Clostridium difficile TcdB toxin differentially regulates pyrin via differences in activity of RhoA between proliferating and non-proliferating mammalian cells

Abstract

Abstract Background Pyrin is a member of the TRIM family of proteins. Pyrin senses RhoA posttranslational modifications that inactivate RhoA, resulting in the formation of a pyrin inflammasome. Clostridium difficile TcdB toxin inactivates RhoA by glycation of threonine 37. Previous studies have shown that active RhoA predominates in proliferating cells. Thus, TcdB may differentially regulate the pyrin inflammasome in proliferating versus non-proliferating mammalian cells. Methods We used a RhoA activation assay (Rhotekin) to test this hypothesis in proliferating monocytic cells (THP1), PMA-differentiated THP1 cells and non-proliferating human monocytes. Pyrin function was determined by its interaction with 14-3-3ɛ and IL-1β release. Results We noted that TcdB did not inactivate RhoA in proliferating THP1 cells. However, the toxin drastically reduced the level of active RhoA in non-proliferating human monocytes and PMA-differentiated THP1 cells. TcdB synergized with LPS to induce IL1β release from TcdB- and LPS-stimulated THP1 and PMA-differentiated cells. In contrast, TcdB alone was sufficient to generate a robust IL1β release from TcdB-stimulated human monocytes. TcdB disrupted pyrin’s interaction with 14-3-3ɛ in human monocytes and differentiated THP1 cells, suggesting a decrease in the level of phosphorylated pyrin. Conclusion Our data indicate that TcdB is capable of turning off active RhoA in non-proliferating cells but not in proliferating cells. Hence, cellular proliferation may impair pyrin inflammasome assembly and activation.