Activation of the Extracytoplasmic Function σ factor σ^V by lysozyme in Clostridioides difficile

Abstract

Clostridioides difficile is naturally resistant to high levels of lysozyme an important component of the innate immune defense system. C. difficile encodes both constitutive as well as inducible lysozyme resistance genes. The inducible lysozyme resistance genes are controlled by an alternative σ factor σ^V that belongs to the Extracytoplasmic function σ factor family. In the absence of lysozyme, the activity of σ^V is inhibited by the anti-σ factor RsiV. In the presence of lysozyme RsiV is destroyed via a proteolytic cascade that leads to σ^V activation and increased lysozyme resistance. This review highlights how activity of σ^V is controlled.

Background

Lysozyme is a muramidase that cleaves the β-(1,4)-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) residues of peptidoglycan, a cell wall polymer that protects bacteria from lysis due to turgor pressure [1]. Lysozymes are produced by many organisms and is an important component of the innate immune response [1]. Lysozymes fall into three broad families C-type (Chicken), G-type (Goose) and I-type (invertebrate) [1]. The most well-studied of these is hen egg white Iysozyme (HEWL) which belongs the C-type lysozyme family. HEWL is homologous to Human C-type lysozyme which is produced by humans in secretions like tears, saliva, and breast milk as well as the lysosomal granules of neutrophils and macrophages [1,2]. This review will focus on the sensing of C-type lysozymes by C. difficile and related organisms.

C. difficile is highly resistant to C-type lysozyme [3,4]. Studies have found that C. difficile is ~1000 times more resistant to lysozyme than the non-pathogenic endospore-forming relative Bacillus subtilis [4–6]. C. difficile is resistant to levels of lysozyme similar to those seen in other opportunistic pathogens and human commensals like Enterococcus faecalis [7,8]. While multiple factors contribute to the high level of lysozyme resistance in C. difficile [3,4,9,10], recent data suggests that peptidoglycan deacetylation is the single most important mechanism [4,11]. Removal of the acetyl group from NAG to produce glucosamine is a well-characterized lysozyme resistance mechanism [1,12], but in C. difficile the peptidoglycan deacetylases PdaV and PgdA exhibit redundant function. Loss of either gene results in a modest increase in lysozyme resistance, while loss of both renders C. difficile ~1000 fold more sensitive to lysozyme [4,11]. Expression of pdaV is highly induced by lysozyme and is dependent upon σ^V an Extracytoplasmic function (ECF) σ factor encoded as part of the pdaV operon [3,13]. Control of pgdA expression is not well understood but has been found to be induced ~2-fold by Iysozyme in a mostly σ^Vindependent manner suggesting there may be other factors which contribute to control of pgdA expression [11].

ECF σ factors belong to the σ⁷⁰ family and are defined by the presence of only the σ₂ and σ_4.2 domains, which bind the -35 and -10 regions of target promoters [14–17]. Like most ECF σ factors, the activity of σ^V is inhibited by an anti-σ factor, RsiV (Regulator of σ^V), which is encoded within the σ factor operon [13]. ECF σ factors are a large and diverse group of σ factors with over a 150 distinct families [18,19]. σ^V belongs to the ECF30 family of σ factors which, is found almost exclusively within Firmicutes [18,19].

Activation of σ^V

In C. difficile σ^V is required for resistance to C-type lysozyme including both HEWL and Human lysozyme [3,4,20,21]. However, σ^V is not required for resistance to cell wall active antibiotics like ampicillin, bacitracin, colistin, nisin or vancomycin [3]. Like other ECF σ factors σ^V is held inactive by the transmembrane anti-σ factor RsiV [13,22,23]. σ^V is activated by C-type lysozymes including HEWL and Human neutrophil lysozyme [3,4,10,11]. However, σ^V is not activated by antimicrobial peptides like bacitracin and polymyxin [10,13]. A thorough analysis of σ^V activation was carried out in B. subtilis, and it was found that only C-type lysozyme was able to activate σ^V but not any cell wall active antibiotics [5]. Surprisingly, other muramidases like mutanolysin and T4 lysozyme failed to activate σ^V [24]. Interestingly, enzymatically inactive human lysozyme activates σ^V in B. subtilis [24]. These data argue that it is not the enzymatic activity but rather the presence of C-type lysozyme that induces activation of σ^V. In E. faecalis, starvation, heat shock, and SDS were reported to increase activation of σ^V based on northern blots [25]. However the levels of induction observed are low when compared to lysozyme [25]. This suggests that while other conditions can have small effects on σ^V activity the physiologically relevant inducer for σ^V activity is C-type lysozymes.

