SigV Mediates Lysozyme Resistance in Enterococcus faecalis via RsiV and PgdA

ABSTRACT

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.

IMPORTANCEEnterococcus 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.

INTRODUCTION

Enterococcus faecalis is a commensal bacterium that is known to be prevalent in the gastrointestinal tracts of animals, including humans (1). However, under antibiotic selective pressure or in the presence of indwelling medical devices, E. faecalis is positioned to cause a wide range of infections, including catheter-associated urinary tract infection, bloodstream infection, wound infection, and endocarditis (2). E. faecalis has gained importance as a nosocomial pathogen because of its ability to endure harsh disinfectant cleaning regimens in health care settings as well as exposure to antiseptics, including chlorhexidine (3). Apart from antibiotics and disinfectants, E. faecalis is also known for its high intrinsic resistance to lysozyme, a key component of the innate defense barriers of host immune systems.

Lysozyme is a major component of the mucosal innate immune system and is a key component of neutrophil granules and a major secretory product of macrophages (4). Lysozyme is usually found in mammalian secretions, including tears and saliva, and principally targets Gram-positive bacteria via two known mechanisms. Lysozyme is a muramidase that cleaves peptidoglycan, targeting the glycosidic bond between β-1,4-linked residues of N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) (5). In addition to this enzymatic role, lysozyme, because of its cationic charge, acts as a cationic antimicrobial peptide (CAMP) and inserts itself into the negatively charged bacterial cell membrane. This leads to membrane destabilization and bacterial cell death due to osmotic lysis (6, 7). Because of its dual activity as both a muramidase and membrane inserting protein, lysozyme is considered to be one of the most effective antibacterial agents. Despite this potent activity, several pathogenic bacteria, including E. faecalis, have developed mechanisms to counter the bactericidal activity of lysozyme.

SigV belongs to a family of extracytoplasmic function (ECF) sigma factors that are normally known to be functionally important during stress conditions. Deletion of sigV was shown to render E. faecalis more susceptible to lysozyme, suggesting that SigV played a major role in its lysozyme resistance phenotype (8). SigV, in E. faecalis, is encoded as part of a bicistronic operon along with an anti-sigma factor, RsiV, a membrane-bound protein that sequesters SigV at the membrane. This sequestration prevents SigV from interacting with the core RNA polymerase and prevents activation of SigV-dependent target genes in the absence of lysozyme stimuli. Work out of the Ellermeier lab has shown that RsiV from several Gram-positive bacteria, including Bacillus subtilis, Clostridioides difficile, and E. faecalis, directly binds to lysozyme and can stoichiometrically inhibit lysozyme in a 1:1 ratio (9–11). In B. subtilis, upon exposure to lysozyme, RsiV is actively cleaved by membrane-bound proteases (12, 13). Our prior work with E. faecalis established a role for the integral membrane site 2 protease, Eep, in the regulated degradation of RsiV (14). This regulated proteolysis releases SigV from RsiV and makes SigV available to activate target genes to provide resistance to lysozyme.

Other gene products that have been shown to play a contributing role in enterococcal lysozyme resistance include OatA (15), Dlt proteins (d-alanylation) (16), and PgdA (17). Each of these proteins makes limited contributions to overall lysozyme resistance in E. faecalis, as individual gene mutants display minimal change with respect to the overall lysozyme resistance phenotype. OatA is an O-acetyltransferase and acetylates the peptidoglycan at the C-6 hydroxyl group of N-acetylmuramic acid (18, 19). OatA was shown to be a major contributor to lysozyme resistance in Staphylococcus aureus (20). The oatA gene is also part of the three-gene B. subtilis sigV operon (sigV, rsiV, and oatA) and along with the SigV-dependent dlt operon contributes to lysozyme resistance (21). Streptococcus pneumoniae displays resistance to lysozyme by deacetylation of its peptidoglycan layer by producing a peptidoglycan deacetylase, PgdA (22). PgdA acts to deacetylate the C-2 N-acetyl group of GlcNac to render S. pneumoniae resistant to lysozyme. The PgdA homolog, in E. faecalis, is known to be regulated by SigV (8) but exhibits a lysozyme susceptibility phenotype only when mutated in conjunction with oatA and dltA (15, 17). The combined deletions of major cell wall-modifying enzymes encoded by pgdA, oatA, and dltA along with the alternative sigma factor gene sigV was seen to have the most pronounced effect on lysozyme resistance (17). Despite growing knowledge of factors that contribute to lysozyme resistance in E. faecalis, little is known in regard to what factors are actually induced upon lysozyme exposure. It was therefore of interest to determine what transcriptional changes take place in response to lysozyme exposure and to determine what gene loci comprise the SigV regulon.

In this study, we identified the genes that are induced upon lysozyme exposure in E. faecalis V583 as well as the subset of genes comprising the SigV regulon. We demonstrate that the SigV regulon is comprised of two transcripts, sigV-rsiV and pgdA, and that this cellular response is dependent on lysozyme exposure. Furthermore, the combined deletion of rsiV and pgdA hinders lysozyme resistance to the levels observed with a sigV deletion. We also report that overexpression of PgdA alters the cell wall in a manner that induces extensive chain formation, suggesting that cell autolytic activity is also regulated by PgdA activity.

RESULTS

SigV regulon induced upon lysozyme exposure.

