Activation of the Extracytoplasmic Function σ Factor σ V in Clostridioides difficile Requires Regulated Intramembrane Proteolysis of the Anti-σ Factor RsiV

Abstract

Clostridioides difficile is a major cause of hospital-acquired diarrhea and represents an urgent concern due to the prevalence of antibiotic resistance and the rate of recurrent infections. We previously showed that σ V and the regulon under its control were involved in lysozyme resistance. Clostridioides ( Clostridium ) difficile is one of the leading causes of nosocomial diarrhea. Lysozyme is a common host defense against many pathogenic bacteria. C. difficile exhibits high levels of lysozyme resistance, which is due in part to the extracytoplasmic functioning (ECF) σ factor, σ V . It has been previously demonstrated that genes regulated by σ V are responsible for peptidoglycan modifications that provide C. difficile with high lysozyme resistance. σ V is not unique to C. difficile however, and its role in lysozyme resistance and its mechanism of activation has been well characterized in Bacillus subtilis where the anti-σ, RsiV, sequesters σ V until lysozyme directly binds to RsiV, activating σ V . However, it remains unclear if the mechanism of σ V activation is similar in C. difficile . Here, we investigated how activation of σ V is controlled in C. difficile by lysozyme. We found that C. difficile RsiV was degraded in the presence of lysozyme. We also found that disruption of a predicted signal peptidase cleavage site blocked RsiV degradation and σ V activation, indicating that the site-1 protease is likely a signal peptidase. We also identified a conserved site-2 protease, RasP, that was required for site-2 cleavage of RsiV and σ V activation in response to lysozyme. Combined with previous work showing RsiV directly binds lysozyme, these data suggested that RsiV directly binds lysozyme in C. difficile , which leads to RsiV destruction via cleavage at site-1 by signal peptidase and then at site-2 by RasP, ultimately resulting in σ V activation and increased resistance to lysozyme. IMPORTANCE Clostridioides difficile is a major cause of hospital-acquired diarrhea and represents an urgent concern due to the prevalence of antibiotic resistance and the rate of recurrent infections. We previously showed that σ V and the regulon under its control were involved in lysozyme resistance. We have also shown in B. subtilis that the anti-σ RsiV acts as a direct sensor for lysozyme. which results in the destruction of RsiV and activation of σ V . Here, we described the proteases required for degradation of RsiV in C. difficile in response to lysozyme. Our data indicated that the mechanism is highly conserved between B. subtilis and C. difficile .