Most ECF σ factors are inactive and require an activation step [14,16–18]. The activity of different families of ECF σ factors are controlled by a variety of mechanisms including; phosphorylation, conformational change of the anti-σ factor which releases the σ factor, partner switching in which an anti-anti-σ factor binds to the anti-σ factor inducing σ factor release, or proteolytic destruction of the anti-σ factor [14,16–18] (Fig. 1). In C. difficile and related organisms, the anti-σ factor RsiV inhibits the activity of σ^V in the absence of lysozyme [13,23,25] (Fig. 1A). In B. subtilis, C. difficile, and E. faecalis activation of σ^V is dependent upon the proteolytic destruction of RsiV via two sequential proteolytic steps [20,26,27] (Fig. 1C and 1D). In the presence of lysozyme, RsiV is cleaved at site-1 by signal peptidases [24,26] (Fig. 1C). Once cleaved at site-1, the intramembrane protease RasP cleaves RsiV at site-2 [21,24] (Fig. 1D). This releases the N-terminal ~50 amino acids of RsiV (Fig. 1E). This RsiV fragment is degraded by unidentified cytosolic protease(s) (Fig. 1E). This degradation results in free σ^V which, can interact with RNA polymerase and direct RNA polymerase to target promoters in the σ^V regulon (Fig. 1).

Figure 1. Model of σ^V activation.

The σ factor σ^V is shown in green, the anti-σ factor RsiV is shown in red with cartoon cylinders representing the unsolved residues 1–75. Signal peptidase (Sip) is shown in light gray, the site two protease RasP is shown in yellow, and lysozyme is shown in blue. A) In the absence of lysozyme, RsiV is resistant to signal peptidase cleavage via the interaction of two amphipathic helices with the membrane that restrict access to the cleavage site. In this state RsiV sequesters σ^V activity and prevents transcription of σ^V-dependent genes. B) Once RsiV binds lysozyme, the amphipathic helices are forced out of the membrane. C) Signal peptidase is able to access the cleavage site and cleave RsiV to site-1. D) The remaining transmembrane portion of RsiV is cleaved within the membrane by the site-2 protease RasP. E) RsiV is further degraded by cytosolic proteases. FThis allows σ^V to interact with RNA polymerase dark grey and promote transcription of σ^V-dependent genes.

This proteolytic signaling cascade is directly activated by lysozyme, with RsiV tightly binding to lysozyme with a binding affinity of ~50 nM. Thus, RsiV functions as a lysozyme receptor, as evidenced by the co-crystal structure of B. subtilis RsiV bound to HEWL. This structure reveals that RsiV binds the active site and substrate-binding site of HEWL [24]. This argues that RsiV is acting as a lysozyme receptor. This was further supported by the co-crystal structure showing RsiV bound to HEWL [28]. In this structure RsiV binds to the active site and substrate-binding site of HEWL, the most conserved regions of C-type lysozyme [28]. Mutational analysis of RsiV revealed that mutations which disrupt the RsiV-HEWL interaction blocked degradation of RsiV and thus blocked activation of σ^V [28]. Thus, the binding of RsiV to HEWL is the signal that leads to σ^V activation. While this work was done using the B. subtilis RsiV homolog, both C. difficile and E. faecalis RsiV directly bind HEWL [24].

The binding of lysozyme to RsiV initiates site-1 cleavage and degradation of RsiV. In B. subtilis RsiV is cleaved after the classic A-X-A motif [24,26,29,30]. Changing the alanine of the signal peptidase recognition site at position -1 to a tryptophan blocks site-1 cleavage of RsiV [24]. SipS and SipT are two redundant but essential signal peptidases that are required for site-1 cleavage of RsiV in B. subtilis [26]. Notably, SipS and cleave RsiV only weakly at site-1 in vitro [26]. However, in the presence of lysozyme, signal peptidase cleavage of RsiV at site-1 is greatly enhanced [26]. This is dependent upon the concentration of lysozyme suggesting that the binding of lysozyme to RsiV allows signal peptidase to cleave RsiV at site-1 and initiate σ^V activation. Interestingly, in C. difficile, the cleavage site appears shifted compared to B. subtilis and mutational analysis reveals the cleavage site is likely V-X-A [24]. In contrast in E. faecalis the cleavage site is more similar to B. subtilis [24].

Evidence from B. subtilis, C. difficile, and E. faecalis shows that site-1 cleavage of RsiV by signal peptidase is the rate-limiting step for σ^V activation. Cleavage at site-1 is controlled by binding of RsiV to lysozyme. This binding allows signal peptidase to cleave RsiV. However, activity of signal peptidases is not known to be regulated. Thus, RsiV must avoid cleavage by signal peptidase in the absence of lysozyme. Recent evidence suggests RsiV is able to evade cleavage in the absence of lysozyme because the signal peptidase cleavage site is embedded in the membrane as it is part of an amphipathic helix [31]. Indeed placing charged residues on the hydrophobic face of the amphipathic helix resulted in activation of σ^V in the absence of lysozyme [31]. Importantly substituted cysteine accessibility labeling revealed that the amphipathic face of the helix is inaccessible to labelling by a membrane impermeable reagent [31]. However, upon binding to lysozyme the amphipathic surface was able to be labeled. This argues that RsiV avoids signal peptidase cleavage in the absence of lysozyme because the cleavage site is embedded in the membrane. Upon binding lysozyme, the cleavage site is displaced from the membrane allowing signal peptidase to cleave RsiV at site-1 (Fig. 1). The structure of the extracellular lysozyme binding domain of RsiV bound the HEWL has been solved however the structure of full length RsiV in the presence or absence of lysozyme is not known [28]. Thus, the conformational change that occurs in RsiV when it binds to lysozyme to allow site-1 cleavage and σ^V activation remains an unanswered question.