Several gene products have been previously reported to be involved in the enterococcal lysozyme resistance pathway, including the alternative sigma factor SigV. Deletion of the sigV gene alone renders the bacteria more sensitive to lysozyme, with an MIC of <5 mg/ml, in contrast to the >64 mg/ml exhibited by wild-type strains (8, 14). As the direct regulon of SigV that is involved in the lysozyme resistance mechanism has not been fully characterized, we sought to identify the genes regulated by SigV in response to lysozyme. This led us to perform a global comparative transcriptomic analysis on the V583 strain and its isogenic sigV deletion mutant exposed to 1 mg/ml of lysozyme. A stringent statistical cutoff of 0.05 for the q value (adjusted to 2 significant figures) was used to identify genes that were differentially expressed in response to lysozyme. As shown in Fig. S1 in the supplemental material, the expression of only three genes was significantly induced upon lysozyme exposure in the parental strain compared to the ΔsigV mutant strain. In addition to the absence of sigV expression in the ΔsigV mutant, rsiV expression was also reduced 450-fold in the ΔsigV strain (Table 1). This result corroborates the bicistronic sigV-rsiV genetic organization and autoregulation of this operon (8). Apart from the sigV and rsiV transcript, pgdA was the only other gene that was significantly affected in the ΔsigV mutant, as its expression was lowered 23-fold (Table 1) compared to expression in the parental V583 strain. This also confirms the previous observation that lysozyme exposure induces pgdA expression in a SigV-dependent manner (17). It was noteworthy to discover that apart from the sigV-rsiV and pgdA transcripts, none of the other genes (oatA, dltA, and epaB) whose protein products have previously been shown to contribute to lysozyme resistance in E. faecalis were significantly induced upon exposure to lysozyme, as shown in Fig. S1 and Table S1.

TABLE 1.

Genes that were seen to be significantly induced upon exposure to 1 mg/ml of lysozyme in V583 strain compared to V583ΔsigV

Gene locus	Fold change	P value	q value
ef_rs15045 (ef3179) (rsiV)	452	5e−05	0.05
ef_rs15050 (ef3180) (sigV)	376	5e−05	0.05
ef_rs08840 (ef1843) (pgdA)	23	5e−05	0.05

The effect of lysozyme exposure on the global transcriptome of the cell to identify putative SigV-independent factors was also determined by performing transcriptome sequencing (RNA-seq) on the V583 parental strain exposed to lysozyme and comparing that response to that with a no-lysozyme control. Apart from the SigV regulon, we observed additional transcripts that were upregulated upon exposure to lysozyme (Fig. S2 and Table S2). Independent of the SigV regulon, 8 additional genes were found to be upregulated in response to lysozyme exposure; 3 are annotated to encode functions in cell wall or cell envelope processes: EF_RS01780 (EF0355), a putative LysM-containing peptidoglycan binding protein; EF_RS04430 (EF0932), a phosphoethanolamine transferase predicted to be important for cell wall/envelope biogenesis; and EF_RS15340 (EF3245), a two-domain-containing protein possessing a cell envelope-associated acid phosphatase (PAP2 domain) and a LytR-CpsA-Psr (LCP) domain. Two gene products, EF_RS00115 (EF0026) and EF_RS06820 (EF1412), are predicted membrane proteins. The remaining three genes, ef_rs01725 (ef0344), ef_rs01730 (ef0345), and ef_rs15320 (ef3239), are annotated as encoding hypothetical proteins. We used reverse transcription-quantitative PCR (qRT-PCR) to confirm that a subset of these genes (ef0355, ef0932, and ef3245) were differentially expressed upon lysozyme exposure (Fig. S4), with ef3245 found to be ∼5-fold upregulated and ef0932 and ef0355 shown to be ∼2-fold upregulated compared to the case under the no-lysozyme control condition.

Deletion of SigV regulon causes lysozyme susceptibility.

As it is known that deletion of sigV affects lysozyme resistance in E. faecalis, we wanted to assess the contribution of the other members of the SigV regulon identified in the transcriptomic studies following lysozyme exposure. MIC assays were performed with serial dilutions of lysozyme (128 mg/ml to 0.25 mg/ml) using the V583 parental strain and the ΔsigV, ΔrsiV, ΔsigV ΔrsiV, ΔpgdA, ΔrsiV ΔpgdA, and ΔsigV ΔrsiV ΔpgdA mutants. The V583 parental strain and ΔrsiV and ΔpgdA mutant strains displayed an MIC of 128 mg/ml of lysozyme, whereas the ΔsigV mutant had an MIC of 4 mg/ml (Table 2). Interestingly, the ΔsigV ΔrsiV and ΔrsiV ΔpgdA double mutants and ΔsigV ΔrsiV ΔpgdA triple mutant phenocopied the ΔsigV strain, displaying an MIC of 4 mg/ml. These observations were consistent with a growth curve analysis in which the ΔsigV, ΔsigV ΔrsiV, ΔrsiV ΔpgdA, and ΔsigV ΔrsiV ΔpgdA strains displayed no growth at 8 mg/ml of lysozyme (Fig. 1B) but did not have any growth defects when grown without lysozyme (Fig. 1A). We also observed the same trend in a lysozyme solid-medium assay (LB agar) in which dilutions of the V583, ΔrsiV, and ΔpgdA strains were able to form colonies efficiently at 10 mg/ml of lysozyme, whereas the ΔsigV, ΔsigV ΔrsiV, ΔrsiV ΔpgdA, and ΔsigV ΔrsiV ΔpgdA mutants were unable to grow efficiently past the 10⁻³ dilution at 10 mg/ml of lysozyme, as shown in Fig. 1D, despite showing no difference in growth compared to other strains in the absence of lysozyme (Fig. 1C).

TABLE 2.

Bacterial strains used in this study and their corresponding MIC values for lysozyme

Strain	Description	Reference	Lysozyme MIC (mg/ml)
V583	Parental strain	41	128
SV07	V583 ΔsigV	14	4
SV14	V583 ΔrsiV	14	128
EJH01	V583 ΔsigV rsiV	This study	4
VI50	V583 ΔpgdA	14	128
KU01	V583 ΔrsiV ΔpgdA	This study	4
EJH02	V583 ΔsigV rsiV ΔpgdA	This study	4
SP40	VI50 (pML28) (ΔpgdA); vector only	This study	128
SP41	VI50 (pKC05) (ΔpgdA); PgdA+	This study	128
SP42	SV07 (pML28) (ΔsigV); vector only	This study	4
SP43	SV07 (pKC05) (ΔsigV); PgdA+	This study	128
SV37	SV07 (pSV17) (ΔsigV); RsiV+	This study	8
SP45	KU01 (pML28) (ΔrsiV ΔpgdA); vector only	This study	4
SP46	KU01 (pKC05) (ΔrsiV ΔpgdA); PgdA+	This study	128
SP47	KU01 (pSV17) (ΔrsiV ΔpgdA); RsiV+	This study	8

FIG 1.

