Cytolethal Distending Toxin Intoxication of Macrophages is Dependent upon Cellugyrin

Objectives: Cytolethal distending toxins (Cdt) are a group of protein toxins produced by several human pathogens, such as Aggregatibacter actinomycetemcomitans (Aa), Haemophilus ducreyi, and Escherichia coli. Internalization of the active Cdt subunit, CdtB is required to induce toxicity: cell cycle arrest and apoptosis in lymphocytes and pro-inflammatory cytokine responses in macrophages. We have demonstrated that cellugyrin, a protein that mediates retrograde transport, is critical for CdtB internalization in lymphocytes. The aim of this study is to determine whether cellugyrin is necessary for internalization and toxicity of CdB from other species.
Methods: Lentiviral vector was employed to silence cellugyrin expression from human monocytic cell line, THP-1 (THP ^Cg-). THP^WT and THP^cg- cells were differentiated into macrophages, treated with E. coli Cdt, and supernatants analyzed for TNFα, and IL-1β.
Results: THP^WT and THP^Cg- cells exposed to medium alone expressed 640±505 and 499±352pg/mL TNFα, respectively. THP^WT cells treated with 5 ng/mL of E. coli CdtA and CdtC and 5, 10, 50ng/mL CdtB, cells released 4415±1570, 5587±1879, and 8642±3020pg/mL TNFα, respectively. THP^Cg- cells treated with Cdt released 1077±766, 975±650, 1104±873ng/mL TNFα in the presence of 5, 10 and 50ng/mL CdtB, respectively. THP^WT and THP^Cg- cells exposed to medium alone expressed 54±12 and 156±66pg/mL of IL-1β, respectively. THP^WT cells treated with Cdt released 988±269, 1065±296, and 1773±556pg/mL IL-1β in the presence of 5, 10, 50ng/mL CdtB. THP^Cg- cells treated with Cdt released 186±62, 162±56, 173±51 ng/mL IL-1β in the presence of 5, 10 and 50ng/mL CdtB.
Conclusions: Results demonstrate that E. coli Cdt toxicity is dependent on the ability of macrophage to express cellugyrin, a putative component of intracellular transport vesicles. Our results are consistent with the hypothesis that cellugyrin is necessary for CdtB internalization.