Articles

Functional Dissection of the CroRS Two-Component System Required for Resistance to Cell Wall Stressors in Enterococcus faecalis

Stephanie L. Kellogg, Christopher J. Kristich
T. J. Silhavy, Editor
DOI: 10.1128/JB.00995-15

ABSTRACT

Bacteria use two-component signal transduction systems (TCSs) to sense and respond to environmental changes via a conserved phosphorelay between a sensor histidine kinase and its cognate response regulator. The opportunistic pathogen Enterococcus faecalis utilizes a TCS comprised of the histidine kinase CroS and the response regulator CroR to mediate resistance to cell wall stresses such as cephalosporin antibiotics, but the molecular details by which CroRS promotes cephalosporin resistance have not been elucidated. Here, we analyzed mutants of E. faecalis carrying substitutions in CroR and CroS to demonstrate that phosphorylated CroR drives resistance to cephalosporins, and that CroS exhibits kinase and phosphatase activities to control the level of CroR phosphorylation in vivo. Deletion of croS in various lineages of E. faecalis revealed a CroS-independent mechanism for CroR phosphorylation and led to the identification of a noncognate histidine kinase capable of influencing CroR (encoded by OG1RF_12162; here called cisS). Further analysis of this TCS network revealed that both systems respond to cell wall stress.

IMPORTANCE TCSs allow bacteria to sense and respond to many different environmental conditions. The opportunistic pathogen Enterococcus faecalis utilizes the CroRS TCS to mediate resistance to cell wall stresses, including clinically relevant antibiotics such as cephalosporins and glycopeptides. In this study, we use genetic and biochemical means to investigate the relationship between CroRS signaling and cephalosporin resistance in E. faecalis cells. Through this, we uncovered a signaling network formed between the CroRS TCS and a previously uncharacterized TCS that also responds to cell wall stress. This study provides mechanistic insights into CroRS signaling and cephalosporin resistance in E. faecalis.

INTRODUCTION

Although Enterococcus faecalis is a normal commensal of the gut microbiome, it poses a serious risk to humans as an opportunistic pathogen. A major risk factor for the pathogenic transition of enterococci is therapy with broad-spectrum cephalosporin antibiotics. Cephalosporins are commonly used to treat bacterial infections; however, nearly all enterococcal isolates are resistant to these compounds. This intrinsic resistance allows for the expansion of resident enterococcal populations during cephalosporin treatment, which is thought to promote dissemination from the gastrointestinal tract, enabling enterococci to establish infections (1, 2). With the emergence of multidrug-resistant isolates of E. faecalis, few options for treating infections are available. Therefore, understanding the mechanisms that support cephalosporin resistance in E. faecalis is crucial for designing new therapies that limit growth during cephalosporin treatment or that can be used as adjuvants with cephalosporins to treat E. faecalis infections. An essential determinant of cephalosporin resistance in E. faecalis is the CroRS two-component system (TCS), consisting of CroS (a transmembrane sensor-histidine kinase) and CroR (an OmpR-family response regulator) (3–5).

TCSs allow organisms to adapt to changing environments using a highly conserved phosphoryl relay between cognate histidine kinase (HK) and response regulator (RR) pairs. Upon sensing a signal, HKs autophosphorylate a conserved histidine residue which participates in phosphoryl transfer to a conserved aspartate residue on the cognate RR, resulting in the activation of the signaling system. Many RRs, including CroR, have DNA binding capabilities and elicit a biological response in the form of gene regulation. Many HKs are thought to be bifunctional and can facilitate dephosphorylation of the RR through the action of a conserved threonine or asparagine residue located near the phosphoryl-accepting His (6–10). Although phosphoryl transfer from CroS to CroR has been observed in vitro (3), the extent or importance of CroS phosphatase activity remains unclear. The biological role of the CroRS TCS (originally described as HK05/RR05 [4]) has been examined in E. faecalis JH2-2, a laboratory strain whose parent originally was isolated from a patient in the 1970s (3, 11, 12), and in a vancomycin-resistant clinical isolate, V583 (5, 13). Studies of JH2-2 demonstrated that a mutant lacking croRS exhibits growth and cell morphology defects and is more susceptible to ampicillin and ceftriaxone than the parent JH2-2 strain. A variety of cell wall-targeting antimicrobial agents are capable of stimulating transcription from the autoregulated croR promoter in a CroR-dependent manner, leading to the hypothesis that the CroRS TCS responds to cell wall stress. Studies of V583 demonstrated that the absence of CroR renders V583 more sensitive to broad-spectrum cephalosporins, vancomycin, and bacitracin. However, changes in the morphology of the V583 croR mutant were not described. Although CroRS signaling has been proposed to respond to and elicit the repair of cell wall damage (3, 14), molecular details of the output of CroRS signaling remain a mystery. CroR is capable of gene regulation, but thus far only three CroR-dependent genes have been described. These include croR itself, a gene encoding a secreted protein (salB), and the first gene of a predicted glutamine transport system (glnQ), although only croR plays a role in cephalosporin resistance (3, 15, 16). Therefore, although CroS and CroR seem to comprise a prototypical TCS involved in the response to cell wall stress, little is known about the CroRS-dependent molecular events that promote resistance to cell wall-targeting agents in E. faecalis.

