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

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.