The extracellular segment of CroS is not required for sensing but fine-tunes the magnitude of CroS signaling to regulate cephalosporin resistance in Enterococcus faecalis

Sarah B Timmler; Christopher J Kristich

doi:10.1128/jb.00274-24

. 2024 Oct 24;206(11):e00274-24. doi: 10.1128/jb.00274-24

The extracellular segment of CroS is not required for sensing but fine-tunes the magnitude of CroS signaling to regulate cephalosporin resistance in Enterococcus faecalis

Sarah B Timmler ¹, Christopher J Kristich ^1,^✉

Editor: Michael J Federle²

PMCID: PMC11580428 PMID: 39445796

ABSTRACT

Enterococci are Gram-positive bacteria that colonize the gastrointestinal tract. Clinically relevant enterococci are intrinsically resistant to antibiotics in the cephalosporin family, and prior therapy with cephalosporins is a major risk factor for the acquisition of an enterococcal infection. One important determinant of intrinsic cephalosporin resistance in enterococci is the two-component signal transduction system CroS/R. The CroS sensor kinase senses cephalosporin-induced cell wall stress to become activated and phosphorylates its cognate response regulator CroR, thereby enhancing CroR-dependent gene expression to drive cephalosporin resistance. CroS possesses a short (~30 amino acids) extracellular segment between its two transmembrane domains near the N-terminus, but whether this extracellular segment is important for sensing cephalosporin stress, or possesses any other function, has remained unknown. Here, we explored the role of the CroS extracellular segment through mutagenesis and functional studies. We found that mutations in the CroS extracellular segment biased CroS to adopt a more active state during ceftriaxone stress, which led to an increase in CroR-dependent gene expression and hyper-resistance to ceftriaxone. Importantly, these mutants still responded to ceftriaxone-mediated stress by enhancing CroS activity, indicating that the extracellular segment of CroS does not directly bind a regulatory ligand. Overall, our results suggest that although the extracellular segment of CroS does not directly bind a regulatory ligand, it can modulate the magnitude of CroS signaling for phosphorylation of CroR to regulate cephalosporin resistance through the resulting changes in CroR-dependent gene expression.

IMPORTANCE

Clinically relevant enterococci are intrinsically resistant to antibiotics in the cephalosporin family. The CroS sensor kinase senses cephalosporin-induced cell wall stress to trigger signaling that drives cephalosporin resistance, but the mechanism by which CroS senses stress is unknown. We report the first functional characterization of the CroS extracellular segment, revealing that mutations in the extracellular segment did not prevent CroS from responding to cell wall stress but instead biased CroS to adopt a more active state during cephalosporin stress that led to an increase in CroR-dependent gene expression and hyper-resistance to ceftriaxone. Overall, our results suggest that the extracellular segment of CroS does not directly bind to a regulatory ligand but that it can modulate the magnitude of CroS signaling.

KEYWORDS: sensor histidine kinase, cephalosporin resistance

INTRODUCTION

Enterococci are Gram-positive bacteria that are commensals of the gastrointestinal tract of humans. However, enterococci have emerged as significant opportunistic pathogens that can cause life-threatening nosocomial infections. Enterococcus faecalis and Enterococcus faecium are the most clinically significant enterococcal species, and infections with these bacteria can be difficult to treat due to the intrinsic resistance of enterococci to several antibiotics as well as the ability of enterococci to rapidly acquire resistance to many antibiotics used in hospital settings, resulting in dangerous multidrug-resistant strains.

A major risk factor for the development of enterococcal infections is prior treatment with cephalosporin antibiotics. Cephalosporins target the bacterial cell wall by preventing cross-linking of peptidoglycan, resulting in bacterial lysis. However, enterococci are intrinsically resistant to cephalosporins allowing them to proliferate when patients are treated with these drugs. One known cephalosporin resistance determinant in enterococci is the CroS/R two-component signal transduction system (TCS). Therefore, having a better understanding of how CroS/R functions would provide insights into the mechanisms that drive resistance in enterococci, potentially enabling the development of improved prevention and treatment options against recalcitrant enterococcal infections.

