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

Bruce J Shenker; Lisa M Walker; Ali Zekavat; Robert H Weiss; Kathleen Boesze-Battaglia

doi:10.3390/pathogens9010038

The Cell-Cycle Regulatory Protein p21^CIP1/WAF1 Is Required for Cytolethal Distending Toxin (Cdt)-Induced Apoptosis

Pathogens. 2020 Jan 2;9(1):38. doi: 10.3390/pathogens9010038.

Authors

Bruce J Shenker¹, Lisa M Walker¹, Ali Zekavat¹, Robert H Weiss², Kathleen Boesze-Battaglia³

Affiliations

¹ Department of Pathology, University of Pennsylvania School of Dental Medicine, Philadelphia, PA 19104, USA.
² Department of Medicine, University of California at Davis School of Medicine, Sacramento, CA 95616, USA.
³ Department of Biochemistry, University of Pennsylvania School of Dental Medicine, Philadelphia, PA 19104, USA.

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 p21^CIP1/WAF1 is critical to Cdt-induced apoptosis. Cdt induces increases in the levels of p21^CIP1/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 p21^CIP1/WAF1 were accompanied by a significant decline in the levels of phosphorylated p21^CIP1/WAF1. The significance of Cdt-induced p21^CIP1/WAF1 increase was assessed by preventing these changes with a two-pronged approach; pre-incubation with the novel p21^CIP1/WAF1 inhibitor, UC2288, and development of a p21^CIP1/WAF1-deficient cell line (Jurkat^p21-) using clustered regularly interspaced short palindromic repeats (CRISPR)/cas9 gene editing. UC2288 blocked toxin-induced increases in p21^CIP1/WAF1, and Jurkat^WT cells treated with this inhibitor exhibited reduced susceptibility to Cdt-induced apoptosis. Likewise, Jurkat^p21- cells failed to undergo toxin-induced apoptosis. The linkage between Cdt, p21^CIP1/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 p21^CIP1/WAF1 as these changes were not observed in Jurkat^p21- cells. Finally, we determined that the p21^CIP1/WAF1 increases were dependent upon toxin-induced increases in the level and activity of the chaperone heat shock protein (HSP) 90. We propose that p21^CIP1/WAF1 plays a key pro-apoptotic role in mediating Cdt-induced toxicity.

Keywords: Keywords Aggregatibacter actinomycetemcomitans; apoptosis; cytolethal distending toxin; lymphocytes; virulence.

Abstract

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