The toxicity of theAggregatibacter actinomycetemcomitanscytolethal distending toxin correlates with its phosphatidylinositol-3,4,5-triphosphate phosphatase activity

Abstract

The Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) induces G2 arrest and apoptosis in lymphocytes and other cell types. We have shown that the active subunit, CdtB, exhibits phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase activity, leading us to propose that Cdt toxicity is the result of PIP3 depletion and perturbation of phosphatidylinositol-3-kinase (PI-3K)/PIP3/Akt signalling. To further explore this relationship, we have focused our analysis on identifying residues that comprise the catalytic pocket and are critical to substrate binding rather than catalysis. In this context, we have generated several CdtB mutants and demonstrate that, in each instance, the ability of the toxin to induce cell cycle arrest correlates with retention of phosphatase activity. We have also assessed the effect of Cdt on downstream components of the PI-3K signalling pathway. In addition to depletion of intracellular concentrations of PIP3, toxin-treated lymphocytes exhibit decreases in pAkt and pGSK3β. Further analysis indicates that toxin-treated cells exhibit a concomitant loss in Akt activity and increase in GSK3β kinase activity consistent with observed changes in their phosphorylation status. We demonstrate that cell susceptibility to Cdt is dependent upon dephosphorylation and concomitant activation of GSK3β. Finally, we demonstrate that, in addition to lymphocytes, HeLa cells exposed to a CdtB mutant that retains phosphatase activity and not DNase activity undergo G2 arrest in the absence of H2AX phosphorylation. Our results provide further insight into the mode of action by which Cdt may function as an immunotoxin and induce cell cycle arrest in target cells such as lymphocytes.

The Cytolethal Distending Toxin Contributes to Microbial Virulence and Disease Pathogenesis by Acting As a Tri-Perditious Toxin

“…However, as discussed earlier, Cdt binding to cells and subsequent CdtB internalization is dependent upon association and binding to cholesterol, a ubiquitous membrane component. Consistent with this finding, we have also demonstrated that Cdt binding and CdtB internalization occurs in cells that are both Cdt-susceptible as well as Cdt-resistant (Shenker et al, 2016a). Thus, restricted susceptibility to Cdt intoxication must be governed by a mechanism independent of binding and subunit internalization.…”

“…Specifically, inhibition of the apoptotic cascade by over expression of Bcl-2 or with the use of a caspase-3 inhibitor results in suppression of the DDR with no effect on Cdt-induced cell cycle arrest. Moreover, under conditions optimized to expose cells to the minimal amount of Cdt necessary to induce maximal G2 arrest within 24 h (and apoptosis at 48 h), we were unable to demonstrate DDR activation in lymphoid cells at 4 h. DDR activation could only be induced following longer exposures to higher Cdt concentrations for both lymphocytes and HeLa cells (Shenker et al, 2016a). These results collectively argue for DDR activation, at least in lymphocytes, to be the result of late events, and likely associated with enzymatic degradation of DNA due to activation of the apoptotic cascade.…”

“…The six conserved residues in the catalytic site of CdtB (Yamada et al, 2006) and IP5P (Tsujishita et al, 2001) superimpose with an RMSD of 0.26 Å (Figure 2), which is slightly better than the alignment of those same residues between CdtB and DNase I. This is not surprising since CdtB, IP5P, and DNase are all phosphoesterases (Dlakić, 2000; Dlakic, 2001); the specific function of each of these enzymes is largely dependent upon accommodation of substrates within their respective active site (Shenker et al, 2016a). Over the past several years, we have generated evidence for a new paradigm to account for the molecular mode of action by which A. actinomycetemcomitans CdtB intoxicates cells.…”

“…However, several investigators have shown that CdtB isolated from multiple bacteria is able to denature or relax supercoiled plasmid DNA in vitro (Elwell and Dreyfus, 2000a; Elwell et al, 2001; Mao and DiRienzo, 2002; Frisan et al, 2003; Nesic et al, 2004). Furthermore, mutations in putative catalytic amino acids have given rise to proteins with both reduced enzymatic activity and toxicity (Elwell and Dreyfus, 2000b; Lara-Tejero and Galán, 2000; Nesic et al, 2004; Hu and Stebbins, 2006; Shenker et al, 2007, 2016a). …”

Functional characterization of Aggregatibacter actinomycetemcomitans QSEBC : a bacterial adrengeric receptor and global regulator of virulence.

“…In addition, A. actinomycetemcomitans produces other virulence factors such as a cytolethal distending toxin that causes apoptosis of lymphocytes [38], lipopolysaccharide (LPS)…”