Because TCS signaling uses a highly conserved phosphoryl relay mechanism for signal propagation, and given that most prokaryotic organisms have multiple TCS loci within their genome, mechanisms to ensure signaling fidelity between cognate HK/RR pairs are essential to maintain specificity in stimulus-response coupling. The intervention of one TCS with the signal transduction of another, termed cross talk between TCSs, can be minimized through the phosphatase activity of HKs, through temporal/spatial regulation, and through specific molecular recognition between cognate HKs and RRs (17). The latter has been extensively explored, with specificity between cognate HK/RR pairs attributed to specific coevolving residues that dictate if a phosphoryl transfer event can occur efficiently (18–20). With this molecular specificity, an HK can use its kinase and phosphatase functions to control the level of phosphorylated cognate RR without interfering with the signaling of other HK/RR pairs. Although these mechanisms are thought to minimize cross talk between TCSs, a few examples of cross talk have been reported. Most studies describe a scenario in which a noncognate HK constitutively phosphorylates an RR in the absence of the cognate kinase (21–26). This accumulation of phosphorylated RR due to the activity of a noncognate kinase often is attributed to the absence of phosphatase activity of the cognate HK, which normally acts to maintain the RR in the unphosphorylated state in the absence of a specific stimulus. Few examples exist where an HK influences a noncognate RR in response to the HK's natural signal, representing what is thought of as genuine cross-regulation between TCSs, which presumably offers an adaptive advantage for the organism (27–30).

In this study, we probed the mechanism of CroRS signal transduction to understand how it promotes cephalosporin resistance in E. faecalis. We report that the phosphorylation state of CroR influences the magnitude of cephalosporin resistance and that CroS has an important role in regulating CroR in vivo. Through these studies we unexpectedly discovered a TCS network formed between CroRS and a previously uncharacterized TCS in E. faecalis (referred to here as CisRS, for CroRS interacting system).

MATERIALS AND METHODS

Bacterial strains, growth media, and chemicals.Bacterial strains and plasmids used in the study are listed in Table 1. E. faecalis strains were grown in half-strength brain heart infusion (hBHI) medium for routine maintenance. Escherichia coli strains were grown in lysogeny broth (LB) or hBHI. Erythromycin was used at 100 μg/ml (E. coli) and 10 μg/ml (E. faecalis) in hBHI, and chloramphenicol was used at 10 μg/ml (E. coli and E. faecalis). All cultures were grown aerobically with shaking.

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TABLE 1

Strains and plasmids used in this work

Plasmid construction.The plasmid pJRG8 (35) was used to express various alleles of croRS or cisR in E. faecalis cells. For each croRS allele, the P23s promoter and multiple-cloning site of pJRG8 was replaced with the promoter region of the croRS locus and the croRS genes (3). The expression of cisR with a C-terminal hemagglutinin (HA) epitope tag was achieved using the P23s promoter of pJRG8. All plasmids were constructed using a BsaI-based seamless cloning strategy with primer-encoded restriction sites or Gibson assembly (38).

Construction of E. faecalis mutants.In-frame deletion mutants in various E. faecalis lineages were constructed using markerless allelic exchange as previously described (36, 37). Mutant alleles were constructed and introduced into pCJK218 or pCJK245 using either a BsaI-based seamless cloning strategy with primer-encoded restriction sites or Gibson assembly (38). Each deletion allele retains codons at the 5′ and 3′ ends of the gene in an attempt to avoid perturbing the expression of adjacent genes.

Antibiotic susceptibility determinations and growth curves.The MICs of antibiotics were determined in aerobic liquid cultures using a microtiter plate serial dilution method in a Bioscreen C plate reader (Oy Growth Curves Ab, Ltd.). Twofold serial dilutions of antibiotics in MHB (supplemented with 10 μg/ml erythromycin for plasmid selection when necessary) were prepared in the wells of a 100-well honeycomb microtiter plate. Bacteria from stationary-phase cultures in MHB (plus 10 μg/ml erythromycin for plasmid-carrying strains) were inoculated into each well to a concentration of ∼105 CFU/ml. Plates were incubated at 37°C for 24 h with brief shaking and measurement of optical density at 600 nm (OD600) at 15-min intervals. The lowest concentration of antibiotic that prevented growth was recorded as the MIC.