CroS (sensor histidine kinase) is activated by exposure to cell wall stressors (including cephalosporins) resulting in CroS autophosphorylation and subsequent phosphorylation of CroR (cognate response regulator for CroS) (1, 2). Phosphorylated CroR regulates gene expression for dozens of genes, including enhancing expression of the croRS operon and genes involved in peptidoglycan synthesis, to mediate resistance to cell wall targeting antimicrobials (1 –3). Consistent with what has been observed for other histidine kinases (HK), CroS exhibits both kinase and phosphatase activity toward CroR (1), such that the balance of the kinase-phosphatase activities of CroS determines the extent of CroR phosphorylation inside the cell and therefore the extent of CroR-dependent gene expression in response to cell wall stress. Recent studies have investigated the CroR regulon to better understand how the CroS/R system contributes to antibiotic tolerance and resistance, identifying CroR-dependent regulation of cell membrane and cell wall synthesis genes (3 –7). However, the mechanism(s) by which CroS senses cell wall stress to modulate its enzymatic activities remains unknown.

The domain architecture of CroS (393 amino acids) includes the canonical conserved cytoplasmic catalytic domains, a HisKA family DHp (8 –11) (dimerization and histidine phosphotransfer) domain and a CA (catalytic and ATP-binding) domain. In addition, the predicted CroS structure (Fig. 1) consists of two transmembrane domains and an extracellular segment (~30 amino acids) (9 –11). As with other HKs, there is evidence that the purified cytoplasmic domain of CroS forms a homodimer (2). It is well known that HK homodimerization results in the formation of four-helix bundles of the transmembrane domains and the HisKA DHp domains in the cytoplasm independent of exposure to activating conditions (12 –14). HK binding to regulatory ligands via an N-terminal input domain is thought to result in a conformational change that is transmitted through these bundles to modulate the catalytic activity of the cytoplasmic domain.

A figure presents an alpha-fold model of E. faecalis CroS, highlighting regions N-terminal, C-terminal, membrane, cytoplasm, A68, E70, A66, W61, A57, V59, and transmembrane domains. The model confidence levels are very high, high, low, and very low. — Alpha-fold model of *E. faecalis* CroS (UniProt accession: Q82YZ0). Alpha-fold generates a per-residue model confidence score (pLDDT), color code shown. Depiction of the Alpha-fold model of CroS embedded in the membrane (*center panel*) is based on the prediction of CroS transmembrane domains (Phobius and TMHMM). The boxed region of CroS is the predicted extracellular segment (wild-type amino acid sequence shown) that was replaced with the flexible linker sequence, with zoomed in view shown to the left. *Right panel* shows “top down” view (rotated 90° from center panel). Mutated CroS residues are labeled.

The domain architecture of CroS most closely resembles that of intramembrane-histidine kinases (IM-HKs), a group of HKs that are predominantly found in Firmicutes and are largely associated with sensing cell envelope stress. IM-HKs are classified by their size (<400 amino acids) and their N-terminal sensing domain consisting of two transmembrane helices connected by an extracellular segment of less than 25 amino acids, often ≤10 amino acids (12, 15). The short nature of the extracellular domain in IM-HKs has led to the hypothesis that this segment primarily acts as a passive linker between the transmembrane helices. Mechanisms of signal perception by IM-HKs vary and are incompletely understood, but certain subgroups of IM-HKs have been associated with sensing activating stimuli via protein-protein interactions with a third component or an accessory protein (15 –18).

The predicted extracellular segment of CroS is slightly longer (~ 30 amino acids) than those found in IM-HKs, and based on genomic context and sequence homology, CroS is formally not classified as an IM-HK. PmrB (extracellular segment of 30–35 amino acids), and VanS (extracellular segment of 25–30 amino acids) are other HKs that, like CroS, resemble IM-HKs based on their overall domain architecture but have extracellular segments that are ≥25 amino acids in length. The extracellular segment of PmrB has been shown to directly bind a regulatory ligand (19). Similarly, some studies suggest that the extracellular segment of VanS can directly bind vancomycin, but other studies are inconsistent (20 –24). The function of the extracellular segment of CroS remains unknown; hence, it is unclear if the extracellular segment plays an active role in CroS sensor function by binding ligands, if the extracellular segment simply acts as a passive linker between transmembrane helices, or if the extracellular segment acts in some other as-yet-unknown role.