Both RsiV and PgdA contribute to lysozyme resistance. (A) Growth curve of the V583, ΔsigV, ΔrsiV, ΔsigV ΔrsiV, ΔpgdA ΔrsiV ΔpgdA, and ΔsigV ΔrsiV ΔpgdA strains grown in LB medium supplemented with glucose at a 0.5% concentration. (B) Growth curve of the V583, ΔsigV, ΔrsiV, ΔsigV ΔrsiV, ΔpgdA ΔrsiV ΔpgdA, and ΔsigV ΔrsiV ΔpgdA strains grown in LB medium supplemented with lysozyme at 8 mg/ml and glucose at a 0.5% concentration. Statistical analysis of three replicates was performed using one-way ANOVA followed by Bonferroni’s multiple-comparison posttest: ***, P value < 0.001; **, 0.001 < P value < 0.005. (C) Lysozyme sensitivity assay performed with the V583, ΔsigV, ΔrsiV, ΔsigV ΔrsiV, ΔpgdA, ΔrsiV ΔpgdA, and ΔsigV ΔrsiV ΔpgdA strains grown on LB agar containing no lysozyme. (D) Lysozyme sensitivity assay performed with the V583, ΔsigV, ΔrsiV, ΔsigV ΔrsiV, ΔpgdA ΔrsiV ΔpgdA, and ΔsigV ΔrsiV ΔpgdA strains grown on LB agar containing 10 mg/ml of lysozyme. The image shown is representative of three independent biological replicates.

Overexpression of SigV regulon restores lysozyme resistance.

The lysozyme MIC assays suggested that the absence of both RsiV and PgdA renders the cells more susceptible to lysozyme. Therefore, we assessed the expression of rsiV and pgdA in the lysozyme-susceptible strains (ΔsigV and ΔrsiV ΔpgdA) to determine the effect that each of these components had on the overall lysozyme resistance of E. faecalis. Overexpression of rsiV and pgdA in a SigV-independent manner was achieved by transforming the V583 parental and mutant strains with the pML28-derived plasmids, which allows for overexpression of cloned genes from the constitutive aph promoter (23). The expression levels of pgdA and rsiV were assessed in overexpressed strains by quantitative real-time PCR. As shown in Fig. 2B, transformation of mutant strains with the pgdA overexpression plasmid resulted in a 10⁴-fold increase for both the ΔrsiV ΔpgdA and ΔsigV mutant backgrounds compared to the unstimulated V583 control. We also measured the expression of pgdA in the rsiV mutant and found expression to increase 150-fold (Fig. 2A). As shown in Fig. 2C, the expression of rsiV was approximately 200-fold more in the ΔsigV and ΔrsiV ΔpgdA strains with the overexpression plasmid compared to the V583 control.

FIG 2.

Quantification of RsiV and PgdA overexpression from a SigV-independent promoter. (A) qPCR to quantify the levels of sigV, rsiV, and pgdA in the parental V583 strain induced with 1 mg/ml of lysozyme and the ΔrsiV strain in the absence of lysozyme. (B) qPCR performed to quantify pgdA expression on the V583, ΔsigV, ΔrsiV, and ΔrsiV ΔpgdA strains and the ΔsigV strain containing pgdA-overexpressing plasmid. (C) qPCR performed to quantify rsiV expression on the V583, ΔsigV, and ΔsigV strains and the ΔrsiV ΔpgdA strain containing rsiV-overexpressing plasmid. RQ was calculated using threshold cycle (ΔΔC_T) method normalized to parental V583 expression levels without lysozyme induction, and gyrB was used as the endogenous control. RQ, relative quantification. Statistical analysis of the expression values was performed using 2-way ANOVA for panel A and using Student’s two-tailed unpaired t test using confidence intervals of 95% for panels B and C based on 3 biological replicates. t test significance representations: *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; ***, P < 0.001.

Upon inspection of the growth kinetics of the pgdA- and rsiV-overexpressing strains from a 1:100 dilution of an overnight culture grown in the absence of lysozyme, it was found that pgdA overexpression was able to restore growth of the lysozyme-sensitive ΔsigV and ΔrsiV ΔpgdA strains to parental levels in both liquid and solid media (Fig. 3). However, rsiV overexpression was only able to restore the growth of these strains at 2 mg/ml and with slower growth kinetics (Fig. 3B). At 8 mg/ml, only pgdA overexpression was able to rescue growth of the ΔsigV and ΔrsiV ΔpgdA mutants (Fig. 3A and B). On solid media, growth of the ΔrsiV ΔpgdA mutant containing the rsiV overexpression plasmid allowed growth at 1 additional 10-fold dilution compared to the ΔrsiV ΔpgdA mutant with the empty vector when grown at 5 mg/ml of lysozyme in solid medium but did not enhance growth in 10 mg/ml of lysozyme (Fig. 3D and E), consistent with the reported MIC data. No difference in growth was observed between strains containing the rsiV- and pgdA-overexpressing plasmids and their corresponding empty vector in the absence of lysozyme (Fig. 3C).

FIG 3.