Phos-tag SDS-PAGE and immunoblotting.Stationary-phase cultures of E. faecalis strains were diluted in fresh MHB (supplemented with 10 μg/ml erythromycin for plasmid-containing strains) and grown to exponential phase at 37°C and 225 rpm (OD600 of approximately 0.2). Vancomycin (3 μg/ml) was added to stimulate signaling for 30 min. Bacteria were collected by centrifugation after mixing with an equal volume of cold ethanol-acetone (1:1) mixture to rapidly kill the bacteria and prevent any further signaling events. Pellets were washed with water and normalized based on the OD600 before lysozyme treatment and lysis with 5× SDS Laemmli sample buffer. Without boiling, samples were loaded on 10% SDS-PAGE gels with or without 20 μM Phos-tag, 40 μM MnCl2 (for CroR) or 25 μM Phos-tag, 50 μM MnCl2 (for CisR-HA). Gels were run at 4°C and 200 V in Laemmli's buffer system until the dye front reached the bottom of the gel. After electrophoresis, gels were soaked in a 5 mM EDTA solution before transferring to polyvinylidene difluoride (PVDF) membrane using a Bio-Rad semidry transfer apparatus. Blocking was done using 5% milk in Tris-buffered saline. CroR was detected using custom rabbit polyclonal antiserum, and CisR-HA was detected using a polyclonal rabbit anti-HA antibody (Abcam). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody was used for detection (Invitrogen). Descriptions by Kinoshita et al. and Barbieri and Stock were used to optimize Phos-tag SDS-PAGE for CroR and CisR (39, 40). Acrylamide-pendant Phos-tag was obtained from Wako Chemicals.

Analysis of gene expression by quantitative reverse transcription-PCR (qRT-PCR).For gene expression analyses, a minimum of three independent cultures were analyzed for each E. faecalis strain of interest. E. faecalis strains were grown in MHB and collected before and after exposure to 3 μg/ml vancomycin for 60 min during exponential growth. Bacteria were collected after mixing with an equal volume of cold ethanol-acetone (as described above) and RNA extracted using a total RNA minikit (Midsci). DNase Turbo was used to remove any carryover DNA, and cDNA was made using SuperScript III first-strand synthesis SuperMix (Invitrogen). A Bio-Rad iCycler and SsoAdvanced SYBR green supermix (Bio-Rad) were used to obtain amplification and melting curves. Primer efficiencies were determined using serial dilutions of E. faecalis genomic DNA. Calculations of fold changes in gene expression used the Pfaffl method and gyrB as a reference gene.

RESULTS

Altering the phosphorylation state of CroR influences cephalosporin resistance.The CroRS two-component system is required for cephalosporin resistance in E. faecalis, but the mechanism of signal transduction has not been probed in vivo. To monitor signaling through CroR, Phos-tag SDS-PAGE was optimized to separate phosphorylated CroR (CroR-P) from nonphosphorylated CroR, as has been done for other RRs (40–44). Figure 1A demonstrates that two species of CroR can be detected after Phos-tag SDS-PAGE when a strain carrying wild-type (WT) CroRS is treated with vancomycin, a known stimulant of CroRS activity (lanes 2 to 3). The more slowly migrating species represents CroR-P, and this species disappears upon boiling of the samples (not shown), consistent with the known lability of phosphoryl-Asp linkages. Moreover, standard SDS-PAGE confirms that this upper species is dependent on separation by Phos-tag (Fig. 1A) and that our constructs for expressing CroRS variants produce levels of CroR similar to that of the wild-type OG1 strain (Fig. 1B).

FIG 1
FIG 1

Analysis of CroR phosphorylation by Phos-tag SDS-PAGE. (A and C) Phos-tag and standard SDS-PAGE analysis of the OG1 ΔcroRS mutant carrying various alleles of croRS (indicated). Whole-cell lysates were collected with (+) or without (−) exposure to vancomycin. (B) Whole-cell lysates subjected to standard SDS-PAGE and immunoblotted for CroR using wild-type E. faecalis (OG1) versus our expression system (OG1 ΔcroRS mutant with ectopic expression of croRS under its native promoter). A subunit of RNA polymerase (RpoA) was used as a loading control. Results are representative of ≥2 experiments.

To understand how the flow of phosphoryl groups from CroS to CroR influences cephalosporin resistance, we constructed mutant strains of E. faecalis OG1 (a laboratory strain originally isolated from a human oral cavity in the 1970s [31]) expressing variants of CroRS with substitutions predicted to alter signal transduction capabilities. To determine if the phosphorylation of CroR is required for cephalosporin resistance, we analyzed a variant of CroR with a substitution at the predicted phosphoryl-accepting Asp (D52A). Immunoblot analysis revealed that wild-type CroR and the CroR mutant are expressed at comparable levels (Fig. 1B). However, Phos-tag SDS-PAGE analysis revealed that the CroR D52A variant does not fractionate as two species in response to vancomycin, indicating that CroR D52A cannot be phosphorylated in vivo (Fig. 1A, lanes 4 and 5). Also in contrast to wild-type CroR, the nonphosphorylatable variant could not provide resistance to the cephalosporin ceftriaxone, yielding a level of resistance mimicking that observed in the absence of CroR (Table 2). Thus, CroR-P is generated in response to cell envelope stress and is required to promote cephalosporin resistance.