In this work, we investigated the function of the CroS extracellular segment through mutagenesis and functional studies. We introduced mutations into the extracellular segment of CroS and assessed the ability of CroS to become activated (i.e., to phosphorylate CroR) in response to stress and drive cephalosporin resistance. We found that mutations in the CroS extracellular segment biased CroS to adopt a more active state during cephalosporin exposure that led to an increase in CroR-dependent gene expression and hyper-resistance to ceftriaxone (a broad-spectrum, representative cephalosporin). Importantly, these mutants still responded to ceftriaxone-mediated stress by enhancing CroS activity, suggesting that the extracellular segment of CroS does not directly bind any regulatory ligand. Overall our results suggest that although the extracellular segment of CroS does not directly bind to a regulatory ligand, it can modulate the magnitude of CroS signaling for phosphorylation of CroR and regulate cephalosporin resistance through the resulting changes in CroR-dependent gene expression.

RESULTS

Cysteine crosslinking suggests that the CroS extracellular segment is not a stable alpha-helix

AlphaFold (25, 26) predicts the extracellular segment of CroS forms an alpha-helix (Fig. 1). To test this, we probed the extracellular segment using cysteine disulfide crosslinking. We reasoned that if the extracellular segment of CroS stably adopts an alpha-helical conformation, then cysteine substitutions along one face of the alpha helix might readily encounter a cysteine on the other monomer in the context of a CroS dimer to enable disulfide bond formation, whereas cysteines along the opposite face of the alpha helix would be much less prone to intermolecular contact with the partner monomer and thus not form disulfide crosslinks readily. We therefore constructed a series of CroS variants by introducing unique cysteine substitutions into residues located on opposite faces of the predicted alpha-helix (V59C, A66C, and E70C along one face; and A57C, W61C, and A68C along the other; Fig. 1) and assessed the ability of the resulting mutants to become crosslinked via disulfide bonds.

Because we have previously shown that in the absence of CroS, a separate histidine kinase (CisS) can influence CroR phosphorylation (1), we performed our studies by co-expressing CroS mutants along with CroR in a ∆croRS∆cisRS strain to ensure that phosphorylation and functional output of CroR is solely due to CroS activity. We used a version of CroS with a C-terminal HA-tag for this work, enabling us to detect CroS via immunoblot (we do not possess an antibody that recognizes CroS). C-terminal HA-tagged CroS co-expressed with wild-type croR largely restores ceftriaxone resistance to wild-type levels in a ∆croRS∆cisRS strain of E. faecalis (Table 1), demonstrating that the HA-tag does not significantly interfere with CroS function. Whole-cell lysates were prepared from exponentially growing cells and subjected to SDS-PAGE and immunoblotting. Wild-type CroS does not contain any cysteine residues and resolved as a monomer (~45 kDa) (Fig. 2). All of the single-Cys CroS variants behaved similarly and resolved as two forms, the ~45 kDa monomeric form and a high-molecular-weight form consistent with the size of two CroS monomers (~90 kDa) that disappeared upon treatment with DTT (Fig. 2). From this, we concluded that disulfide crosslinking of CroS occurred during cell growth for all CroS cysteine variants, regardless of the Cys location in the extracellular segment. Given the similar propensity of Cys substitutions on both “faces” of the predicted alpha helix to form intermolecular disulfide crosslinks, these results do not support the existence of a stable alpha helix in the CroS extracellular segment.

TABLE 1.

Ceftriaxone resistance of CroS extracellular segment variants

Plasmid^a	Minimal Inhibitory Concentration (MIC) ceftriaxone (µg/mL)^b
Vector P-croR P-croR-croS P-croR-croS-HA	8 8 128 64
P-croR-croS A66C-HA P-croR-croS E70C-HA P-croR-croS V59C-HA P-croR-croS A68C-HA P-croR-croS W61C-HA P-croR-croS A57C-HA	>512 32 >512 32 >512 64
P-croR-croS (G4S)₆ HA P-croR-croS (G4S)₂ HA	1,024 >1,024
P-croR-croS 7 A-HA	>512

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^{^a}

The strain analyzed was E. faecalis OG1 ∆croRS∆cisRS (SB105) carrying indicated plasmids. The plasmids analyzed were as follows: wild type P-croR-croS-HA, pSLK134; vector, pJRG8; wild type P-croR-croS, pJLL59; wild type P-croR, pSLB31; P-croR-croS A66C-HA, pSBT82; P-croR-croS E70C-HA, pSBT90; P-croR-croS V59C-HA, pSBT80; P-croR-croS A68C-HA, pSBT89; P-croR-croS W61C-HA, pSBT81; P-croR-croS A57C-HA, pSBT88; P-croR-croS (G4S)₆ HA, pSBT123; P-croR-croS (G4S)₂ HA, pSBT124; and P-croR-croS 7 A-HA, pSBT103.