Overexpression of RsiV and PgdA confers lysozyme resistance in sensitive strains. (A) Growth curve of V583 and ΔsigV and ΔrsiV ΔpgdA mutants with and without overexpression of RsiV and PgdA grown in LB medium supplemented with lysozyme at 8 mg/ml, glucose at 0.5%, and spectinomycin at 0.5 mg/ml. (B) Growth curve of V583 and ΔsigV and ΔrsiV ΔpgdA mutants with and without overexpression of RsiV and PgdA grown in LB medium supplemented with lysozyme at 2 mg/ml, glucose at 0.5%, and spectinomycin at 0.5 mg/ml. Statistical analysis was done using one-way ANOVA and Bonferroni’s multiple-comparison posttest. Asterisks representing P values of strains, comparing their growth to parental V583, are included beside the strain names (***, P < 0.001). (C) Lysozyme sensitivity assay performed with ΔsigV and ΔrsiV ΔpgdA strains overexpressing PgdA and RsiV grown on LB agar containing no lysozyme. (D) Lysozyme sensitivity assay performed with ΔsigV and ΔrsiV ΔpgdA strains overexpressing PgdA and RsiV grown on LB agar containing 5 mg/ml of lysozyme. (E) Lysozyme sensitivity assay performed with ΔsigV and ΔrsiV ΔpgdA strains overexpressing PgdA and RsiV grown on LB agar containing 10 mg/ml of lysozyme. Data are representative of those from three independent biological replicates.

PgdA overexpression affects cell wall architecture.

In a previous study, we showed that constitutive expression of sigV as a consequence of rsiV deletion caused extensive cellular chaining (Fig. 4Aii), leading to a settling phenotype in the absence of lysozyme (Fig. 4B) (14). This led us to hypothesize that this chaining and settling phenotype were due to an adverse effect on cell wall remodeling by causing the constitutive expression of sigV. As pgdA expression is dependent on SigV and PgdA is also known to deacetylate the C-2 N-acetyl group of GlcNac, we hypothesized that PgdA overexpression would alter the cell wall architecture to cause the cell chaining and settling phenotype. We tested this hypothesis by microscopic examination of the ΔpgdA strain transformed with the plasmid overexpressing PgdA. Compared to the parental strain, the ΔrsiV mutant alone and the ΔpgdA mutant complemented with the PgdA overexpression plasmid were capable of forming extensive chains even in the absence of lysozyme, as shown in Fig. 4Aii and v, and not in the parental V583 strain (Fig. 4Ai) or the ΔpgdA strain transformed with the empty vector (Fig. 4Aiv). The chaining phenotype observed in the ΔrsiV strain was absent in a ΔrsiV ΔpgdA strain (Fig. 4Aiii), providing additional support that PgdA expression is responsible for the chaining phenotype. As we have shown that extensive chaining leads to a cell settling phenotype (14), a cell settling index was calculated on parental and mutant strains and directly correlated with cellular chaining using an inverse correlation model of the optical density at 600 nm (OD₆₀₀) of the top third layer from liquid overnight cultures (Fig. S3). As shown in Fig. 4B, only the ΔrsiV strain and strains containing the PgdA overexpression plasmid displayed a settling phenotype. This suggests that expression of PgdA likely alters the cellular muramidase activity (autolysin) to also promote chain formation and that expression of PgdA protects the cell wall both from lysozyme attack and from its own autolytic enzymes.

FIG 4.

PgdA overexpression causes chaining phenotype. (A) Microscopic imaging of overnight-grown cultures of V583 (i), the ΔrsiV mutant (ii), the ΔrsiV ΔpgdA mutant (iii), the ΔpgdA mutant plus empty vector (EV), and the ΔpgdA mutant plus PgdA stained with crystal violet and imaged at a magnification of ×100 (v). (B) Chaining phenotype graphed based on a mathematical calculation correlating the chaining of cells to the settling observed upon overnight planktonic growth (Fig. S3) of the shown cultures. Statistical analysis was performed using Student’s two-tailed unpaired t test with confidence intervals of 95%. **, 0.001 < P < 0.01; ***, P < 0.001.

PgdA and RsiV overexpression disrupts lysozyme binding.

Restoration of lysozyme resistance in the susceptible ΔsigV and ΔrsiV ΔpgdA strains upon overexpression of PgdA and RsiV led us to hypothesize that the altered cell wall architecture of these strains might affect the amount of lysozyme able to bind to the cell surface. To test this, the lysozyme-susceptible ΔsigV strain and the lysozyme-resistant V583 strain, along with the ΔsigV strain overexpressing either PgdA or RsiV, were exposed to 1 mg/ml of lysozyme. As seen in Fig. 5, we corroborated our previous finding that the ΔsigV strain accumulated more lysozyme, explaining its susceptibility (14). In contrast, we observed decreased amounts of lysozyme from lysates of the ΔsigV strain overexpressing PgdA or RsiV (Fig. 5A), suggesting that overexpression of both of these proteins alters lysozyme binding to the bacterial cell surface. Conversely, the amount of unbound lysozyme available in the supernatant increased in the ΔsigV strain overexpressing PgdA and RsiV to parental levels, in contrast to the ΔsigV strain and the ΔsigV strain containing the empty vector, in which the unbound lysozyme was reduced significantly compared to the case with the parental strain, as shown in Fig. 5B. This observation further supports the notion that overexpression of PgdA and RsiV disrupts lysozyme binding to the bacterial cell wall, contributing to the observed lysozyme resistance.

FIG 5.

PgdA and RsiV overexpression disrupts lysozyme binding. (A) SDS-PAGE Tris-Tricine (16%) gel run with normalized lysates of V583, the ΔsigV mutant, the ΔsigV mutant plus PgdA, and the ΔsigV mutant plus RsiV grown and exposed to or not exposed to 1 mg/ml of lysozyme and stained with Coomassie brilliant blue dye. The corresponding lysozyme peak intensities were quantified using densitometry by ImageJ, normalized to the V583 levels without lysozyme exposure, and are representative of three separate biological replicates. “+” or “−” represents presence or absence of lysozyme, and the arrow indicates the band corresponding to lysozyme. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s multiple-comparison posttest. (B) SDS-PAGE Tris-Tricine (16%) gel run with normalized culture supernatants of V583, the ΔsigV mutant, the ΔsigV mutant plus PgdA, the ΔsigV mutant plus RsiV, and the ΔsigV mutant plus EV grown and exposed to 1 mg/ml of lysozyme and stained with Coomassie brilliant blue dye. The corresponding lysozyme peak intensities were quantified using densitometry by ImageJ, normalized to the V583 levels, and are representative of three separate biological replicates. The arrow indicates the band corresponding to lysozyme. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s multiple-comparison posttest.

