Dataset Information

The Anti-sigma Factor RsiV Is a Bacterial Receptor for Lysozyme: Co-crystal Structure Determination and Demonstration That Binding of Lysozyme to RsiV Is Required for ?V Activation.

ABSTRACT: ? factors provide RNA polymerase with promoter specificity in bacteria. Some ? factors require activation in order to interact with RNA polymerase and transcribe target genes. The Extra-Cytoplasmic Function (ECF) ? factor, ?V, is encoded by several Gram-positive bacteria and is specifically activated by lysozyme. This activation requires the proteolytic destruction of the anti-? factor RsiV via a process of regulated intramembrane proteolysis (RIP). In many cases proteases that cleave at site-1 are thought to directly sense a signal and initiate the RIP process. We previously suggested binding of lysozyme to RsiV initiated the proteolytic destruction of RsiV and activation of ?V. Here we determined the X-ray crystal structure of the RsiV-lysozyme complex at 2.3 Å which revealed that RsiV and lysozyme make extensive contacts. We constructed RsiV mutants with altered abilities to bind lysozyme. We find that mutants that are unable to bind lysozyme block site-1 cleavage of RsiV and ?V activation in response to lysozyme. Taken together these data demonstrate that RsiV is a receptor for lysozyme and binding of RsiV to lysozyme is required for ?V activation. In addition, the co-structure revealed that RsiV binds to the lysozyme active site pocket. We provide evidence that in addition to acting as a sensor for the presence of lysozyme, RsiV also inhibits lysozyme activity. Thus we have demonstrated that RsiV is a protein with multiple functions. RsiV inhibits ?V activity in the absence of lysozyme, RsiV binds lysozyme triggering ?V activation and RsiV inhibits the enzymatic activity of lysozyme.

SUBMITTER: Hastie JL

PROVIDER: S-EPMC5014341 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Publications

The Anti-sigma Factor RsiV Is a Bacterial Receptor for Lysozyme: Co-crystal Structure Determination and Demonstration That Binding of Lysozyme to RsiV Is Required for σV Activation.

Hastie Jessica L JL Williams Kyle B KB Bohr Lindsey L LL Houtman Jon C JC Gakhar Lokesh L Ellermeier Craig D CD

PLoS genetics 20160907 9

σ factors provide RNA polymerase with promoter specificity in bacteria. Some σ factors require activation in order to interact with RNA polymerase and transcribe target genes. The Extra-Cytoplasmic Function (ECF) σ factor, σV, is encoded by several Gram-positive bacteria and is specifically activated by lysozyme. This activation requires the proteolytic destruction of the anti-σ factor RsiV via a process of regulated intramembrane proteolysis (RIP). In many cases proteases that cleave at site-1 ...[more]

PMID: 27602573

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Project description: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, σV. It has been previously demonstrated that genes regulated by σV are responsible for peptidoglycan modifications that provide C. difficile with high lysozyme resistance. σV 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 σV until lysozyme directly binds to RsiV, activating σV. However, it remains unclear if the mechanism of σV activation is similar in C. difficile. Here, we investigated how activation of σV 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 σV 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 σV 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 σV 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 σV 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 σV. 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.