^{^b}

Median MIC is reported from a minimum of three independent replicates.

A figure presents alpha-HA immunoblot images of CroS cysteine disulfide crosslinking for wild type, vector, A66C, A68C, V59C, E70C, A57C, and W61C. — CroS cysteine disulfide crosslinking. Cell lysates were prepared from exponentially growing *E. faecalis* OG1 ∆*croRS*∆*cisRS* cells carrying the empty vector (*vector*), expressing wild-type *croR-croS-HA* (*wildtype*), or expressing *croR-croS cysteine mutant-HA* as indicated. Aliquots of cell lysates were treated with DTT to reduce disulfide bonds where indicated. Cells were treated with N-ethylmaleimide for 5 minutes prior to cell collection. Immunoblotting was performed to detect the HA tag on CroS.

Amino acid residues in the extracellular segment of CroS impact function.

The impact, if any, of the extracellular segment on CroS function was previously unknown. To determine if the cysteine substitutions affected CroS function, we measured CroS-dependent cephalosporin resistance by assessing antimicrobial susceptibility against ceftriaxone. Ceftriaxone resistance of E. faecalis ∆croRS∆cisRS expressing only croR from the native croR promoter without croS mimicked that of the empty vector, demonstrating that functional CroS is required to drive wild-type levels of ceftriaxone resistance (Table 1). Among the cysteine mutants, two phenotypic classes were observed. Three of the CroS cysteine variants (A57C, A68C, E70C) exhibited wild-type or near wild-type levels of resistance to ceftriaxone (Table 1), indicating that CroS function remained essentially intact. However, CroS variants with cysteine substitutions located more centrally in the predicted extracellular segment (A66C, V59C, and W61C) resulted in substantial hyper-resistance (Table 1), the first evidence to our knowledge that amino acid residues in the extracellular segment of CroS impact its function.

To determine if the cysteine mutations in the CroS extracellular segment impacted ceftriaxone resistance by altering CroS signaling, we monitored CroR phosphorylation in response to ceftriaxone exposure using Phos-tag SDS-PAGE, as we have done previously (1). During Phos-tag SDS-PAGE, phosphorylated proteoforms of proteins migrate more slowly than unphosphorylated proteoforms, enabling specific detection of phosphorylated proteoforms.

The CroS W61C and A57C mutants were chosen for analysis as representatives of the two different ceftriaxone resistance phenotypic classes. In ∆croRS∆cisRS cells expressing wild-type croR/croS-HA, CroR was minimally phosphorylated in the absence of ceftriaxone, and phosphorylation was enhanced upon ceftriaxone exposure (Fig. 3A). Similar to the control with wild-type CroS, CroR was mostly unphosphorylated in strains expressing CroS W61C or A57C mutants in the absence of ceftriaxone. Exposure of the mutants to ceftriaxone resulted in enhanced CroR phosphorylation, demonstrating that the CroS A57C and W61C mutants are capable of recognizing ceftriaxone-induced stress to become activated and phosphorylate CroR. The hyper-resistant CroS W61C mutant appeared to contain slightly more phosphorylated CroR upon ceftriaxone exposure compared with either wild type or the CroS A57C mutant (Fig. 3A), suggesting that the W61C substitution biased CroS towards an activated state during ceftriaxone exposure.

A figure shows alpha-CroR immunoblot images of cell lysates at wild type, vector, A57C, and W61C treated with ceftriaxone, and total protein. A bar graph with error bars depicts beta galactosidase activity in wildtype, vector, A57C, and W61C. — The hyper-resistant CroS W61C mutant enhances CroR phosphorylation and CroR-dependent transcription during ceftriaxone stress. (A) Cell lysates were prepared from exponentially growing *E. faecalis* OG1 ∆*croRS*∆*cisRS* cells carrying empty vector (*vector*), expressing wild-type *croR-croS-HA* (*wildtype*), or expressing *croR-croS cysteine mutant-HA* as indicated. Where indicated, ceftriaxone was included at 256 µg/mL for wild-type CroS and CroS A57C or 512 µg/mL for CroS W61C. Cell lysates were subjected to phos-tag SDS PAGE with immunoblotting to detect CroR. The arrow indicates migration of the phosphorylated proteoform of CroR. Data are representative of two biological replicates. (B) β-galactosidase activity was determined from *lacZ* fusion to the *croR* promoter. Where indicated, ceftriaxone was included at 256 µg/mL for wild-type CroS and CroS A57C, 8 µg/mL for empty vector, or 512 µg/mL for CroS W61C. Error bars represent the standard deviation from three biological replicates. Statistical significance (*, P value < 0.001) was determined by a two-tailed t test. ns, not statistically significant (P value > 0.05).