DISCUSSION

In this study, we have identified the direct regulon of SigV that is required for lysozyme resistance in E. faecalis. It has been shown that SigV is essential for lysozyme resistance and that deletion of the sigV gene renders E. faecalis more susceptible to lysozyme attack (8, 16). Hence, genes comprising the SigV regulon are expected to contribute to lysozyme resistance. We have identified the genes (rsiV and pgdA) that are regulated by SigV upon lysozyme exposure, and the deletion of rsiV combined with pgdA renders that mutant sensitive to lysozyme at levels observed for the sigV mutant. To the best of our knowledge, this is the first study identifying the complete SigV regulon using transcriptomics.

Our global transcriptomic analysis also revealed that sigV-rsiV and pgdA were the most abundantly upregulated genes upon lysozyme exposure that met the statistical cutoff established to reveal genes differentially expressed compared to the uninduced control. In addition to members of the SigV regulon, we also observed a number of transcripts predicted to encode functions related to cell wall/envelope processes that were significantly upregulated under lysozyme-inducing conditions. EF_RS04430 (EF0932), a putative phosphoethanolamine transferase, is predicted to be important for cell wall/envelope biogenesis. However, the sequence similarity between EF0932 and known phosphoethanolamine transferases only extends to the transmembrane region and not to the enzymatic domain. Thus, the potential function of EF0932 in responding to lysozyme-induced stress awaits further characterization. EF_RS15340 (EF3245), a putative dual-domain protein consisting of a cell envelope-associated acid phosphatase (PAP2 domain) and wall teichoic acid transferase (LCP domain), was found to be upregulated ∼5-fold in response to lysozyme stress. PAP2 domain-containing proteins are involved in the recycling of undecaprenyl pyrophosphate to undecaprenyl phosphate (24), whereas the LCP domain has been recently shown to be responsible for wall teichoic acid transferase activity (25, 26). Interestingly, Abranches et al. showed that ef3245, a gene that is unique to enterococci, is activated upon exposure to antibiotics that affect cell wall biogenesis (27). Further, deletion of ef3245 in the OG1RF strain background resulted in a 4-fold increased sensitivity to bacitracin and attenuated its virulence in a Galleria mellonella animal model, suggesting the importance of EF3245 in responding to cell wall stress (27). In this study, we found that exposure to lysozyme, another potent cell wall stressor, significantly upregulates the expression of ef3245. In combination with the previous studies, this finding leads us to predict that EF3245 plays an important role in cell wall biogenesis, likely through its role in modulating cell wall assembly and recycling the lipid carrier needed for peptidoglycan synthesis, as well as the synthesis of accessory cell wall polymers. However, the relevance of EF3245 with respect to enterococcal resistance to lysozyme and other cell membrane-targeting antibacterial agents will require further investigation. Intriguingly, ef_rs01725 (ef0344), ef_rs01730 (ef0345), and ef_rs01780 (ef0355) are part of the prophage-like element pp-1 (28). Prophage elements pp-1 and pp-7 were shown to form a composite phage in the V583 strain and confer a competitive advantage to the V583 strain over other E. faecalis strains in vivo and in vitro (29). Whether lysozyme exposure induces phage activation is unclear, but the elevated expression of some pp-1 phage genes in response to lysozyme exposure in this study warrants further investigation.

Previous research has identified several genes, including oatA, dltA, and the core region of the epa operon, in lysozyme resistance (15, 16, 30). Even though these gene products were seen to affect lysozyme resistance only in combination, none of these genes were shown to be induced by SigV activation. Likewise, in our RNA-seq data, oatA, dltA, and the epa operon were not seen to be upregulated in a SigV-dependent or independent manner upon lysozyme exposure. It is possible that general cell wall alterations caused by the effect of OatA, DltA, and Epa alter the overall charge of the E. faecalis cell wall. OatA, in pathogenic staphylococci, contributes to lysozyme resistance by affecting lysozyme binding to peptidoglycan (20). OatA is known to O-acetylate the C-6 OH of the N-acetylmuramic acid residue of the peptidoglycan layer, which is hypothesized to sterically hinder lysozyme binding to that residue (20). Similarly, DltA, in E. faecalis, has been shown to reduce the net negative charge of the bacterial cell surface, making the cells less susceptible to attack by cationic antimicrobial peptides (31). More recently, Smith et al. showed that transposon insertions in the variable region of epa in the quadruple mutant background of an oatA, pgdA, dltA, and sigV mutant, designated OPDV, resulted in a restoration to wild-type levels of lysozyme resistance (32). These transposon insertion strains were also shown to have a decreased net negative charge on the bacterial cell surface. This suggests that the loss of the variable region of epa might alter lysozyme affinity for the cell wall by altering the overall charge. It was recently shown by Guerardel et al. that the epa variable region encodes a teichoic acid polymer that is covalently linked to the Epa rhamnopolysaccharide backbone (33). Thus, the loss of teichoic acid would be predicted to restore lysozyme resistance in the OPDV mutant. Despite the contributions made by OatA, DltA, and Epa in lysozyme resistance, only pgdA has been shown to be controlled by SigV (14, 17) and, importantly, in the present study was one of only two transcripts dependent on SigV and induced by lysozyme exposure.

