Clostridium difficile (C. difficile) is an anaerobic gram-positive pathogen that is the leading cause of nosocomial bacterial infection globally. C. difficile infection (CDI) typically occurs after ingestion of infectious spores by a patient that has been treated with broad-spectrum antibiotics. While CDI is a toxin-mediated disease, transmission and pathogenesis are dependent on the ability to produce viable spores. These spores must become metabolically active (germinate) in order to cause disease. C. difficile spore germination occurs when spores encounter bile salts and other co-germinants within the small intestine, however, the germination signaling cascade is unclear. Here we describe a signaling role for Ca2+ during C. difficile spore germination and provide direct evidence that intestinal Ca2+ coordinates with bile salts to stimulate germination. Endogenous Ca2+ (released from within the spore) and a putative AAA+ ATPase, encoded by Cd630_32980, are both essential for taurocholate-glycine induced germination in the absence of exogenous Ca2+. However, environmental Ca2+ replaces glycine as a co-germinant and circumvents the need for endogenous Ca2+ fluxes. Cd630_32980 is dispensable for colonization in a murine model of C. difficile infection and ex vivo germination in mouse ileal contents. Calcium-depletion of the ileal contents prevented mutant spore germination and reduced WT spore germination by 90%, indicating that Ca2+ present within the gastrointestinal tract plays a critical role in C. difficile germination, colonization, and pathogenesis. These data provide a biological mechanism that may explain why individuals with inefficient intestinal calcium absorption (e.g., vitamin D deficiency, proton pump inhibitor use) are more prone to CDI and suggest that modulating free intestinal calcium is a potential strategy to curb the incidence of CDI.

Bacteria encounter numerous environmental stresses which can delay or inhibit their growth. Many bacteria utilize alternative factors to regulate subsets of genes required to overcome different extracellular assaults. The largest group of these alternative factors are the extracytoplasmic function (ECF) factors. In this paper, we demonstrate that the expression of the ECF factor V in Bacillus subtilis is induced specifically by lysozyme but not other cell wall-damaging agents. A mutation in sigV results in increased sensitivity to lysozyme killing, suggesting that V is required for lysozyme resistance. Using reverse transcription (RT)-PCR, we show that the previously uncharacterized gene yrhL (here referred to as oatA for O-acetyltransferase) is in a four-gene operon which includes sigV and rsiV. In quantitative RT-PCR experiments, the expression of oatA is induced by lysozyme stress. Lysozyme induction of oatA is dependent upon V . Overexpression of oatA in a sigV mutant restores lysozyme resistance to wild-type levels. This suggests that OatA is required for Vdependent resistance to lysozyme. We also tested the ability of lysozyme to induce the other ECF factors and found that only the expression of sigV is lysozyme inducible. However, we found that the other ECF factors contributed to lysozyme resistance. We found that sigX and sigM mutations alone had very little effect on lysozyme resistance but when combined with a sigV mutation resulted in significantly greater lysozyme sensitivity than the sigV mutation alone. This suggests that sigV, sigX, and sigM may act synergistically to control lysozyme resistance. In addition, we show that two ECF factor-regulated genes, dltA and pbpX, are required for lysozyme resistance. Thus, we have identified three independent mechanisms which B. subtilis utilizes to avoid killing by lysozyme.The majority of genes in actively growing bacteria are transcribed by RNA polymerase using the general "housekeeping" factor 70 . Bacteria often utilize alternative factors to regulate subsets of genes required for specific environmental conditions (18). The largest group of these alternative factors are the extracytoplasmic function (ECF) factors (18, 39). ECF factors represent the "third pillar" of bacterial signal transduction and are often involved in response to extracytoplasmic stress (18,39). ECF factors are members of the 70 family of factors and are characterized by the presence of only two regions of 70 , regions 2 and 4.2 (18). Bacillus subtilis encodes seven known ECF factors (18). ECF factors are often required for their own transcription; thus, the expression of an ECF factor promoter is often indicative of activity of the ECF factor (18, 39). The signals which induce the activity of several ECF factors are known. For instance, the expression of sigW is induced by antimicrobial peptides and pH change (6, 9, 14, 32, 45), while sigM expression is induced by inhibitors of cell wall biosynthesis, heat shock, paraquat, and ethanol stress (12,40). Like sigM, sigX is induced by in...

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.

During growth in the environment, bacteria encounter stresses which can delay or inhibit their growth. To defend against these stresses, bacteria induce both resistance and repair mechanisms. Many bacteria regulate these resistance mechanisms using a group of alternative factors called extracytoplasmic function (ECF) factors. ECF factors represent the largest and most diverse family of factors. Here, we demonstrate that the activation of a member of the ECF30 subfamily of ECF factors, V in Bacillus subtilis, is controlled by the proteolytic destruction of the anti-factor RsiV. We will demonstrate that the degradation of RsiV and, thus, the activation of V requires multiple proteolytic steps. Upon exposure to the inducer lysozyme, the extracellular domain of RsiV is removed by an unknown protease, which cleaves at site 1. This cleavage is independent of PrsW, the B. subtilis site 1 protease, which cleaves the anti-factor RsiW. Following cleavage by the unknown protease, the N-terminal portion of RsiV requires further processing, which requires the site 2 intramembrane protease RasP. Our data indicate that the Nterminal portion of RsiV from amino acid 1 to 60, which lacks the extracellular domain, is constitutively degraded unless RasP is absent, indicating that RasP cleavage is constitutive. This suggests that the regulatory step in RsiV degradation and, thus, V activation are controlled at the level of the site 1 cleavage. Finally, we provide evidence that increased resistance to lysozyme decreases V activation. Collectively, these data provide evidence that the mechanism for V activation in B. subtilis is controlled by regulated intramembrane proteolysis (RIP) and requires the site 2 protease RasP.