σ^V Regulon

Like most cell stress response systems, σ^V is induced by a stress and in turn induces genes required for responding to this stress. The regulon of σ^V has been defined in several Gram-positive bacteria including B. subtilis, E. faecalis and C. difficile [3,6,27,32] and a consensus sequence (-35 tgaAAC and -10 CGTC) has been identified for ECF30-dependent promoters, which includes σ^V[19]. In each of these organisms the regulon is small (3–5 operons depending on the organism), and the most highly induced genes are those in the sigV-rsiV operons [3,6,27]. In E. faecalis activation of σ^V induces expression of the sigV-rsiV operon as well as pgdA which encodes a peptidoglycan deacetylase [7,27]. In B. subtilis σ^V activation leads to a large increase in expression of oatA (O-acetyltransferase) and yrhK (unknown function) which are part of the sigV-rsiV operon [6,32]. OatA increases lysozyme resistance likely by adding an acetyl group to the O6 position of NAM [33,34]. C. difficile does not encode homologs of OatA however like B. subtilis the C. difficile sigV (csfV) operon is the mostly highly induced and encodes genes which modify peptidoglycan. These include pdaV (peptidoglycan deacetylase), prsA2 (peptidyl-prolyl cis-trans isomerase), rsiV (anti-σ factor) and lbpA (lysozyme-binding protein). LbpA is a homolog of RsiV that lacks the σV binding domain. LbpA can directly bind to and inhibit lysozyme activity much like RsiV [4,28]. In response to lysozyme these genes are induced >10-fold in wild type C. difficile. In a csfV mutant induction lysozyme fails to induce expression of these genes and the basal level of expression of these genes decreases ~10-fold. There are a few additional genes outside the csfV operon which are induced by σ^V including two genes of unknown function cd0738 and cd0739, as well as an operon cd1606–1611, which encodes a predicted GntR-like regulator (CD1606) and a ABC transporter system (CD1607–1611) [3]. In addition, Woods et al found that expression of the dlt operon was induced by lysozyme in σ^V-dependent manner [10]. Of these genes only pdaV, rsiV, lbpA and dlt have been implicated in lysozyme resistance [3,4,10,11]. Consistent with activation of σ^V being controlled by lysozyme, the σ^V regulons from multiple organisms show that the major σ^V-regulated genes encode either deacetylases of NAG or O-acetylases of NAM which can increase lysozyme resistance.

Role of Noise in σ^V activation

Recent work in B. subtilis shows that activation of σ^V at the single-cell level is heterogenous. This heterogeneity is more pronounced under in response to low levels of lysozyme and at higher levels activation is more uniform [35]. Interestingly in B. subtilis it was found that cells with higher basal levels of σ^V resulted in faster and more consistent σ^V activation [35]. In C. difficile the basal level of expression of csfV operon is higher than in B. subtilis [4,36]. Because of the autoregulatory nature of ECF σ factors like σ^V, this increased level of basal expression may lead to reduced noise. This is consistent with observations showing generally uniform responses at the single cell level in C. difficile [36]. It is unclear why basal levels of expression are higher in C. difficile but could be due to changes near the signal peptidase (site-1) cleavage site [24].

Unanswered questions

In addition to not understanding how lysozyme displaces the amphipathic helices to allow for signal peptidase to cleave RsiV and activate σ^V, it is also not known how the σ^V system is turned off in the absence of lysozyme. There are two possibilities, neither of which have been addressed yet. The first is that RsiV is produced at higher levels compared to σ^V and newly synthesized RsiV binds to and inhibits free σ^V. Another possibility is that free σ^V is inherently unstable and thus is turned over rapidly by cytosolic proteases. In this case newly synthesized RsiV binds to newly synthesized σ^V inhibiting its activity and the activated σ^V is destroyed over time by cytosolic proteases.

The role of the other ECF σ factors in C. difficile is not known. ECF σ factors have been implicated in resistance to many antimicrobials [37–39]. Most C. difficile strains encode three ECF σ factors although some strains carry four ECF σ factors [18]. Of these two ECF σ factors are conserved amongst nearly all sequenced C. difficile isolates csfT and sigV (csfV) [13]. To date no role for the other ECF σ factors has been described. Understanding the role of these other C. difficile ECF σ factors will provide insight into the stresses encountered by C. difficile during its life cycle and may provide targets for future therapeutic interventions.

Highlights:

• The anti-sigma factor RsiV is a receptor for lysozyme

• Binding of Lysozyme to RsiV triggers degradation of RsiV and activation of σ^V

• Activation of σ^V increases lysozyme resistance