This study identified the limited regulon of SigV that affects lysozyme resistance in E. faecalis. Even though the involvement and importance of SigV in lysozyme resistance are well established (8), the actual regulon directly regulated by SigV remained unknown. Benachour et al. predicted four direct targets of SigV, namely, ef0159, ef0315, pgdA, and ef1934, based on computational prediction of the SigV binding site in their promoter region (8). However, none of those genes, other than pgdA, were determined to be directly targeted by SigV. Transcriptomic analysis in this study revealed that pgdA and rsiV are the only genes that constitute the exclusive regulon of SigV. Importantly, none of the other genes mentioned above were affected in their expression upon sigV deletion and exposure to lysozyme, suggesting that SigV governs a limited regulon to control resistance to lysozyme.

Peptidoglycan GlcNAc deacetylase (PgdA), in E. faecalis, has been shown to catalyze peptidoglycan deacetylation in a SigV-dependent manner in response to lysozyme (17). In this paper, we show that overexpression of PgdA alone is sufficient to restore lysozyme resistance in sensitive strains (ΔsigV and ΔrsiV ΔpgdA). This leads to the hypothesis that cell wall deacetylation is a key determinant in the bacterium’s ability to ward off lysozyme attack. Polysaccharide deacetylase catalyzes the hydrolysis of the C-6 N-linked acetyl group from GlcNAc residues. PgdA was first described for S. pneumoniae, in which the cells became susceptible to lysozyme upon pgdA inactivation (22). This was the only annotated deacetylase in the pneumococcal genome and is responsible for roughly 90% of the deacetylating activity (22). Bacillus anthracis encodes 5 homologs of polysaccharide deacetylases, from which only 1, BA1977, has been shown to be contribute to lysozyme resistance and virulence (34). Recently, two polysaccharide deacetylases, PgdA and PdaV, were shown to be responsible for complete peptidoglycan deacetylation and lysozyme resistance in C. difficile (35). Enterococcal strains with an inherent resistance to antibiotics such as vancomycin possess a distinct deacetylation pattern in their cell wall, which further increases upon exposure to cell wall-active antibiotics (36). Intriguingly, deletion of pgdA alone did not significantly affect the lysozyme resistance in E. faecalis (Table 2). It is noteworthy that 8 gene products were found to be induced upon lysozyme exposure and were not part of the SigV regulon. Whether any of these gene products contribute to lysozyme resistance will require additional investigation. As was observed in B. anthracis and C. difficile, the possibility of additional cell wall deacetylases that could modify the peptidoglycan in the absence of PgdA should be considered. The V583 strain contains several homologs of cell wall/polysaccharide deacetylases, and their involvement in peptidoglycan deacetylation and lysozyme resistance remains an active topic of ongoing research.

Our data from SDS-PAGE indicate that ΔsigV mutants overexpressing pgdA and rsiV bind less lysozyme on their cell walls than the parental V583 strain and the isogenic sigV mutant. It has previously been shown that the ΔsigV mutant binds more lysozyme than the V583 strain (14), possibly explaining its increased susceptibility to lysozyme. However, the actual mechanistic explanation for this observation is unknown. As a part of the overall lysozyme resistance model, we propose that PgdA, expressed upon lysozyme exposure, leads to increased deacetylation of GlcNAc residues of peptidoglycan (17) and blocks lysozyme binding to the cell wall (Fig. 5). This could happen by possible steric hindrance caused by exposure of the amine group of GlcNAc residue upon deacetylation to binding by positively charged amino acids of lysozyme (37). RsiV is known to directly bind lysozyme and act as a suicide inhibitor of lysozyme, resulting in a conformational change in the RsiV structure exposing a proteolytically labile site that is cleaved by a site 1 membrane protease in B. subtilis (9, 10, 12, 13). Further cleavage of RsiV has been shown to follow a regulated intramembrane proteolysis (RIP) by Eep in E. faecalis (14) or RasP in B. subtilis (13). In our present model, we propose that following RIP, the RsiV C-terminal domain bound to lysozyme diffuses away from the membrane, thus disrupting the CAMP activity of lysozyme and partially rescuing the cell from the bactericidal activity of lysozyme. In B. subtilis, overexpression of RsiV results in a 1.5-fold incremental increase in lysozyme resistance (7.5 μg/ml versus 5 μg/ml) compared to that of a sigV rsiV mutant (9), when RsiV is expressed from a SigV-independent promoter, and our results for E. faecalis are consistent with these observations, as we observed only a slight increase (4 to 8 mg/ml) in the lysozyme MIC when we overexpressed RsiV from a SigV-independent promoter in either the sigV or rsiV pgdA mutant background. Although we showed a dramatic increase in rsiV transcript abundance from the overexpression system, it is likely that the abundance and tolerance of overexpressing a membrane protein may be less tolerated than the expression of PgdA, which is predicted to be a secreted protein by Signal P 5.0. RsiV is known to possess two main functions in responding to lysozyme stress: (i) it regulates the availability of SigV to initiate transcription on SigV-dependent promoters through its anti-sigma factor activity, and (ii) it serves as a bacterial receptor for lysozyme (9). Our data clearly establish its regulatory function, as an rsiV mutant displayed a significant increase in sigV as well as pgdA expression. For its binding activity, we show that overexpression of RsiV decreases the amount of lysozyme bound to the cell surface, presumably through the process of regulated intramembrane proteolysis that occurs on RsiV upon lysozyme binding (9, 10, 14).

Figure 6 depicts the overall model by which the cell responds to exposure to lysozyme. Having shown that sigV rsiV and pgdA constitute the sole regulon of SigV, we propose that exposure to lysozyme leads to activation of SigV via a putative site 1 protease and Eep-mediated processing of the anti-sigma factor RsiV. We show that upon activation, SigV activates rsiV and pgdA expression, leading to increased levels of these proteins, and confers resistance to lysozyme by preventing both its enzymatic cleavage of peptidoglycan by the PgdA cell wall deacetylase activity and the likely contributions of both PgdA and RsiV toward the CAMP activity associated with lysozyme on the bacterial cell membrane. The contributions of the additional factors known to contribute to lysozyme resistance independent of the SigV regulon are also shown, including OatA, Dlt, and Epa. Overall, lysozyme resistance in E. faecalis appears to be multifactorial, but with SigV regulating the lion’s share of the response, and importantly, the SigV regulon comprises the key components (RsiV and PgdA) functionally induced upon lysozyme exposure.

