The Cell-Cycle Regulatory Protein p21CIP1/WAF1 Is Required for Cytolethal Distending Toxin (Cdt)-Induced Apoptosis

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.

The Active Subunit of the Cytolethal Distending Toxin, CdtB, Derived From Both Haemophilus ducreyi and Campylobacter jejuni Exhibits Potent Phosphatidylinositol-3,4,5-Triphosphate Phosphatase Activity

“…Intracellular levels of p21 CIP1/WAF1 are controlled transcriptionally, as well as post-translationally (Warfel and El-Deiry, 2013;Dutto et al, 2015). Previously, we demonstrated that p21 CIP1/WAF1 mRNA levels were not significantly altered in AaCdt-treated lymphocytes (Shenker et al, 2020); instead, we observed that the increases were likely dependent upon posttranslational modification as we have demonstrated a requisite role for the chaperone protein, HSP90 (Shenker et al, 2020). HSP90 has been shown to stabilize p21 CIP1/WAF1 and prevent proteasomal degradation (Jascur et al, 2005;Diehl et al, 2009).…”

“…Increased pH2AX has been interpreted by several investigators as not only an indicator of DDR activation, but also as a surrogate for Cdt-mediated DNA damage (Bezine et al, 2014;Frisan, 2016). It should be noted, however, that we previously demonstrated that AaCdt-mediated p21 CIP1/WAF1 upregulation in human lymphocytes is not linked to activation of the DDR; the doses of AaCdt required to induce cell cycle arrest (and apoptosis) do not induce increases of pH2AX (Shenker et al, 2006;Shenker et al, 2020). Therefore, we also evaluated the effect HdCdt and CjCdt at the doses employed in this study for their ability to induce increases in pH2AX in lymphocytes.…”

“…Indeed, several investigators have reported that Cdt-treated cells exhibit DDR activation (i.e, phosphorylation of H2AX), and further, they have interpreted these findings as evidence of toxin-induced DNA damage (Bezine et al, 2014;Frisan, 2016). However, we have repeatedly demonstrated that the DDR is not activated (ie., we do not observe increases in pH2AX) in lymphocytes under conditions of AaCdt-treatment (Shenker et al, 2006;Shenker et al, 2020). In these studies, we employed the minimum doses of toxin required to achieve maximum cell cycle arrest and apoptosis.…”

Internalization and Intoxication of Human Macrophages by the Active Subunit of the Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Is Dependent Upon Cellugyrin (Synaptogyrin-2)

“…We have proposed an alternative paradigm which is based upon our observation that CdtB, derived from A. actinomycetemcomitans aggregatibacter Cdt, is a potent PIP3 phosphatase; Cdt treated cells exhibit depletion of PIP3 and downstream PI-3K blockade: decreased phosphorylation of Akt (inactivation) and GSK3β (activation). Furthermore, we have demonstrated that CdtB toxicity, which we have observed in both proliferating cells and non-proliferating cells, is dependent upon its PIP3 phosphatase activity and not DNase activity (24,(62)(63)(64)(65). It is likely that the operative molecular mode of action relates to the amount of toxin employed, the bacterial source of Cdt and the host target cell.…”

Editorial Comments to the Special Issue: “Aggregatibacter actinomycetemcomitans—Gram-Negative Bacterial Pathogen”

“…As described above, the CDT is the other exotoxin produced by A. actinomycetemcomitans [ 2 ]. In their paper in the present Special Issue, Shenker and co-workers [ 12 ] report on the mechanism for CDT-induced apoptosis in T-lymphocytes. Their data demonstrate that toxin-induced apoptosis is dependent upon increased levels of specific intracellular signaling, and conclude that that the ability of CDT to impair lymphocyte proliferation and promote cell death therefore compromises the host response to CDT-producing organisms.…”