To determine how the CroS substitutions impacted CroR-dependent gene expression, we analyzed transcriptional activation of the CroR-dependent croR promoter using a lacZ reporter (Fig. 3B). Under the conditions of this experiment, we observed a modest increase in CroR-dependent transcriptional activation in cells expressing wild-type or A57C CroS upon exposure to ceftriaxone that was not observed in the absence of CroS/R (empty vector). During exposure to ceftriaxone, CroS W61C led to enhanced CroR-dependent transcription compared with wild type (Fig. 3B), consistent with the presence of higher levels of phosphorylated CroR. Taken together, these results are consistent with the model that substitutions in the extracellular segment of CroS can impact ceftriaxone resistance by biasing signaling through CroS to promote enhanced activation of CroR.

The extracellular segment of CroS does not bind a regulatory ligand for activation of CroS

To determine if the CroS extracellular segment is important for the recognition of cell wall stress that activates CroS, we replaced the extracellular segment (Fig. 1) with flexible linker sequences (GGGGS)_x, abbreviated here as (G4S)_x. The transmembrane segments of CroS have not been experimentally defined, and prediction of the transmembrane segments by Phobius and TMHMM yields similar but slightly different results: amino acid residues 21 through 46 and 82 through 100 (Phobius), or 21 through 43 and 75 through 97 (TMHMM). For our studies, we replaced residues 47 through 77 (30 amino acids) with either a (G4S)₆ or (G4S)₂ flexible linker. The (G4S)₆ flexible linker replaced the CroS extracellular segment with a flexible linker of the same length as the natural CroS extracellular segment, whereas the (G4S)₂ flexible linker replaced the CroS extracellular segment with a flexible linker similar in size to canonical intramembrane-histidine kinases. Immunoblot analyses on the flexible linker mutants revealed that the (G4S)₂ mutant migrated at lower MW (as expected) and exhibited increased abundance relative to wild-type CroS (Fig. 4), whereas the (G4S)₆ mutant exhibited substantially reduced abundance relative to wild type. The underlying reasons for the difference in abundance of the (G4S)_x mutants remain unknown. Nevertheless, antimicrobial susceptibility assays against ceftriaxone (Table 1) revealed that both flexible linker replacement mutants exhibited a hyper-resistant phenotype compared with wild type, with the CroS (G4S)₂ flexible linker mutant exhibiting higher ceftriaxone resistance compared with the CroS (G4S)₆ flexible linker mutant.

A figure shows immunoblot images of cell lysates from wildtype and vector for ceftriaxone, and total protein. A bar graph with error bars shows beta-galactosidase activity for untreated, 8, 256, and 512 µg per mL ceft. — Replacement of the CroS extracellular segment with (GGGGS)_x flexible linkers enhances phosphorylation of CroR and CroR-dependent transcription during ceftriaxone exposure. (**A-B)** Cell lysates were prepared from exponentially growing *E. faecalis* OG1 ∆*croRS*∆*cisRS* cells carrying the empty vector (*vector*), expressing wild-type *croR-croS-HA* (*wildtype*), or expressing *croR-croS (GGGGS)_x-HA* as indicated. Where indicated, ceftriaxone was included at 256 µg/mL for wild type or 512 µg/mL for CroS (GGGGS)_x mutants. Cell lysates were subjected to phos-tag SDS PAGE with immunoblotting to detect CroR. The arrow indicates migration of the phosphorylated proteoform of CroR. Data are representative of a minimum of two biological replicates. (C) β-galactosidase activity was determined from *lacZ* fusion to the *croR* promoter. Error bars represent the standard deviation from a minimum of three biological replicates. Statistical significance (*, P value < 0.001) was determined by a two-tailed t test. ns, not statistically significant (P value > 0.05).