FIG 6.

Model of SigV regulated lysozyme resistance. SigV regulates lysozyme resistance via two of its direct targets, RsiV and PgdA. 1. Lysozyme attacks the bacterial cell wall by cleaving the glycosidic linkage between N-acetylglucosamine and N-acetylmuramic acid. 2. Based on results seen in Fig. 4 and 5 and previous studies by Benachour et al. (17), bacterial cell wall deacetylation by PgdA protects the cells from lysozyme-mediated peptidoglycan degradation. 3. Upon exposure to lysozyme, the RsiV C-terminal domain is cleaved by a putative yet-unknown site 1 protease, as observed in Bacillus subtilis (42). 4. This cleaved RsiV is further processed via regulated intramembrane proteolysis (RIP) by a transmembrane-localized site 2 protease Eep (14). 5. The series of RsiV cleavage events, coupled with a putative cytoplasmic proteolytic processing as was observed in B. subtilis (43), is predicted to relieve SigV sequestration from the anti-sigma factor RsiV. 6. SigV as part of the RNA polymerase holoenzyme directs recruitment to the promoter elements of its regulon (sigV-rsiV and pgdA), leading to expression of target genes.

MATERIALS AND METHODS

Bacterial growth.

V583 parental and mutant strains (Table 2) were grown in LB broth containing 0.5% glucose (LBG) or LB agar for growth curve and lysozyme sensitivity assays, respectively. V583 strains were grown in Todd-Hewitt broth (THB) for all other assays and were grown in the presence of 500 μg/ml of spectinomycin for growth of strains containing pML28-derived overexpression plasmids (see Table in the supplemental material).

Construction of isogenic deletion mutants and overexpression strains.

To create KU01 (V583 ΔrsiV ΔpgdA), strain SV14 (V583 ΔrsiV) (14) was transformed with pVI15 (pLT06 derivative harboring the pgdA deletion construct) (14). To create EJH01 (V583 ΔsigV rsiV), strain V583 was transformed with pLEH300 (pLT06 derivative harboring the sigV rsiV deletion construct). To create EJH02 (V583 ΔsigV rsiV ΔpgdA), strain VI50 (V583 ΔpgdA) (14) was transformed with pLEH300. The process of plasmid integration and excision to yield the various deletion strains was followed as described previously (38), and the presence of the desired deletion was confirmed by either RsiVUp and RsiVDown primer pairs for the ΔrsiV ΔpgdA deletion or SigVUp and RsiVDown primer pairs for the ΔsigV rsiV and ΔsigV rsiV ΔpgdA deletions. Plasmid pLEH300 was constructed by amplifying an ∼1-kb region upstream of the sigV gene using primer pairs SigVP1 and SigVP2. A region ∼1 kb downstream of rsiV was amplified using primer pairs RsiVP3c and RsiVP4b. Each amplicon was digested with BamHI and then ligated to each other to create the sigV rsiV deletion construct. This ligated product was reamplified with SigVP1 and RsiVP4b. The resulting PCR product was digested with EcoRI and PstI and ligated into the markerless deletion vector pLT06, similarly digested with EcoRI and PstI restriction enzymes. This ligated product was electroporated into the Escherichia coli cloning host ElectroTen Blue and selected on LB agar containing 10 μg/ml of chloramphenicol. Positive clones were initially confirmed by PCR using primer pairs OriF and SeqR. Plasmid pLEH300 was purified and subjected to restriction digest analysis and confirmed by DNA sequencing.

To create the overexpression plasmid for pgdA, plasmid ML28 (23) was used to clone the pgdA complete open reading frame along with its upstream ribosome binding site generated by PCR using primers PgdA 5′ and PgdA 3′ (Table S3). The PCR amplicon was digested with BamHI and SalI and cloned into similarly digested pML28. The ligated product was electroporated into E. coli ElectroTen Blue cells and selected on LB agar containing 150 μg/ml of spectinomycin. The resulting plasmid was designated pKC05, confirmed by restriction digest analysis, and sequenced. Plasmids pKC05 (pgdA overexpression) and pSV17 (rsiV overexpression) along with the pML28 empty vector control were electroporated into the various strain backgrounds as described in Table 3.

TABLE 3.

Plasmid constructs used in this study

Plasmid	Description	Reference
pML28	pAT28 derivative containing the aph promoter	23
pKC05	pML28 derivative containing pgdA	This study
pSV17	pML28 derivative containing rsiV with 3× N-terminal FLAG tag	14
pVI15	pLT06 containing engineered pgdA deletion (∼2-kb EcoRI/PstI fragment)	14
pLEH300	pLT06 containing engineered sigV rsiV deletion (∼2-kb EcoRI/PstI fragment)	This study

Lysozyme MIC.

An MIC of lysozyme was determined as previously described (14) from a hen egg white lysozyme (Sigma-Aldrich) stock at 256 mg/ml in 10 mM Tris (pH 8.5), with final lysozyme concentrations ranging from 128 mg/ml to 0.5 mg/ml in LBG broth.

Growth curves in the presence of lysozyme.

