Dataset Information

Signal Peptidase Is Necessary and Sufficient for Site 1 Cleavage of RsiV in Bacillus subtilis in Response to Lysozyme.

ABSTRACT: Extracytoplasmic function (ECF) ? factors are a diverse family of alternative ? factors that allow bacteria to sense and respond to changes in the environment. ?V is an ECF ? factor found primarily in low-GC Gram-positive bacteria and is required for lysozyme resistance in several opportunistic pathogens. In the absence of lysozyme, ?V is inhibited by the anti-? 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 ?V 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 ?V 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) ? factors are a diverse family of alternative ? factors that respond to extracellular signals. The ECF ? factor ?V is present in many low-GC Gram-positive bacteria and induces resistance to lysozyme, a component of the innate immune system. The anti-? factor RsiV inhibits ?V activity in the absence of lysozyme. Lysozyme binds RsiV, which initiates a proteolytic cascade leading to destruction of RsiV and activation of ?V 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.

SUBMITTER: Castro AN

PROVIDER: S-EPMC5952393 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Signal Peptidase Is Necessary and Sufficient for Site 1 Cleavage of RsiV in Bacillus subtilis in Response to Lysozyme.

Castro Ana N AN Lewerke Lincoln T LT Hastie Jessica L JL Ellermeier Craig D CD

Journal of bacteriology 20180509 11

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. σ<sup>V</sup> 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, σ<sup>V</sup> is inhibited by the anti-σ factor RsiV. In response to lysozyme, RsiV is degraded via the process of regulated intramembrane proteolysis (RI ...[more]

PMID: 29358498

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Project description:Escherichia coli signal peptidase I (LepB) has been shown to inefficiently cleave secreted proteins with aromatic amino acids at the second position after the signal peptidase cleavage site (P2'). The Bacillus subtilis exported protein TasA contains a phenylalanine at P2', which in B. subtilis is cleaved by a dedicated archaeal-organism-like signal peptidase, SipW. We have previously shown that when the TasA signal peptide is fused to maltose binding protein (MBP) up to the P2' position, the TasA-MBP fusion protein is cleaved very inefficiently by LepB. However, the precise reason why the TasA signal peptide hinders cleavage by LepB is not known. In this study, a set of 11 peptides were designed to mimic the inefficiently cleaved secreted proteins, wild-type TasA and TasA-MBP fusions, to determine whether the peptides interact with and inhibit the function of LepB. The binding affinity and inhibitory potential of the peptides against LepB were assessed by surface plasmon resonance (SPR) and a LepB enzyme activity assay. Molecular modeling of the interaction between TasA signal peptide and LepB indicated that the tryptophan residue at P2 (two amino acids before the cleavage site) inhibited the active site serine-90 residue on LepB from accessing the cleavage site. Replacing the P2 tryptophan with alanine (W26A) allowed for more efficient processing of the signal peptide when the TasA-MBP fusion was expressed in E. coli. The importance of this residue to inhibit signal peptide cleavage and the potential to design LepB inhibitors based on the TasA signal peptide are discussed. IMPORTANCE Signal peptidase I is an important drug target, and understanding its substrate is critically important to develop new bacterium-specific drugs. To that end, we have a unique signal peptide that we have shown is refractory to processing by LepB, the essential signal peptidase I in E. coli, but previously has been shown to be processed by a more human-like signal peptidase found in some bacteria. In this study, we demonstrate how the signal peptide can bind but is unable to be processed by LepB, using a variety of methods. This can inform the field on how to better design drugs that can target LepB and understand the differences between bacterial and human-like signal peptidases.

Project description:To define the ECF sigma sigV - regulated genes during log growth phase in LB media under induction conditions for sigV The seven extracytoplasmic function (ECF) sigma (σ) factors of Bacillus subtilis are broadly implicated in resistance to antibiotics and other cell envelope stressors mediated, in part, by regulation of cell envelope synthesis and modification enzymes. We here define the regulon of σV as including at least 20 operons many of which are also regulated by σM, σX, or σW. The σV regulon is strongly and specifically induced by lysozyme and this induction is key to the intrinsic resistance of B. subtilis to lysozyme. Strains with null mutations in either sigV or in all seven ECF σ factor genes (Δ7ECF) have essentially equal increases in sensitivity to lysozyme. Induction of σV in the Δ7ECF background restores lysozyme resistance, whereas induction of σM, σX or σW does not. Lysozyme resistance results from the ability of σV to activate the transcription of two operons: the autoregulated sigV-rsiV-oatA-yrhK operon and dltABCDE. Genetic analyses reveal that oatA and dlt are largely redundant with respect to lysozyme sensitivity: single mutants are not affected in lysozyme sensitivity whereas a double oatA dltA mutant is as sensitive as a sigV null strain. Moreover, the triple sigV oatA dltA mutant is no more sensitive than the oatA dltA double mutant, indicating that there are no other σV-dependent genes necessary for lysozyme resistance. Thus, σV confers lysozyme resistance by activation of two cell wall modification pathways: O-acetylation of peptidoglycan catalyzed by OatA and D-alanylation of teichoic acids by DltABCDE. Strains Δ7Pxyl-sigV + xylose vs. Δ7Pxyl-sigV - xylose, 168 + lysozyme vs. 168 - lysozyme. Each experiment was conducted 6 times using three independent total RNA preparations (biologlical triplicates). For each paired comparison, one sample was labeled with Alexa Fluor 555 and the other was with Alexa Fluor 647. For each comparison, three replicates were performed with dyeswap with the same RNA preparation.

Dataset Information

Signal Peptidase Is Necessary and Sufficient for Site 1 Cleavage of RsiV in Bacillus subtilis in Response to Lysozyme.

Publications

Signal Peptidase Is Necessary and Sufficient for Site 1 Cleavage of RsiV in Bacillus subtilis in Response to Lysozyme.

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