To assess signaling by the CroS (G4S)_x mutants, we analyzed CroR phosphorylation in response to stress by Phos-tag SDS-PAGE (Fig. 4). Despite the lower abundance of CroS (G4S)₆ protein, we found that strains expressing either (G4S)_x mutant contained slightly more phosphorylated CroR compared with wild type in the absence of ceftriaxone treatment, suggesting slight activation of CroS signaling even in the absence of stress. Importantly, CroR phosphorylation increased in response to ceftriaxone stress for both (G4S)_x mutants and did so to a greater extent in (G4S)_x mutants than observed in cells expressing wild-type CroS. This effect was more pronounced for the CroS (G4S)₂ mutant, correlating with the increased ceftriaxone resistance of this mutant. Thus, these results indicate that CroS is capable of becoming activated to enhance phosphorylation of CroR in response to ceftriaxone stress despite the absence of all the natural amino acid residues throughout its entire predicted extracellular segment. Moreover, consistent with observations from the CroS W61C mutant above, residues in the extracellular segment appear to influence the propensity of CroS to adopt a signaling state that leads to enhanced phosphorylation of CroR.

To determine if CroR-dependent transcriptional activation was impacted by replacement of the CroS extracellular segment with a flexible linker, we assessed transcriptional activation of the croR-lacZ promoter fusion in the CroS (G4S)₂ strain cultured in the presence of varied ceftriaxone concentrations. Wild-type cells exhibited a dose-dependent increase in CroR transcriptional activation of the croR promoter, demonstrating that increasing concentrations of ceftriaxone, and presumably cell wall stress, result in increasing levels of activation of the CroS/R system. croR expression was elevated in the (G4S)₂ mutant relative to wild type in the absence of ceftriaxone (Fig. 4C), consistent with the presence of some phosphorylated CroR in untreated cells observed via Phos-tag SDS-PAGE and autoregulation of transcription from the croR promoter. Exposure of the (G4S)₂ mutant to ceftriaxone resulted in increased croR expression, also consistent with the increase in phosphorylated CroR observed via Phos-tag SDS-PAGE (and with the elevated abundance of CroR observed via standard immunoblot, Fig. 4). Moreover, in contrast to wild-type CroS, the response of the (G4S)₂ mutant appeared saturated at 256 µg/mL ceftriaxone. Of note, cells expressing wild-type CroS grew more slowly at 256 µg/mL ceftriaxone than those expressing CroS (G4S)₂, suggesting that the wild-type cells are experiencing greater ceftriaxone-induced stress than the (G4S)₂ mutant; however, the response of the mutant is already saturated. Collectively, these results indicate that the ceftriaxone hyper-resistant phenotype of the (G4S)_x mutants is driven by CroR-dependent gene expression and is consistent with the model that residues in the extracellular segment appear to influence the propensity of CroS to adopt a signaling state in the presence of ceftriaxone stress that leads to enhanced phosphorylation of CroR.

Identification of functionally important residues in the CroS extracellular segment

We previously reported that E. faecium CroS/R restores cephalosporin resistance to the E. faecalis ∆croRS strain (27). However, comparison of the sequences of the extracellular segments from the E. faecalis and E. faecium CroS orthologs reveals significant variation (Fig. 5). Because E. faecium CroS complements the E. faecalis deletion, we hypothesized that conserved residues in the extracellular segment of CroS from both species might be functionally important. In particular, we focused on a stretch of conserved residues located at one end of the extracellular segment (L₇₁N₇₂T₇₃D₇₄L₇₅F₇₆W₇₇), and introduced an alanine-stretch mutation replacing all seven residues of E. faecalis CroS with alanines (CroS 7A mutant) to assess the significance of these residues on the ability of CroS to drive ceftriaxone resistance and enhance CroR phosphorylation in response to ceftriaxone stress.

The figure illustrates the amino acid sequence alignment of E. faecalis and E. faecium CroS. The highlighted sequences are GQLLSENSPLAGV, SSVINSSPSLTNA, IWATKDAVAKELNTDLFW, and IWDSKNIFAERLNTDLFW. — *E. faecalis* and *E. faecium* (UniProt accession: Q3XYJ6) CroS amino acid sequence alignment, residues 1 through 119 (*E. faecalis*) and 120 (*E. faecium*). Highlighted underlined sequence corresponds to the predicted extracellular segment.

Overall, the CroS 7A mutant exhibited the same phenotypes we observed with the CroS (G4S)₂ mutant. Namely, the CroS 7A mutant exhibited hyper-resistance to ceftriaxone (Table 1), and in the presence of cell wall stress, the CroS 7A mutant exhibited higher levels of CroR phosphorylation than wild type (Fig. 6A) with a corresponding increase in CroR-dependent gene expression (Fig. 6B). Thus, one or more residues in the conserved seven-residue cluster of the CroS extracellular segment is functionally important to modulate CroS signaling during ceftriaxone stress.

