Abstract
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.
Highlights
The cytolethal distending toxin (Cdt) is a putative virulence factor that is produced by a wide range of human pathogens capable of colonizing mucocutaneous tissue, resulting in disease characterized by persistent infection and inflammation
Cdt derived from A. actinomycetemcomitans, Haemophilus ducreyi, and Helicobacter hepaticus were shown to induce increases in p21CIP1/wild-type p53-activated fragment 1 (WAF1) within 24–48 h in several cell lines including fibroblasts, lymphocytes, enterocytes, and hepatocytes [16,34,35,36,37,38]
We demonstrate that A. actinomyetemcomitans Cdt induces increases in p21CIP1/WAF1 levels in Jurkat cells in a time- and dose-dependent manner
Summary
The cytolethal distending toxin (Cdt) is a putative virulence factor that is produced by a wide range of human pathogens capable of colonizing mucocutaneous tissue, resulting in disease characterized by persistent infection and inflammation (reviewed in References [1,2]). Pathogens 2020, 9, 38 holotoxin functions as an AB2 toxin; the cell binding unit (B) is responsible for toxin association with the cell surface and is composed of subunits CdtA and CdtC These subunits deliver the active subunit (A), CdtB, to intracellular compartments. Over the past several years, our studies suggested an alternative paradigm to account for Aggregatibacter actinomycetemcomitans Cdt-mediated toxicity which is based upon a novel molecular mode of action for CdtB. In this regard, we demonstrated that, in addition to exhibiting DNase activity, CdtB is a potent lipid phosphatase capable of converting the signaling lipid phosphatidylinositol (PI)-3,4,5-triphosphate (PIP3) to PI-3,4-diphosphate [24,25,26,27,28]
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