Evidence of a Bacterial Receptor for Lysozyme: Binding of Lysozyme to the Anti-σ Factor RsiV Controls Activation of the ECF σ Factor σV

Abstract

σ factors endow RNA polymerase with promoter specificity in bacteria. Extra-Cytoplasmic Function (ECF) σ factors represent the largest and most diverse family of σ factors. Most ECF σ factors must be activated in response to an external signal. One mechanism of activation is the stepwise proteolytic destruction of an anti-σ factor via Regulated Intramembrane Proteolysis (RIP). In most cases, the site-1 protease required to initiate the RIP process directly senses the signal. Here we report a new mechanism in which the anti-σ factor rather than the site-1 protease is the sensor. We provide evidence suggesting that the anti-σ factor RsiV is the bacterial receptor for the innate immune defense enzyme, lysozyme. The site-1 cleavage site is similar to the recognition site of signal peptidase and cleavage at this site is required for σV activation in Bacillus subtilis. We reconstitute site-1 cleavage in vitro and demonstrate that it requires both signal peptidase and lysozyme. We demonstrate that the anti-σ factor RsiV directly binds to lysozyme and muramidase activity is not required for σV activation. We propose a model in which the binding of lysozyme to RsiV activates RsiV for signal peptidase cleavage at site-1, initiating proteolytic destruction of RsiV and activation of σV. This suggests a novel mechanism in which conformational change in a substrate controls the cleavage susceptibility for signal peptidase. Thus, unlike other ECF σ factors which require regulated intramembrane proteolysis for activation, the sensor for σV activation is not the site-1 protease but the anti-σ factor.

The Clostridium difficile Dlt Pathway Is Controlled by the Extracytoplasmic Function Sigma Factor σ V in Response to Lysozyme

“…Moreover, V is necessary for lysozyme resistance in C. difficile (21). In fact, the C. difficile V anti-sigma factor RsiV binds lysozyme and may serve as a direct lysozyme receptor, as it does in B. subtilis (22). An ortholog of X has not been identified in any sequenced C. difficile isolate, but C. difficile strains encode an additional ECF sigma factor, T (csfT or sigT).…”

The atypical two-subunit σ factor from Bacillus subtilis is regulated by an integral membrane protein and acid stress

“…Under environmental conditions that elevate ECF s factor activity, many integral membrane anti-s factors undergo RIP, as is the case for anti-s factors regulating s E from E. coli and s W and s V from B. subtilis (Chaba et al, 2007;Grigorova et al, 2004;Hastie et al, 2014;Ho & Ellermeier, 2012). RIP is initiated by a highly specific site-1-protease (S1P).…”

“…It has been suggested that PrsW, the site-1 protease, is the sensor of the environmental stressor (Ellermeier & Losick, 2006), but further research into the mechanism suggests the involvement of a negative regulator of proteolysis in sensing the stress signal (Heinrich et al, 2009). More recently, the regulation of s V by its anti-s factor RsiV has been investigated (Hastie et al, 2013(Hastie et al, , 2014). Like the s W stress response, the s V stress response chemically remodels cell envelope structure providing resistance to its inducer, lysozyme (Guariglia-Oropeza & Helmann, 2011;Ho et al, 2011).…”

“…Like the s W stress response, the s V stress response chemically remodels cell envelope structure providing resistance to its inducer, lysozyme (Guariglia-Oropeza & Helmann, 2011;Ho et al, 2011). RsiV is a receptor for lysozyme, and when bound to lysozyme, the site-1 cleavage site on RsiV is exposed to the activity of the peptidase SipS (Hastie et al, 2014).…”

Activation without Proteolysis of Anti-<i>σ</i> Factor RsiV of the Extracytoplasmic Function <i>σ</i> Factor <i>σ</i><sup>V</sup> in a Glucolipid-Deficient Mutant of <i>Bacillus subtilis</i>

“…Lysozyme directly binds to RsiV and induces the cleavage of its C-terminal extracytoplasmic portion by signal peptidase (site-1 cleavage). Subsequently the N-terminal portion is processed by the intramembrane protease RasP (site-2 cleavage), and σ V is released [6] [7].…”