34.  Activation of the Inflammasome by bacterial protein toxins targeting the cytoskeleton

The inflammasome is a complex present in the cytosol of stimulated immune cells that leads to the activation of pro-inflammatory caspase-1. Active caspase-1 is responsible for the release of the pro-inflammatory interleukin-1β (IL-1β) and IL-18, and cleavage of Gasdermin-D, which ultimately results in a form of lytic cell death known as pyroptosis. Clostridium difficile is an anaerobe, spore-forming bacterium that can cause antibiotic-related diarrhea and pseudomembranous colitis. Major virulence factors of this pathogen are the Rho-glycosylating toxins A and B (TcdA, TcdB), which inactivate Rho-GTPases. Hyper virulent strains of Clostridium difficile also produce the binary toxin C. difficile transferase (CDT). CDT ADP-ribosylates monomeric G-actin resulting in complete depolymerization of the actin cytoskeleton. It has been previously studied the influence of TcdA and TcdB over inflammasome activation. Recently it was identified that Pyrin, a protein capable of assembling the inflammasome, acts as a sensor responsible for detecting the inactivation of Rho GTPases. Since no direct binding of the modified GTPases to Pyrin was observed, the authors conclude that Pyrin likely senses the effects of GTPase inactivation on the actin cytoskeleton. Interactions of pyrin and other receptors activating the inflammasome with components of the cytoskeleton indicate that alteration of cytoskeletal dynamics plays an important role in inflammasome activation. Considering this, we decided to evaluate if CDT could activate the inflammasome, and to further characterize inflammasome activation by TcdB. Our results show that both TcdB and CDT are capable of inducing ASC speck formation and release of IL- 1β from competent immune cells. TcdB also induced activation of caspase-1 and pyroptosis, and we found evidence that TcdB influences several receptors involved in inflammasome assembly. This data confirm the important role of clostridial toxins that taget the cytoskeleton in exerting inflammatory responses in the host.