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

Abstract

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.

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

“…As noted earlier, several investigators have reported that Cdt-treated cells exhibit DDR activation exemplified by phosphorylation of H2AX; these findings have also been interpreted as indirect evidence of toxin-induced DNA damage due to CdtB-associated DNase activity [ 68 , 69 , 70 ]. However, we, and others, have demonstrated that the DDR is not activated (i.e., we do not observe increases in pH2AX) in lymphocytes under conditions of Cdt-treatment that involve exposure to optimal levels of toxin required to induce both cell cycle arrest and apoptosis [ 15 , 45 , 47 , 51 ]. Higher doses of toxin nominally increased pH2AX levels; however, these increases appeared to be the result of activation of the apoptotic cascade rather than a direct effect of Cdt on DNA damage [ 51 ].…”

“…Exposure to Cdt not only leads to decreases in pGSK3β and, in turn, kinase activation, but we have also shown that Cdt’s toxicity on other cell types was dependent upon activation of this kinase [ 24 , 42 , 47 ]. As shown in Figure 6 B, TIGK cells exposed to Cdt alone (blue bars), exhibited cell cycle arrest as the percentage of G2/M cells was significantly increased over control cell (no Cdt) values.…”

“…Additionally, we have established that induction of G2/M arrest is dependent upon GSK3β activation while apoptosis is additionally reliant upon the cycle-dependent kinase inhibitor known as CDK-interacting protein 1 (Cip1) and wild-type p53-activated fragment 1 [(WAF1), p21 CIP1/WAF1 ] [ 46 ]. These observations have been extended to Cdts produced by Haemophilus ducreyi ( Hd Cdt) as well as Campylobacter jejuni ( Cj Cdt) and demonstrated that CdtB contained in these toxins are also potent PIP3 phosphatases capable of inducing PI-3K blockade in lymphocytes [ 47 ].…”

Campylobacter jejuni Cytolethal Distending Toxin Induces GSDME-Dependent Pyroptosis in Colonic Epithelial Cells

“…revealed that A. actinomycetemcomitans CDT-treated macrophages exhibited GSDMD-mediated pyroptosis for its phosphatidylinositol-3,4,5-trisphosphate (PIP 3 ) phosphatase activity ( Shenker et al., 2020 ). Recently, PIP 3 phosphatase activity of C. jejuni CDT was discovered ( Huang et al., 2021 ). In the present study, however, no relationship was found between GSDMD and C. jejuni CDT-induced pyroptosis.…”

Campylobacter jejuni virulence factors: update on emerging issues and trends

“…CdtB has been known for a while to function as type I deoxyribonuclease (DNAse I) that cleaves double stranded DNA within host cells [ 126 , 127 ]. However, a recent study has led to a novel paradigm for the CdtB function distinct from its DNAse I activity: CdtB exhibits phosphatidylinositol 3-4-5 trisphosphate (PIP3) phosphatase activity in lymphocytes, which blocks PI-3 K signaling in lymphocytes, and results in toxin-induced cell cycle arrest and apoptosis [ 128 ]. In addition, blockade of the PI-3 K signaling has a pro-inflammatory effect, inducing the production of IL-1β, TNFα and IL-6 by macrophages [ 129 ].…”