Internalization and Intoxication of Human Macrophages by the Active Subunit of the Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Is Dependent Upon Cellugyrin (Synaptogyrin-2)

Abstract

The Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is a heterotrimeric AB 2 toxin capable of inducing cell cycle arrest and apoptosis in lymphocytes and other cell types. Recently, we have demonstrated that human macrophages are resistant to Cdt-induced apoptosis but are susceptible to toxininduced pro-inflammatory cytokine response involving activation of the NLRP3 inflammasome. Exposure to Cdt results in binding to the cell surface followed by internalization and translocation of the active subunit, CdtB, to intracellular compartments. Internalization involves hijacking of retrograde pathways; treatment of cells with Retro-2 leads to a decrease in CdtB-Golgi association. These events are dependent upon toxin binding to cholesterol in the context of lipid rich membrane microdomains often referred to as lipid rafts. We now demonstrate that within 1 h of exposure of macrophages to Cdt, CdtB is internalized and found primarily within lipid rafts; concurrently, cellugyrin (synaptogyrin-2) also translocates into lipid rafts. Further analysis by immunoprecipitation indicates that CdtB associates with complexes containing both cellugyrin and Derlin-2. Moreover, a human macrophage cell line deficient in cellugyrin expression (THP-1 Cg−) challenged with Cdt failed to internalize CdtB and was resistant to the Cdt-induced pro-inflammatory response. We propose that lipid rafts along with cellugyrin play a critical role in the internalization and translocation of CdtB to critical intracellular target sites in human macrophages. These studies provide the first evidence that cellugyrin is expressed in human macrophages and plays a critical role in Cdt toxicity of these cells.

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

“…Indeed, we have demonstrated that soon after exposure to Cdt, intracellular molecular complexes form that contain both CdtB and cellugyrin. Moreover, cells deficient in the expression of cellugyrin (Jurkat Cg- ) were resistant to CdtB internalization and intoxication ( Boesze-Battaglia et al, 2017 ; Boesze-Battaglia et al, 2020 ). Therefore, we explored whether this dependence on cellugyrin also extended to HdCdt and CjCdt.…”

“…We have demonstrated previously that shortly after holotoxin binding to human lymphocytes and macrophages, CdtB is internalized via its association with cholesterol rich membrane microdomains ( Boesze-Battaglia et al, 2006 ; Boesze-Battaglia et al, 2009 ; Boesze-Battaglia et al, 2015 ; Boesze-Battaglia et al, 2016 ). Internalization leads to an association of CdtB with cellugyrin, a component of synaptic-like microvesicles (SLMVs) which may participate in intracellular transport ( Boesze-Battaglia et al, 2017 ; Boesze-Battaglia et al, 2020 ). Moreover, reduced expression of cellugyrin protects cells from CdtB internalization and subsequent toxicity.…”

C910 chemical compound inhibits the traffiking of several bacterial AB toxins with cross-protection against influenza virus

“…The following reference appears in the Supplemental Information: Azarnia Tehran et al., 2015 , Boesze-Battaglia et al., 2020 , Buchrieser et al., 2020 , Canton and Kima, 2012 , Harrison et al., 2016 , Kali et al., 2021 , Schnell et al., 2016 , Secher et al., 2015 , Wu et al., 2019 , Wu et al., 2020 , Zilbermintz et al., 2015 .…”

From DNA Damage to Cancer Progression: Potential Effects of Cytolethal Distending Toxin

“…CDT is a prominent virulence factor of CDT-producing bacteria and aids in effective tissue colonization, thereby promoting potent infection by breaking down host defense ( 50 ). The dampened host defense mainly results from: (i) disrupted epithelial barrier, which is caused by CDT-induced cell cycle arrest and subsequent cell death in epithelial cells ( 51 ); and (ii) impaired host immunity, which is caused by the extreme sensitivity of lymphocytes to CDT cytotoxicity and altered macrophage functions ( 52 , 53 ). To perform such sophisticated tasks, the CDT must first enter the cells to exert its activity.…”