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TABLE 2

Complementation of E. faecalis OG1 ΔcroRS and ΔcroRS ΔcisRS mutants with alleles of croRS predicted to be impaired in signal transduction

To further establish the correlation between CroR phosphorylation and cephalosporin resistance, we assessed resistance after genetically increasing the level of CroR-P. To achieve this, we impaired the presumed phosphatase activity of CroS. Many histidine kinases are bifunctional and can facilitate dephosphorylation of their cognate RR through the action of a conserved threonine or asparagine residue located near the phosphoryl-accepting His (T176 in CroS). Phos-tag analysis of a mutant strain carrying a croS allele with substitution T176A (croS T176A) revealed a constitutive population of phosphorylated CroR dependent on CroR D52, suggesting that CroS exhibits phosphatase activity in vivo (Fig. 1A, lanes 6 to 9). The croS T176A mutant strain also exhibited dramatic hyperresistance to ceftriaxone, again dependent on CroR D52 (Table 2). Further, this strain exhibited a CroR-P-dependent growth defect compared to wild-type and ΔcroRS strains (Fig. 2). These data indicate that elevated levels of CroR-P drive enhanced cephalosporin resistance and support the hypothesis that CroS exhibits important phosphatase activity to regulate the amount of phosphorylated CroR in vivo.

FIG 2
FIG 2

Absence of CroS phosphatase activity results in a CroR-P-dependent growth defect. Growth curves of unstressed E. faecalis strains carrying different alleles of croRS: ○, wild-type plus empty vector; ▼, ΔcroRS plus empty vector; ▽, ΔcroRS plus croRS; ◆, ΔcroRS plus croR D52A croS T176A; ♢, ΔcroRS plus croR croS T176A. Curves are representative of ≥3 experiments.

To probe the contribution of CroS kinase activity toward CroR phosphorylation and cephalosporin resistance, we introduced substitutions at the conserved histidine predicted to be the site of autophosphorylation (H172) or the adjacent acidic residue (D173) which is critical for HK autophosphorylation in other TCS kinases (9, 10, 45). Phos-tag analysis demonstrated that the CroS H172A variant resulted in some CroR-P that can be detected in a culture without CroRS stimulation, while the CroS D173A variant cannot produce CroR-P even upon stimulation of CroS (Fig. 1C, lanes 4 to 7). Cephalosporin resistance provided by these variants correlated with the CroR-P profile: expression of croS H172A resulted in hyperresistance compared to resistance of the wild type, while croS D173A did not provide cephalosporin resistance (Table 2). These opposing phenotypes suggest that the H172A and D173A substitutions affect CroS activity differently. Previous studies with other HKs reported that substitutions at the phosphoryl-accepting His not only abolished phosphoryl transfer but also impaired phosphatase activity (10, 46–49), which likely explains our results (discussed further below). Our attempt to test this in vitro was unsuccessful, as we were unable to obtain soluble truncations of CroS that were catalytically active on CroR. Nevertheless, the observation that CroR-P is present when CroS is unable to serve as a phosphoryl donor (CroS H172A mutant) led us to explore alternative mechanisms for CroR phosphorylation.

Some E. faecalis lineages possess a CroS-independent mechanism for CroR phosphorylation.To explore CroS-independent phosphorylation of CroR, we introduced an in-frame deletion of croS into two distantly related lineages of E. faecalis: OG1 and T1 (isolated prior to 1950 from an unknown source [33]). As observed with the phosphatase-defective CroS mutant, deletion of croS in both of these lineages resulted in a population of CroR-P that could be detected from a culture in the absence of CroRS stimulants (Fig. 3). Because the CroRS TCS is known to autoregulate its own expression (3), we verified that deletion of croS results in an increase in CroR signaling by monitoring croR transcription. In the OG1 lineage, deletion of croS resulted in a 20-fold increase in croR transcription compared to that of the wild type (Fig. 4A). This increase in CroR signaling in the absence of CroS correlates with the croS mutants being hyperresistant to ceftriaxone compared to the respective wild-type strain (Table 3). However, elevated ceftriaxone resistance is not the phenotype described by Comenge et al. for a croS deletion in E. faecalis JH2-2, in which the croS deletion mutant was found to be much less resistant to ceftriaxone than JH2-2 (3). We introduced our deletion allele of croS into E. faecalis JH2 (the parent strain of JH2-2) and into a fourth distinct lineage, E. faecalis CK221 (an erythromycin-sensitive variant of V583). In contrast to our results with E. faecalis OG1 and T1, mutants of both of the latter lineages display reduced resistance to ceftriaxone upon deletion of croS (Table 3). Moreover, Phos-tag analysis revealed that CroR-P is not present in these mutants (Fig. 3), and expression of croS complemented these phenotypes (Table 4). These results indicate that some lineages of E. faecalis possess CroS-independent phosphoryl donors for CroR.

FIG 3
FIG 3

CroR-P is present in croS deletion mutants of some E. faecalis lineages. Shown is Phos-tag SDS-PAGE and immunoblot analysis of CroR from whole-cell lysates of wild-type and ΔcroS (Δ) strains of E. faecalis in the absence of CroRS stimulation. Results are representative of >2 experiments.

FIG 4
FIG 4

Analysis of gene expression in E. faecalis OG1 and cro-cis mutants. (A) croR gene expression in wild-type OG1 versus mutant derivatives in the absence of CroRS stimulation. (B) Expression of croR, croS, and OG1RF_12161 in response to vancomycin treatment. Error bars represent standard deviations from a minimum of three independent cultures analyzed in triplicate.