To monitor growth kinetics in the presence of lysozyme, LBG broth containing either 2 mg/ml (0.5× the MIC for the ΔsigV and ΔrsiV ΔpgdA mutants) or 8 mg/ml (2× the MIC for the ΔsigV and ΔrsiV ΔpgdA mutants) of lysozyme for the V583, ΔsigV, ΔrsiV, ΔsigV rsiV, ΔpgdA, ΔrsiV ΔpgdA, and ΔsigV rsiV ΔpgdA strains as well as the ΔsigV and ΔrsiV ΔpgdA strains harboring the empty vector pML28, the pgdA overexpression (pKC05), and the rsiV overexpression (pSV17) was used. For strains harboring the spectinomycin resistance plasmids (pML28, pKC05, and pSV17), spectinomycin at 500 μg/ml was included in the growth medium to ensure plasmid maintenance. Growth was monitored as OD₆₀₀ every 15 min for 8 to 10 h at 37°C using an Infinite M200 Pro plate reader (Tecan Instruments).

Lysozyme sensitivity assay.

Lysozyme sensitivity assays were performed on LBG agar medium as previously described (15, 17), with slight modifications. Briefly, LB agar plates supplemented with 0.5% glucose alone or with lysozyme concentrations of 5 and 10 mg/ml were poured in a square petri plate. Overnight cultures of E. faecalis grown in LB agar plus 0.5% glucose were serially diluted 10-fold, and 5-μl volumes of the dilutions were plated. For strains harboring pML28 or its derivatives, spectinomycin was added at 500 μg/ml to ensure plasmid maintenance. Bacterial growth was monitored after 48 h of growth at 37°C and photographed.

Transcriptomics and analysis.

RNA isolation was performed as previously described (39). Briefly, V583 and derivative strains were grown overnight or to mid-exponential phase and pelleted, resuspended in TRIzol, and lysed using 0.1-mm zirconia beads. Insoluble cell debris was removed by centrifugation, and the clarified lysate was used for RNA isolation using the Direct-zol RNA miniprep plus kit from Zymo Research. Five micrograms of RNA was DNase treated using Ambion’s Turbo DNase kit and subsequently used for RNA-seq and qRT-PCR studies. gyrB was used as the endogenous control for qPCR analysis.

For lysozyme induction and RNA-seq studies, V583 and its isogenic ΔsigV mutant were grown from an overnight culture grown in Todd-Hewitt broth and serially diluted 1:100 into 50 ml of fresh THB medium and grown to an OD₆₀₀ of 0.5 to 0.6. Cultures were then split equally; one half was exposed to a 1-mg/ml final concentration of lysozyme and grown for an additional hour in the presence of lysozyme, and the other half of the culture was left unexposed and grown for an additional hour before harvesting of the cells for RNA isolation. Cells were pelleted and RNA isolation and DNase treatment were performed using the aforementioned protocol. The DNase-treated RNA was ribosomally depleted using the Ribo-zero magnetic kit for Gram-positive bacteria (Epicentre). Library preparation was performed by the University of Kansas Genome Sequencing core facility, and RNA was processed using the TruSeq RNA Library kit (Illumina) with indexing. RNA-seq was performed using an Illumina Hiseq2500 high-output system with a single-read run of 100 bp each.

Raw reads were preprocessed with Scythe version 0.991 (https://github.com/vsbuffalo/scythe) and Sickle version 1.2 (https://github.com/najoshi/sickle) to improve overall read quality. Then, the Tuxedo suite of programs (40) was used for mapping and differential expression analysis. Briefly, trimmed reads were mapped to the reference genome (Enterococcus faecalis V583 uid57669) using Tophat (version 2.0.10) with default options except for –max-intron-length, which was 10. Cuffdiff (version 2.1.1) was followed to identify differentially expressed (DE) genes using a set of 3,257 gene models. Gene-level expression values are represented by fragments per kilobase exon per million reads mapped (FPKM), and genes ranked with false-discovery rates (FDR) of <0.1 were reported as significant between each set of compared samples.

Chaining and cell settling assay.

Cultures of E. faecalis V583, mutant strains and strains containing overexpression plasmids (pML28-derived) were grown overnight in THB, and 5 μl of culture was fixed and simple stained with 1% crystal violet. Bacterial cells were imaged by light microscopy using the 60× objective on an EVOS FL Auto 2 microscope (Thermo Fisher Scientific). Cell settling assays were performed as described previously (14). Relative chaining index (RCI) based on the observed cell settling phenotype was calculated as

$$\text{RCI}=1\hspace{0.17em}-\hspace{0.17em}\frac{{\text{OD}}_{\text{600}}\text{\hspace{0.17em}strain}}{{\text{OD}}_{\text{600}}\text{\hspace{0.17em}V583}}$$

CBB staining of whole-cell lysates and supernatants.

Coomassie brilliant blue (CBB) staining of whole-cell lysates was performed as previously described (14). Briefly, V583 parental strain, the ΔsigV strain, and the ΔsigV strain with the pgdA and rsiV overexpression plasmids were grown in 25 ml of THB to an OD₆₀₀ of 0.7 to 0.8. Lysozyme was added at a concentration of 1 mg/ml and the strains were grown for 2 h. Lysates were prepared and protein concentration was estimated using Bradford assay with Coomassie protein assay reagent. Cell lysates were normalized to total protein, equal amounts were loaded onto 16% SDS-PAGE gels, and samples were run using 1× Tris-Tricine rich cathode buffer (prepared from 10× buffer containing 121.1 g of Tris base, 179.2 g of Tricine, and 100 ml of 10% SDS set to pH 8.3 in a 1-liter final volume made with deionized water) and a Tris-based anode buffer (0.2 M Tris-HCl buffer [pH 8.8]). Supernatants were similarly prepared and were loaded onto 16% SDS-PAGE and run under similar conditions as lysates. Gels were stained using CBB stain, and the lysozyme band intensities were quantified using the Gel-plot densitometry program of ImageJ software. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple-comparison posttest.

Data availability.

The RNA-seq data are deposited in the Sequence Read Archive at NCBI under accession no. SRX9565353, SRX9565354, SRX9565355, SRX9565356, SRX9565357, and SRX9565358. The BioProject accession number is PRJNA679977.