of periodontitis, formerly referred to as localized aggressive periodontitis. The active Cdt subunit, CdtB, is a potent phosphatidylinositol (PI) 3,4,5-triphosphate phosphatase capable of inducing PI-3-kinase signaling blockade, a requisite for Cdt-induced toxicity in lymphocytes. In this study, we extended our observations to include the oral keratinocyte response to AaCdt using cell lines and primary gingival keratinocytes. All three exhibited G2/M arrest when exposed to AaCdt toxin within 24 h. Toxin-treated cells exhibited reduced levels of pAkt and pGSK3β within 6 h. Pre-treatment with GSK3β kinase inhibitors, LY2090314, CHIR99021 and Tideglusib, abrogated Cdt-induced G2/M arrest. None of the oral epithelial cells exhibited evidence of apoptosis. Cells remained arrested in the G2/M phase for at least 72 h without evidence of DNA damage response activation (H2AX phosphorylation). Cdt-treated cells displayed increased phosphorylation of the cyclin dependent kinase 1 (CDK1); moreover, the GSK3 inhibitors blocked this increase and reduced total CDK1 levels. This study further clarifies the potential mechanism(s) contributing to Cdt toxicity and toxin-mediated pathogenesis.

Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Induces Cellugyrin-(Synaptogyrin 2) Dependent Cellular Senescence in Oral Keratinocytes

“…Primary human gingival keratinocytes (PGK) were derived from discarded healthy gingival tissue obtained from patients undergoing crown lengthening procedures as previously described [ 39 , 41 ]. Briefly, tissue was washed in a HAMS F12 nutrient mixture (ThermoFisher Scientific, Waltham, MA, USA) containing 1% penicillin/streptomycin and 1% ampthotericin and then cut into 0.3 cm 2 fragments.…”

“…Recently, we demonstrated that oral-epithelial cells (OE) are unique in their response to Aa Cdt [ 39 ]. Assessment of the effect of Aa Cdt on two immortalized OE lines as well as primary gingival keratinocytes (PGKs) showed that all three cell types were sensitive to Aa Cdt-induced cell cycle arrest as toxin-treated cells accumulated in the G2/M phase within 24 h of exposure to low Cdt concentrations (pg/mL) [ 39 ].…”

“…Recently, we demonstrated that oral-epithelial cells (OE) are unique in their response to Aa Cdt [ 39 ]. Assessment of the effect of Aa Cdt on two immortalized OE lines as well as primary gingival keratinocytes (PGKs) showed that all three cell types were sensitive to Aa Cdt-induced cell cycle arrest as toxin-treated cells accumulated in the G2/M phase within 24 h of exposure to low Cdt concentrations (pg/mL) [ 39 ]. Similar to other cell types, toxin-treated cells also exhibited PI-3K signaling blockade, leading to glycogen synthase kinase 3β (GSK3β) activation, a requirement for Cdt-induced cell cycle arrest.…”

“…Similar to other cell types, toxin-treated cells also exhibited PI-3K signaling blockade, leading to glycogen synthase kinase 3β (GSK3β) activation, a requirement for Cdt-induced cell cycle arrest. However, unlike other cells, which typically undergo apoptosis following Cdt-induced cell cycle arrest, epithelial cell G2/M arrest did not lead to cell death [ 39 ]. We have now extended these observations and demonstrated that Cdt-induced cell cycle arrest in OE is durable.…”

The footprint of gut microbiota in gallbladder cancer: a mechanistic review

“…These substances are also known as genotoxins, including cytolethal distending toxin (CDT), typhoid toxin (TT) and colibactin ( Wang and Li, 2022 ). CDT was the first described bacterial genotoxin and encodes three polypeptides (CdtA, CdtB, and CdtC) ( Shenker et al., 2022 ). CDT induces double-strand breaks (DSBs), activates the ataxia telangiectasia mutated (ATM)-dependent DNA damage response and forms DNA repair complexes, triggering irreversible G2/M arrest of the cell cycle and apoptosis ( Lara-Tejero and Galán, 2000 ; Zhang et al., 2018 ; Tatekawa et al., 2022 ).…”