Evaluation of functionality of type II toxin-antitoxin systems of Clostridioides difficile R20291

Clostridioides difficile is a nosocomial, Gram-positive, strictly anaerobic, spore-forming pathogen capable of colonizing and proliferating in the human intestine. In bacteria, it has been shown that the Toxin-Antitoxin systems mediate the cellular response to external stress by initiating processes such as biofilm formation and programmed cell death. This work aims to evaluate the functionality of four type II TA modules of Clostridioides difficile R20291. We performed bioinformatic analysis to search for putative TA systems using the TADB platform. Then we performed a heterologous expression assay to evaluate the functionality of these systems. Our results showed that the MazEF and RelBE systems were functional, suggesting that their corresponding toxins possess an endoribonuclease activity. In conclusion, MazEF and RelBE systems of C. difficile R20291 are functional in a heterologous expression system.