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Bacterial sensing: A putative amphipathic helix in RsiV is the switch for activating ?V in response to lysozyme.

ABSTRACT: Extra Cytoplasmic Function (ECF) ? factors are a diverse group of alternate ? factors bacteria use to respond to changes in the environment. The Bacillus subtilis ECF ? factor ?V responds to lysozyme. In the absence of lysozyme, ?V is held inactive by the anti-? factor, RsiV. In the presence of lysozyme RsiV is degraded via regulated intramembrane proteolysis, which results in the release of ?V and thus activation of lysozyme resistance genes. Signal peptidase is required to initiate degradation of RsiV. Previous work indicated that RsiV only becomes sensitive to signal peptidase upon direct binding to lysozyme. We have identified a unique domain of RsiV that is responsible for protecting RsiV from cleavage by signal peptidase in the absence of lysozyme. We provide evidence that this domain contains putative amphipathic helices. Disruption of the hydrophobic surface of these helices by introducing positively charged residues results in constitutive cleavage of RsiV by signal peptidase and thus constitutive ?V activation. We provide further evidence that this domain contains amphipathic helices using a membrane-impermeable reagent. Finally, we show that upon lysozyme binding to RsiV, the hydrophobic face of the amphipathic helix becomes accessible to a membrane-impermeable reagent. Thus, we propose the amphipathic helices protect RsiV from cleavage in the absence of lysozyme. Additionally, we propose the amphipathic helices rearrange to form a suitable signal peptidase substrate upon binding of RsiV to lysozyme leading to the activation of ?V.

SUBMITTER: Lewerke LT

PROVIDER: S-EPMC6066255 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Bacterial sensing: A putative amphipathic helix in RsiV is the switch for activating σV in response to lysozyme.

Lewerke Lincoln T LT Kies Paige J PJ Müh Ute U Ellermeier Craig D CD

PLoS genetics 20180718 7

Extra Cytoplasmic Function (ECF) σ factors are a diverse group of alternate σ factors bacteria use to respond to changes in the environment. The Bacillus subtilis ECF σ factor σV responds to lysozyme. In the absence of lysozyme, σV is held inactive by the anti-σ factor, RsiV. In the presence of lysozyme RsiV is degraded via regulated intramembrane proteolysis, which results in the release of σV and thus activation of lysozyme resistance genes. Signal peptidase is required to initiate degradation ...[more]

PMID: 30020925

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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

Bacterial sensing: A putative amphipathic helix in RsiV is the switch for activating ?V in response to lysozyme.

Publications

Bacterial sensing: A putative amphipathic helix in RsiV is the switch for activating σV in response to lysozyme.

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