ALBERT

Description: The Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) induces lymphocytes to undergo cell-cycle arrest and apoptosis; toxicity is dependent upon the active Cdt subunit, CdtB. We now demonstrate that p21CIP1/WAF1 is critical to Cdt-induced apoptosis. Cdt induces increases in the levels of p21CIP1/WAF1 in lymphoid cell lines, Jurkat and MyLa, and in primary human lymphocytes. These increases were dependent upon CdtB’s ability to function as a phosphatidylinositol (PI) 3,4,5-triphosphate (PIP3) phosphatase. It is noteworthy that Cdt-induced increases in the levels of p21CIP1/WAF1 were accompanied by a significant decline in the levels of phosphorylated p21CIP1/WAF1. The significance of Cdt-induced p21CIP1/WAF1 increase was assessed by preventing these changes with a two-pronged approach; pre-incubation with the novel p21CIP1/WAF1 inhibitor, UC2288, and development of a p21CIP1/WAF1-deficient cell line (Jurkatp21−) using clustered regularly interspaced short palindromic repeats (CRISPR)/cas9 gene editing. UC2288 blocked toxin-induced increases in p21CIP1/WAF1, and JurkatWT cells treated with this inhibitor exhibited reduced susceptibility to Cdt-induced apoptosis. Likewise, Jurkatp21− cells failed to undergo toxin-induced apoptosis. The linkage between Cdt, p21CIP1/WAF1, and apoptosis was further established by demonstrating that Cdt-induced increases in levels of the pro-apoptotic proteins Bid, Bax, and Bak were dependent upon p21CIP1/WAF1 as these changes were not observed in Jurkatp21− cells. Finally, we determined that the p21CIP1/WAF1 increases were dependent upon toxin-induced increases in the level and activity of the chaperone heat shock protein (HSP) 90. We propose that p21CIP1/WAF1 plays a key pro-apoptotic role in mediating Cdt-induced toxicity.

Description: Human lymphocytes exposed to Aggregatibacter actinomycetemcomitans (Aa) cytolethal distending toxin (Cdt) undergo cell cycle arrest and apoptosis. In previous studies, we demonstrated that the active Cdt subunit, CdtB, is a potent phosphatidylinositol (PI) 3,4,5-triphosphate phosphatase. Moreover, AaCdt-treated cells exhibit evidence of PI-3-kinase (PI-3K) signaling blockade characterized by reduced levels of PIP3, pAkt, and pGSK3β. We have also demonstrated that PI-3K blockade is a requisite of AaCdt-induced toxicity in lymphocytes. In this study, we extended our observations to include assessment of Cdts from Haemophilus ducreyi (HdCdt) and Campylobacter jejuni (CjCdt). We now report that the CdtB subunit from HdCdt and CjCdt, similar to that of AaCdt, exhibit potent PIP3 phosphatase activity and that Jurkat cells treated with these Cdts exhibit PI-3K signaling blockade: reduced levels of pAkt and pGSK3β. Since non-phosphorylated GSK3β is the active form of this kinase, we compared Cdts for dependence on GSK3β activity. Two GSK3β inhibitors were employed, LY2090314 and CHIR99021; both inhibitors blocked the ability of Cdts to induce cell cycle arrest. We have previously demonstrated that AaCdt induces increases in the CDK inhibitor, p21CIP1/WAF1, and, further, that this was a requisite for toxin-induced cell death via apoptosis. We now demonstrate that HdCdt and CjCdt also share this requirement. It is also noteworthy that p21CIP1/WAF1 was not involved in the ability of the three Cdts to induce cell cycle arrest. Finally, we demonstrate that, like AaCdt, HdCdt is dependent upon the host cell protein, cellugyrin, for its toxicity (and presumably internalization of CdtB); CjCdt was not dependent upon this protein. The implications of these findings as they relate to Cdt’s molecular mode of action are discussed.