Aggregatibacter actinomycetemcomitans cytolethal distending toxin modulates host phagocytic function

Abstract

Cytolethal distending toxins (Cdt) are a family of toxins produced by several human pathogens which infect mucocutaneous tissue and induce inflammatory disease. Human macrophages exposed to Aggregatibacter actinomycetemcomitans (Aa) Cdt respond through canonical and non-canonical inflammasome activation to stimulate cytokine release. The inflammatory response is dependent on PI3K signaling blockade via the toxin’s phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase activity; converting PIP3 to phosphatidylinsoitol-3,4-diphosphate (PI3,4P2) thereby depleting PIP3 pools. Phosphoinositides, also play a critical role in phagosome trafficking, serving as binding domains for effector proteins during phagosome maturation and subsequent fusion with lysosomes. We now demonstrate that AaCdt manipulates the phosphoinositide (PI) pools of phagosome membranes and alters Rab5 association. Exposure of macrophages to AaCdt slowed phagosome maturation and decreased phago-lysosome formation, thereby compromising macrophage phagocytic function. Moreover, macrophages exposed to Cdt showed decreased bactericidal capacity leading to increase in Aggregatibacter actinomycetemcomitans survival. Thus, Cdt may contribute to increased susceptibility to bacterial infection. These studies uncover an underexplored aspect of Cdt function and provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt-producing organisms such as Aa.

Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Induces Cellugyrin-(Synaptogyrin 2) Dependent Cellular Senescence in Oral Keratinocytes

“…To date, we have demonstrated that Cdt is a tri-perditious toxin that can profoundly affect acquired and innate immune cells as well as epithelial barrier function [ 27 , 28 , 30 , 39 , 43 , 91 , 92 ]. We propose that Aa Cdt-induced OE senescence is a significant contributing factor to A. actinomycetemcomitans infection and the subsequent development of chronic inflammation.…”

“…The cells were permeabilized and blocked in 5% BSA and 0.2% Triton X-100 in PBS (PBST) at 37 • C for 1 h. Cells were then incubated with a 1:100 dilution of anti-ß-catenin antibody (Cell Signaling) diluted in blocking solution at 4 • C overnight, washed three times with PBST, incubated with donkey anti-rabbit Alexa Fluor 488 (1:1000; Invitrogen) and Hoechst 33258 (1:10,000; Invitrogen) at 37 • C for 1 h, washed and mounted in Prolong Gold Antifade reagent (P36930, Invitrogen). Confocal Z-stack images were acquired on a Nikon A1R laser scanning confocal microscope with a 60X (water) objective at 18 • C, and the data were analyzed using Nikon software (NIS Elements AR 5.30.03) [30].…”

“…Cdts, regardless of bacterial origin, cause similar effects on proliferating cells: cell cycle arrest (typically G2/M) and eventually apoptotic cell death [ 10 , 14 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 ]. Recent observations suggest that Cdt is also capable of inducing functional alterations in the absence of cell death in non-proliferating cells [ 27 , 28 , 29 , 30 ].…”

A Synthetic Small Molecule, LGM2605: A Promising Modulator of Increased Pro-Inflammatory Cytokine and Osteoclast Differentiation by Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin

“…The assembly and expression of the plasmid containing the cdt genes encoding the holotoxin (pUCAacdtABChis) is described in prior studies [25]. The plasmid was engineered to incorporate the cdt genes under the regulation of the lac promoter and subsequently introduced into E. coli DH5α.…”

“…After an overnight freezing step followed by thawing, the cells were subjected to sonication. The purification of the histidine-tagged peptide holotoxin was accomplished using nickel affinity chromatography, as described previously [25].…”