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TABLE 3

Cephalosporin resistance of WT strains or isogenic ΔcroS mutants of different E. faecalis lineages

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TABLE 4

Complementation of E. faecalis OG1 and CK221 ΔcroS deletion mutants through ectopic expression of croS

To identify potential CroR phosphoryl donors present in OG1 and T1 but absent from JH2 and CK221, we focused on histidine kinases. Cross talk at the level of phosphoryl transfer has been described in other TCSs and can be predicted based on the identity of specific coevolving residues found in HKs and RRs (18–20). Comparison of the repertoire of histidine kinases encoded by the E. faecalis lineages OG1RF (a nearly isogenic derivative of OG1 whose genome has been sequenced), T1, V583 (the parent of CK221), and JH2-2 (a derivative of JH2 whose genome has been sequenced) reveal that one HK in particular is present in OG1RF and T1, absent from V583 and JH2-2, and shares many coevolving residues with CroS: OG1RF_12162 (renamed CisS for reasons described below). Figure 5 shows an alignment of the dimerization and histidine phosphoryl transfer (DHp) domains of a subset of HKs found in OG1RF along with the genomic architecture of the cisRS TCS. The specificity residues of CisS exhibit the most commonality with CroS, sharing 6 out of the 9 residues implicated in dictating RR specificity. Although another HK, OG1RF_10260, also exhibits some commonality with CroS specificity residues (5 of 9 residues are identical), an ortholog of OG1RF_10260 is found in V583 (EF0373), where we do not observe CroS-independent phosphorylation of CroR. Therefore, we focused on the HK encoded by cisS found in both OG1 and T1 strains of E. faecalis as the most likely candidate for cross talk with CroR.

FIG 5
FIG 5

(A) Clustal W alignment of DHp domains from group III histidine kinases in E. faecalis OG1RF. The sequence of EnvZ from E. coli is provided as a reference for the described specificity residues of HK/RR pairs. The autophosphorylated His is highlighted in light gray, and specificity residues identical to CroS in E. faecalis HKs are highlighted in dark gray. (B) Genomic architecture of the cisRS locus. A three-gene cluster encoding the response regulator CisR (OG1RF_12163), the histidine kinase CisS (OG1RF_12162), and a putative carboxypeptidase (OG1RF_12161) is found in E. faecalis OG1 and T1 but not V583. Homologs of the genes flanking this cluster, OG1RF_12164 and OG1RF_12160, are found in V583 (EF2860 and EF2859, respectively). No obvious features associated with mobile genetic elements are associated with the 3-gene cluster.

Noncognate histidine kinase promotes CroR phosphorylation in the absence of CroS.To determine if CisS is involved in the phosphorylation of CroR in the absence of CroS, we constructed double mutants lacking both croS and cisS and monitored CroR signaling using Phos-tag SDS-PAGE and qRT-PCR. In contrast to the ΔcroS single mutant, the double mutants in either the OG1 or T1 lineage did not exhibit detectable CroR-P in the absence of CroRS stimulants and could not respond to vancomycin (OG1 data are shown in Fig. 6). The analysis of gene expression also indicated that the activation of CroR in the absence of CroS depends on CisS, as the double HK mutant lacked increased croR transcription in the absence of CroRS stimulants (Fig. 4A). Furthermore, ceftriaxone hyperresistance of the croS mutant also is lost upon the deletion of CisS (Table 5). The lack of CroR signaling and cephalosporin resistance in a croS cisS double mutant indicates that CroS and CisS collectively represent the only phosphoryl donors for CroR under these conditions. Moreover, the double HK mutant also exhibited a loss of resistance to vancomycin and bacitracin that mimics a mutant lacking croR (Table 5). Thus, in the absence of CroS, CisS is responsible for constitutive phosphorylation of CroR that promotes high-level cephalosporin resistance, prompting us to rename the OG1RF_ 12162/3 TCS CisRS (for CroRS interacting system). These findings explain the resistance phenotypes of mutants lacking croS in different E. faecalis lineages; cephalosporin hyperresistance only occurs in lineages carrying CisS.

FIG 6
FIG 6

CisS promotes phosphorylation of CroR in the absence of CroS. Phos-tag SDS-PAGE and immunoblot analysis of whole-cell lysates from E. faecalis OG1 derivatives carrying different combinations of CroS/CisS in the presence (+) or absence (−) of vancomycin. A subunit of RNA polymerase (RpoA) was used as a loading control. Results are representative of ≥2 experiments.

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TABLE 5

Antibiotic resistance for E. faecalis OG1 (wild type) and mutant derivatives

To determine if CisRS is sufficient for the constitutive phosphorylation of CroR in the absence of CroS, we attempted to express CisRS from E. faecalis OG1 in ΔcroS mutants of the 2 lineages lacking cisRS (V583 and JH2). Although we obtained transformants carrying the empty vector control, in both cases we were unable to obtain transformants carrying the cisRS expression plasmid. We infer that CisS-mediated phosphorylation of CroR leads to marked growth inhibition of such transformants (consistent with the findings shown in Fig. 2) that prevents their recovery, suggesting that CisS indeed is sufficient for CroR phosphorylation in vivo in the absence of CroS.