Project description:Enterococcus faecalis is a gut commensal but transitions to a pathogenic state as a consequence of intestinal dysbiosis and/or the presence of indwelling medical devices, causing a wide range of infections. One of the unique features of E. faecalis is its ability to display high level resistance to lysozyme, an important host defense of the innate immune response. Lysozyme resistance in E. faecalis is known to be mediated by the extracytoplasmic function (ECF) sigma factor SigV. PgdA and RsiV expression is directly regulated by SigV, but pgdA and rsiV mutants display nominal changes in lysozyme resistance, suggesting that additional gene products in the SigV regulon contribute to lysozyme resistance. Using transcriptome sequencing (RNA-seq) analysis, we compared the transcriptional profile of the parental strain to that of an isogenic sigV mutant and show that apart from sigV, only rsiV and pgdA expression was induced upon lysozyme exposure. The combined deletion mutant of both rsiV and pgdA rendered E. faecalis sensitive to lysozyme at a level comparable to that of the sigV mutant, highlighting the limited SigV regulon. Several additional genes were also induced upon lysozyme exposure, but in a SigV-independent fashion. Overexpression of pgdA from a SigV-independent promoter restored lysozyme resistance in a sigV deletion mutant and also induced cell chaining. Overexpression of rsiV from a SigV-independent promoter only partially restored lysozyme resistance in a sigV mutant. Overall, we provide evidence for a simple adaptation to lysozyme stress, in which SigV controls the expression of rsiV and pgdA, and that both gene products contribute to lysozyme resistance. IMPORTANCE Enterococcus faecalis causes health care-associated infections and displays resistance to a variety of antibiotics and molecules of the innate immune system. SigV has been shown to play an important role in enterococcal lysozyme resistance. Even though several proteins have been implicated in enterococcal lysozyme resistance, a complete SigV-dependent regulon has not been functionally characterized as being responsible for the dramatic increase in lysozyme susceptibility displayed by a sigV mutant. Using RNA-seq, we have identified the SigV regulon to be comprised of two gene loci, sigV-rsiV and pgdA. Deletion of both rsiV and pgdA renders E. faecalis susceptible to lysozyme on par with a sigV mutant. We also demonstrate that overproduction of rsiV and pgdA contributes to lysozyme resistance in susceptible strains.

Project description:Extracytoplasmic function (ECF) σ factors are a diverse family of alternative σ factors that allow bacteria to sense and respond to changes in the environment. σV is an ECF σ factor found primarily in low-GC Gram-positive bacteria and is required for lysozyme resistance in several opportunistic pathogens. In the absence of lysozyme, σV is inhibited by the anti-σ factor RsiV. In response to lysozyme, RsiV is degraded via the process of regulated intramembrane proteolysis (RIP). RIP is initiated by cleavage of RsiV at site 1, which allows the intramembrane protease RasP to cleave RsiV within the transmembrane domain at site 2 and leads to activation of σV Previous work suggested that RsiV is cleaved by signal peptidase at site 1. Here we demonstrate in vitro that signal peptidase is sufficient for cleavage of RsiV only in the presence of lysozyme and provide evidence that multiple Bacillus subtilis signal peptidases can cleave RsiV in vitro This cleavage is dependent upon the concentration of lysozyme, consistent with previous work that showed that binding to RsiV was required for σV activation. We also show that signal peptidase activity is required for site 1 cleavage of RsiV in vivo Thus, we demonstrate that signal peptidase is the site 1 protease for RsiV.IMPORTANCE Extracytoplasmic function (ECF) σ factors are a diverse family of alternative σ factors that respond to extracellular signals. The ECF σ factor σV is present in many low-GC Gram-positive bacteria and induces resistance to lysozyme, a component of the innate immune system. The anti-σ factor RsiV inhibits σV activity in the absence of lysozyme. Lysozyme binds RsiV, which initiates a proteolytic cascade leading to destruction of RsiV and activation of σV This proteolytic cascade is initiated by signal peptidase, a component of the general secretory system. We show that signal peptidase is necessary and sufficient for cleavage of RsiV at site 1 in the presence of lysozyme. This report describes a role for signal peptidase in controlling gene expression.

Dataset Information

The Anti-sigma Factor RsiV Is a Bacterial Receptor for Lysozyme: Co-crystal Structure Determination and Demonstration That Binding of Lysozyme to RsiV Is Required for ?V Activation.

Publications

The Anti-sigma Factor RsiV Is a Bacterial Receptor for Lysozyme: Co-crystal Structure Determination and Demonstration That Binding of Lysozyme to RsiV Is Required for σV Activation.

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