The figure illustrates Phos-tag and standard immunoblot for wild-type and vector, and total protein. The bar graph shows beta-galactosidase activity for the wild-type vector, CroS7A, under untreated, 8 and 512 μg per mL ceft. — The CroS 7A mutant enhances CroR phosphorylation and CroR-dependent transcription. (A) Cell lysates were prepared from exponentially growing *E. faecalis* OG1 ∆*croRS*∆*cisRS* cells carrying empty vector (*vector*), expressing wild-type *croR-croS-HA* (*wildtype*), or expressing *croR-croS 7A-HA* as indicated. Where indicated, ceftriaxone was included at 256 µg/mL for wild type or 512 µg/mL for CroS 7A mutant. Cell lysates were subjected to phos-tag SDS PAGE with immunoblotting to detect CroR. The arrow indicates migration of the phosphorylated proteoform of CroR. Data are representative of a minimum of two biological replicates. (B) β-galactosidase activity was determined from *lacZ* fusion to the *croR* promoter. Error bars represent the standard deviation from three biological replicates. Statistical significance (*, P value < 0.001) was determined by a two-tailed t test.

Because CroS/R is also activated by cell-wall targeting antimicrobials that act at different steps of the peptidoglycan biosynthesis pathway, we tested if mutations in the CroS extracellular segment also led to hyper-resistance to vancomycin, bacitracin, or fosfomycin. However, none of the three CroS variants we tested (CroS W61C, CroS (G4S)₂, or CroS 7A) led to changes in MIC for any of those three antibiotics compared with wild-type CroS.

CroS extracellular segment mutations impact CroR phosphorylation and ceftriaxone resistance through CroS kinase activity

Our data indicate that mutations in the CroS extracellular segment influence the extent of CroR phosphorylation. As with many two-component sensor kinases, CroS exhibits both kinase and phosphatase activity toward CroR. Hence, CroR phosphorylation levels are determined by the balance of the kinase and phosphatase activities of CroS toward CroR. To determine if mutations in the extracellular segment of CroS specifically impact kinase or phosphatase activity, we exploited several previous observations: first, that CroS D173 is required for CroS kinase activity and that a CroS D173A mutant lacks kinase activity but maintains CroS phosphatase activity (1); second, that CroR can be phosphorylated by another enterococcal two-component sensor kinase (CisS), and as a result, in the presence of CisS but the absence of phosphatase activity from CroS, CroR becomes highly phosphorylated leading to ceftriaxone hyper-resistance (1). Thus, by analyzing the effect of a mutation in the extracellular segment of CroS on cephalosporin resistance of the D173A variant in the presence and absence of CisS, we can genetically dissect CroS kinase and phosphatase function.

We therefore constructed a CroS variant that combined the 7A substitutions in the extracellular segment with D173A and expressed this variant in both the ∆croRS∆cisRS strain and the ∆croRS strain. Analysis of ceftriaxone resistance in the ∆croRS∆cisRS mutant (where CroS is the only source of kinase activity for CroR) revealed that hyper-resistance to ceftriaxone of the CroS 7A mutant was eliminated upon introduction of D173A (Table 2). Thus, the D173A mutation is epistatic to the 7A mutation in the extracellular segment, confirming that hyper-resistance to ceftriaxone observed with the 7A mutation is due to CroS-mediated phosphorylation of CroR to drive CroR-dependent gene expression. Analysis of ceftriaxone resistance upon expression of the CroS 7A D173A variant in the ∆croRS mutant (where CisS is present and can phosphorylate CroR) revealed a low level of ceftriaxone resistance (Table 2), indicating that CroR is not phosphorylated. In other words, the phosphatase activity of CroS remains fully intact and functional to maintain CroR in its unphosphorylated state despite the presence of the 7A mutation in the extracellular segment of CroS. We conclude, therefore, that substitutions in the extracellular segment of CroS bias it toward a state of enhanced kinase activity toward CroR in the presence of ceftriaxone stress, driving increased CroR phosphorylation and CroR-dependent gene expression.

TABLE 2.