To determine if the CisRS TCS influences the output of the CroRS TCS in cells containing functional CroS, we first assessed the contribution of CisS to cephalosporin resistance. Deletion of CisS or the entire CisRS TCS did not alter ceftriaxone resistance of E. faecalis OG1 or T1 (OG1 data are shown in Table 5), indicating that the CisRS TCS does not influence CroR-dependent signaling when CroS is present in a way that contributes to the output of cephalosporin resistance. Consistent with this, the expression of CisRS from E. faecalis OG1 in the V583 and JH2 lineages did not alter cephalosporin resistance levels of those hosts. Moreover, the absence of CisS did not impair the production of CroR-P in response to vancomycin treatment (Fig. 6). Because the CroRS TCS may have other as-yet-unknown phenotypic outputs, we also quantified CroR-mediated signal transduction using qRT-PCR to monitor croRS gene expression. Vancomycin induced substantial increases in croR and croS transcription in wild-type E. faecalis OG1 in a CroS- and CroR-dependent manner (Fig. 4B). Vancomycin still induced a robust transcriptional response in mutants lacking CisS or the entire CisRS TCS, indicating that CisS is not required for vancomycin-induced signaling through the CroRS TCS. In fact, deletion of cisS or cisRS resulted in a 4- to 5-fold increase in croR transcription in response to vancomycin beyond that observed in the wild type, indicating that the CisRS TCS attenuates CroRS signaling in the presence of vancomycin.

CroS exhibits phosphatase activity toward CroR in vivo.Substitution of CroS T176 resulted in a large pool of CroR-P during growth in the absence of CroRS stimuli and dramatic hyperresistance to ceftriaxone (Fig. 1A and Table 2). Because CisS contributes to CroR phosphorylation in the absence of CroS, we asked if CisS was responsible for the CroR-P accumulation in the strain carrying the phosphatase-defective (but predicted kinase-competent) CroS T176A variant by expressing croS T176A in an E. faecalis host lacking the CisRS TCS. Hyper-resistance to ceftriaxone still was observed and required CroS H172 (Table 2), suggesting that CroS modulates the CroR-P pool through both kinase and phosphatase activities in vivo. Analysis of additional CroS mutants is consistent with this hypothesis. For example, the expression of the CroS H172A mutant resulted in a population of CroR-P in the absence of stimulation and rendered the strain hyperresistant to ceftriaxone. However, when expressed in the CisRS deletion background, hyperresistance to ceftriaxone was no longer observed (Table 2). This supports the hypothesis that substitution of the conserved phosphoryl-accepting His of CroS (H172) impairs CroS phosphatase activity, allowing CisS to drive the phosphorylation of CroR, leading to cephalosporin hyperresistance. Analysis of the CroS D173A variant also is consistent with the hypothesis that CroS phosphatase activity is essential for controlling cross talk with CisS, as we did not observe CroR-P or hyperresistance in a strain carrying this CroS variant, which is expected to retain wild-type phosphatase activity (Fig. 1A and Table 2). These results indicate that CroS exhibits both kinase and phosphatase activities toward CroR in vivo and that both activities are critical for proper regulation of CroR phosphorylation.

CroS influences the CisR response regulator.The data described above support a model in which CisS constitutively phosphorylates CroR in the absence of CroS. However, because the specificity of phosphoryl transfer between HK/RR pairs is dictated by specific coevolving residues, we reasoned that if the commonality between these residues of CroS and CisS was sufficient to allow phosphoryl transfer from CisS to CroR, the shared-specificity residues also may permit phosphoryl transfer from CroS to CisR. Therefore, we monitored CisRS signaling in the presence and absence of CroRS to determine if reciprocity in cross talk would be observed.

Although a deletion mutant of the CisRS TCS was screened for defects in growth and altered resistance to various stressors, no significant differences between the mutant and isogenic wild-type strains were observed, offering no insight into the output of this system. However, because genes encoding TCSs often are colocalized with members of their target regulon, we investigated the expression of the adjacent gene (OG1RF_12161, encoding a putative carboxypeptidase) located immediately downstream of the CisRS TCS as a readout for CisRS signaling. In response to vancomycin, wild-type E. faecalis OG1 induced a 160-fold increase in transcription of OG1RF_12161 (Fig. 4B). However, rather than being a strictly Cis-mediated response, vancomycin-induced OG1RF_12161 transcription results from a combined effect of the CroRS and CisRS systems. Deletion of either TCS attenuated vancomycin-induced OG1RF_12161 transcription 3- to 6-fold compared to that of the wild-type response, and deletion of both HKs or TCSs led to a 35-fold decrease in transcription compared to that of the wild type, indicating that the CroRS and CisRS TCSs both contribute to OG1RF_12161 regulation under these conditions. When CisS alone was deleted, an increase in OG1RF_12161 transcription in response to vancomycin was observed and was dependent on CroS. This indicates not only that both the CroRS and CisRS TCSs are contributing to OG1RF_12161 regulation but also that CroS is a strong driver of OG1RF_12161 expression in response to vancomycin and that CisS attenuates CroS signaling.

