Lymphoid susceptibility to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin is dependent upon baseline levels of the signaling lipid, phosphatidylinositol-3,4,5-triphosphate.

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 and depletes lymphoid cells of PIP3. Hence we propose that Cdt toxicity results from depletion of this signaling lipid and perturbation of phosphatidylinositol-3-kinase (PI-3K)/PIP3/Akt signaling. We have now focused on the relationship between cell susceptibility to CdtB and differences in the status of baseline PIP3 levels. Our studies demonstrate that the baseline level of PIP3, and likely the dependence of cells on steady-state activity of the PI-3K signaling pathway for growth and survival, influence cell susceptibility to the toxic effects of Cdt. Jurkat cells with known defects in both PIP3 degradative enzymes, PTEN and SHIP1, not only contain high baseline levels of PIP3, pAkt, and pGSK3ß, but also exhibit high sensitivity to Cdt. In contrast, HUT78 cells, with no known defects in this pathway, contain low levels of PIP3, pAkt, and pGSK3ß and likely minimal dependence on the PI-3K signaling pathway for growth and survival, and exhibit reduced susceptibility to Cdt. These differences in susceptibility to Cdt cannot be explained by differential toxin binding or internalization of the active subunit. Indeed, we now demonstrate that Jurkat and HUT78 cells bind toxin at comparable levels and internalize relatively equal amounts of CdtB. The relevance of these observations to the mode of action of Cdt and its potential role as a virulence factor is discussed.

Treponema denticola immunoinhibitory protein induces irreversible G1 arrest in activated human lymphocytes.

Oral spirochetes may contribute to the pathogenesis of a number of disorders including periodontal and periradicular diseases; however, the mechanism (s) by which these organisms act to cause disease is unknown. We have previously shown that extracts of the oral spirochete, Treponema denticola, contain an immunosuppressive protein (Sip) which impairs human lymphocyte proliferation. The objective of this study was to determine the mechanism by which Sip alters the proliferative response of lymphocytes. Human T-cells were activated by PHA in the presence or absence of Sip and cell cycle progression was assessed by flow cytometry. Cell cycle distribution was based upon DNA, RNA and protein content as well as expression of the activation markers; CD69 and IL-2R. Seventy-two hours following activation with PHA, cells were found in the G0, G1, S and G2/M phases of the cell cycle. In contrast, pretreatment with Sip resulted in a significant reduction of cells in the S and G2/M phases and a concomitant increase in the G1 phase. Sip did not alter the expression of the early activation markers CD69 and CD25R. To determine if G1 arrest resulted in activation of the checkpoint and cell death, we also monitored Sip-treated cells for apoptosis. Indeed, treatment with Sip resulted in both DNA fragmentation and caspase activation after 96 h. Our results indicate that Sip induces G1 arrest in human T-cells and, furthermore, that the arrest is irreversible, culminating in activation of the apoptotic cascade. We propose that if cell cycle arrest occurs in vivo, it may result in local and/or systemic immunosuppression and thereby enhance the pathogenicity of spirochetes and/or that of other opportunistic organisms.

Induction of apoptosis in human T cells by Actinobacillus actinomycetemcomitans cytolethal distending toxin is a consequence of G2 arrest of the cell cycle.

We have previously shown that Actinobacillus actinomycetemcomitans produces an immunosuppressive factor that is encoded by the cdtB gene, which is homologous to a family of cytolethal distending toxins (Cdt) expressed by several Gram-negative bacteria. Moreover, we have shown that CdtB impairs lymphocyte function by inducing G(2) arrest of the cell cycle. We now report that both CdtB as well as an extract prepared from an Escherichia coli strain that expresses all three of the A. actinomycetemcomitans cdt genes (rCdtABC) induce apoptosis. Pretreatment of lymphocytes with either CdtB or rCdtABC leads to DNA fragmentation in activated lymphocytes at 72 and 96 h. No DNA fragmentation was induced in nonactivated cells. Flow cytometric analysis of the Cdt-treated lymphocytes demonstrates a reduction in cell size and an increase in nuclear condensation. Mitochondrial function was also perturbed in cells pretreated with either CdtB or rCdtABC. An increase in the expression of the mitochondria Ag, Apo 2.7, was observed along with evidence of the development of a mitochondrial permeability transition state; this includes a decrease in the transmembrane potential and elevated generation of reactive oxygen species. Activation of the caspase cascade, which is an important biochemical feature of the apoptotic process, was also observed in Cdt-treated lymphocytes. Overexpression of the bcl-2 gene in the human B lymphoblastoid cell line, JY, led to a decrease in Cdt-induced apoptosis. Interestingly, Bcl-2 overexpression did not block Cdt-induced G(2) arrest. The implications of our results with respect to the immunosuppressive functions of Cdt proteins are discussed.