To determine if CroS mediates the phosphorylation of the noncognate RR CisR, we constitutively expressed an HA-tagged version of CisR in E. faecalis cells and monitored phosphorylation using Phos-tag SDS-PAGE. Upon stimulation with vancomycin, two species of CisR were detected from wild-type cells (Fig. 7), indicative of CisR phosphorylation. The production of the reduced-mobility isoform requires the predicted phosphoryl-accepting Asp (D53), indicating that the reduced-mobility isoform results from CisR phosphorylation. The production of phosphorylated CisR upon vancomycin stimulation is not completely dependent on its presumed cognate HK, CisS, but also requires CroS. Thus, CroS is capable of influencing the phosphorylation state of not only its cognate RR, CroR, in a stimulus-dependent manner but also a noncognate RR, CisR, in E. faecalis cells.

FIG 7
FIG 7

CroS promotes CisR phosphorylation in response to vancomycin. Phos-tag SDS-PAGE and immunoblot analysis of CisR phosphorylation in different E. faecalis (EF) OG1 mutants. CisR is expressed from the P23s promoter of pJRG8 with a C-terminal HA tag. A subunit of RNA polymerase (RpoA) was used as a loading control. Results are representative of ≥2 experiments.

DISCUSSION

Past studies have used the JH2-2 and V583 lineages of E. faecalis to reveal that mutants lacking the CroRS TCS exhibit growth and cell morphology defects (11) and are more sensitive to certain cell wall-targeting antibiotics, such as β-lactams, bacitracin, and vancomycin (3, 5). However, these studies used gene knockouts and did not explicitly examine how signaling through CroRS influences resistance. Here, we investigated how the flow of phosphoryl groups between CroS and CroR promotes antibiotic resistance using the well-studied OG1 lineage of E. faecalis as a model. Through these studies we unexpectedly uncovered a TCS signaling network involving CroRS and a previously uncharacterized TCS encoded by the OG1 genome (50).

Our investigation of the relationship between phosphorylation of CroR and cephalosporin resistance demonstrated that CroR-P drives cephalosporin resistance in E. faecalis. Because the expression of the CroS T176A variant (or deletion of croS) led to an accumulation of CroR-P and dramatic hyperresistance to ceftriaxone compared to that of the wild type (in the OG1/T1 lineages), cephalosporins themselves do not stimulate the CroRS system to its maximum capacity under our standard conditions. In fact, we were unable to find conditions under which ceftriaxone exposure led to the robust production of CroR-P in E. faecalis OG1; thus, we used vancomycin to stimulate signal transduction. Because a variety of cell wall-targeting agents with different modes of action can stimulate CroRS signaling, the input for CroS and output of CroR are thought to be involved in cell wall homeostasis (3, 14). Specifically, the output of CroR has been proposed to be related to the function of penicillin binding proteins, which mediate resistance to β-lactam antibiotics (3). Our data support this notion, as strains with elevated CroR-P are selectively more resistant to cephalosporin antibiotics. However, this potential benefit is not without a fitness cost: hyperproduction of CroR-P also leads to impaired growth (Fig. 2), providing an evolutionary rationale for careful regulation of CroR phosphorylation.

Identification of the phosphoryl donor for CroR in the absence of CroS revealed a TCS network formed between CroRS and CisRS in some lineages of E. faecalis. To assess the phylogenetic distribution of CisRS, we used BLAST to interrogate the genomes of 18 diverse commensal and clinical E. faecalis isolates for the presence of cisRS. These 18 genomes were previously analyzed in detail by Palmer et al. (51) and represent deep nodes in the E. faecalis phylogeny to provide the greatest diversity. Although croRS is universally present in all 18 genomes, cisRS was found in only 6 of these 18 genomes, distributed sporadically across the phylogenetic tree. The 6 cisRS-containing strains were isolated at various times over the course of >50 years, and no obvious correlations exist between the presence of cisRS and type of isolate (commensal versus clinical), multilocus sequence type, or antibiotic resistance profiles. Therefore, we are uncertain how the CisRS TCS contributes to the biology of E. faecalis as it is not functionally redundant to CroRS, is not ubiquitous in enterococci, and does not significantly alter the biology of OG1 or T1 under the conditions we have examined. However, it is apparent that CisRS interferes with other TCSs when cognate components are missing. CisR was recently implicated in cross talk in another E. faecalis lineage which carries a VanG gene cluster controlled by the VanRSG TCS for vancomycin resistance. In this E. faecalis isolate, CisR was found to promote the production of the VanTG racemase in the absence of VanRG, a process which requires the HK VanSG and vancomycin (52). Thus, it remains unclear if CisRS cross talk is relevant to the biology of wild-type E. faecalis or if this phenomenon only manifests when cognate HK-RR pairs are disrupted.

Is cross talk between CisS and CroR physiologically relevant in wild-type cells? Our results argue that such cross-regulation is not an important feature of this signaling network. For example, CisS-driven CroR phosphorylation is observed only in mutants in which the phosphatase activity of CroS is impaired (ΔcroS and croS H172A) (Fig. 1 and 2). Moreover, CroR-dependent antibiotic resistance is normal in a mutant lacking CisS but containing the cognate HK CroS, and antibiotic resistance of mutants lacking both CroRS and CisRS are no more impaired than a mutant lacking CroRS only (Table 5). Conversely, a mutant in which CroS is phosphatase competent but kinase impaired (croS D173A) that contains wild-type CisS does not generate CroR-P (Fig. 1C) and lacks CroR-dependent ceftriaxone resistance (Table 2), indicating that CisS cannot activate CroR-mediated signaling. Collectively, these results indicate that the CisS-mediated phosphorylation of CroR is not physiologically relevant but likely is enabled by a subset of shared-specificity residues and mitigated by CroS phosphatase activity in wild-type cells. Unregulated hyperproduction of CroR-P impairs growth (Fig. 2), providing an evolutionary selective pressure to maintain insulation between CisS and CroR through CroS phosphatase activity.

In contrast, cross-regulation between CroS and CisR may play a biological role in wild-type cells. CroS phosphorylated CisR in the absence of CisS but only in response to CroS-activating stimuli (vancomycin) (Fig. 7). Hence, this behavior is different from that observed with the reciprocal phosphorylation event (constitutive phosphorylation of CroR by CisS). One possibility for the differing behaviors of CroS and CisS toward their noncognate RR is that CroS exhibits phosphatase activity toward CisR-P in the absence of an activating signal while CisS is unable to dephosphorylate CroR-P at all. The production of CisR-P in the ΔcisS mutant appeared to be attenuated compared to that in wild-type cells, suggesting that both CroS and the cognate HK CisS contribute to CisR-P production in response to vancomycin stress in an otherwise wild-type setting, although we cannot exclude the possibility that phosphatase activity from CisS prevents CroS-mediated phosphorylation of CisR. It remains unclear what the biological role for CisR phosphorylation is, as vancomycin resistance is nearly the same as that of the wild type in mutants lacking cisRS. We speculate that the CisRS TCS contributes to some other, as-yet-unknown biological output for which input from CisS and CroS may be functionally important. Because both CroS and CisS are capable of responding to vancomycin, unambiguously determining if CroRS-CisRS cross-regulation occurs in wild-type cells requires the identification of unique stimuli and outputs for each system. Other than vancomycin, no stimuli that activate CisRS have been identified thus far, and because both CroR and CisR influence OG1RF_12161 transcription (Fig. 4B), a specific CisR-dependent readout is not available. The identification of a biological role for CisRS will allow for further interrogation of this signaling network in wild-type E. faecalis cells.

In the context of stimulation by vancomycin stress, the CisRS TCS appears to attenuate signaling through CroRS, because transcription of croRS (which is CroR dependent) is elevated 5- to 6-fold upon vancomycin stress in mutants lacking CisS or the entire CisRS TCS compared to wild-type cells (Fig. 4B). These results are consistent with the model that stimulus-induced cross-regulation from CisS to CroR does not function to enhance CroR-dependent responses in wild-type cells. The mechanism by which CisRS impairs CroRS signaling is unknown, but one possibility is that CisS sequesters a fraction of the CroR population through protein-protein interaction, thereby rendering it unavailable to participate in signaling via CroS. Thus, in mutants lacking CisS, more CroR would be available for phosphorylation to drive transcription of CroR-dependent genes in response to vancomycin.

In summary, we have shown that phosphorylated CroR is the key driver of cephalosporin resistance in E. faecalis. The cognate HK for CroR, CroS, exhibits both kinase and phosphatase activities in vivo to control phosphorylation of CroR and, as a result, CroR-dependent output (cephalosporin resistance). Nonreciprocal cross-regulation between CroRS and a TCS of unknown function was identified that influences vancomycin-induced gene regulation but not CroRS-dependent cephalosporin resistance in E. faecalis. Additional work is required to define the contribution of this linked TCS signaling network to cell wall integrity in E. faecalis; many E. faecalis lineages lack the CisRS TCS yet remain highly resistant to cell wall stresses. Lastly, further studies to define specific inputs (stimuli) and outputs (regulons) for each of these TCSs is necessary to enable mechanistic dissection of their regulatory interplay.

ACKNOWLEDGMENTS

We thank Jaime Little for the construction of pJLL59.

This work was supported by grants R01 AI081692 and OD006447 from the NIH.

The content of this work is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

FOOTNOTES

    • Received 15 December 2015.
    • Accepted 5 February 2016.
    • Accepted manuscript posted online 16 February 2016.
  • Address correspondence to Christopher J. Kristich, ckristich{at}mcw.edu.

  • Citation Kellogg SL, Kristich CJ. 2016. Functional dissection of the CroRS two-component system required for resistance to cell wall stressors in Enterococcus faecalis. J Bacteriol 198:1326–1336. doi:10.1128/JB.00995-15.

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