Ceftriaxone resistance of CroS extracellular and kinase-impaired variants

Plasmid^a	Ceftriaxone MIC^b (μg/mL) for:
Plasmid^a	∆croRS∆cisRS mutant	∆croRS mutant
vector	4	8
P-croR-croS-HA	32	32
P-croR-croS D173A-HA	8	8
P-croR-croS 7A-HA	>512	>512
P-croR-croS 7A D173A-HA	4	8

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^{^a}

The strains analyzed were E. faecalis OG1 ∆croRS∆cisRS (SB105) or E. faecalis OG1 ∆croRS (SB35) carrying indicated plasmids. The plasmids analyzed were as follows: vector, pJRG8; wild type P-croR-croS-HA, pSLK134; P-croR-croS 7A-HA, pSBT103; P-croR-croS D173A-HA, pSLK136; P-croR-croS 7A D173A-HA, pSBT125.

^{^b}

Median MIC is reported from a minimum of two independent replicates.

DISCUSSION

The CroS sensor histidine kinase is required for cephalosporin resistance in enterococci. The mechanism by which CroS senses stress imposed by cephalosporins and other cell-wall-active antibiotics is unknown. The domain architecture of CroS is such that it possesses only a short extracellular segment of ~30 amino acids between the two transmembrane domains at its N-terminal end. Experimentally determined structures are not available for CroS; hence, it remains unclear if the extracellular segment adopts any stable fold, and no specific role for the extracellular segment in ligand recognition, sensing, or CroS function more generally has previously been defined. Here, we report the first functional analysis of the CroS extracellular segment, using mutagenesis and functional studies to reveal that although the CroS extracellular segment appears to modulate the amplitude of the CroS response to cephalosporin exposure, the extracellular segment is not required for CroS to recognize cephalosporin stress to enhance phosphorylation of its cognate response regulator CroR and drive changes in gene expression that promote cephalosporin resistance. We conclude that the extracellular segment of CroS is not responsible for directly binding to any regulatory signal encountered or produced upon cephalosporin exposure but rather acts to tune the magnitude of the response.

Although the structure of CroS has not been determined experimentally, AlphaFold predicts that the extracellular segment folds, in part, as an alpha-helix (Fig. 1). As one approach to test for the presence of this putative helix, we used cysteine disulfide crosslinking, reasoning that in the context of a CroS homodimer, cysteine substitutions on opposite faces of the putative helix would exhibit differential abilities to become crosslinked with the other monomer. However, we found that all of the single-Cys CroS variants we tested became crosslinked in E. faecalis cells, regardless of their location in the putative extracellular segment helix (Fig. 2). These results suggest that the extracellular segment of CroS does not adopt a stable alpha helical conformation in E. faecalis cells but instead is likely dynamic, perhaps acting as a flexible loop or adopting a helical conformation only transiently. Formally, we cannot rule out the possibility that disulfide crosslinking is occurring between monomers from different CroS dimers, although given that the Cys thiols need to be within only about 2 Å of each other for crosslinking to occur, such a possibility would require very close proximity between CroS dimers. It is perhaps worth noting that AlphaFold3 also had difficulty predicting the existence of the canonical 4-helix bundle of the transmembrane segments of a CroS dimer, possibly in part because the putative extracellular helix introduced constraints in the relative spacing of the transmembrane segments that were incompatible with 4-helix bundle formation in the context of the homodimer, which would be consistent with the notion that prediction of a helix in the CroS extracellular segment is incorrect (28).

We found that some cysteine mutations in the CroS extracellular segment altered signaling by CroS, as reflected by increased CroR phosphorylation, increased CroR-dependent transcription (Fig. 3), and increased cephalosporin resistance (Table 1), suggesting that the extracellular segment of CroS plays an active role in CroS-mediated signal transduction. To explore the requirement for the extracellular segment in more detail, we replaced nearly the entire predicted CroS extracellular segment with flexible linkers of two different lengths and found both flexible linker variants were able to respond to ceftriaxone-induced cell wall stress by enhancing phosphorylation of CroR, leading to elevated CroR-dependent transcription. Thus the extracellular segment of CroS is not specifically required for recognition of a physiologically relevant signal, or for CroS to become activated in response to that signal. We conclude that the extracellular segment of CroS is not an “input” domain for CroS in the classical sense, in that the extracellular segment does not directly bind to regulatory ligands that lead to the activation of CroS. This contrasts with PmrB of Salmonella, a two-component sensor kinase that possesses an extracellular segment of similar length to CroS, where the PmrB extracellular segment physically binds to a regulatory ligand to trigger kinase activation (19). We speculate that sensing of activating signals by CroS could occur via the CroS transmembrane domains within the membrane, possibly through sensing of perturbations of the cell membrane, direct binding of a membrane-embedded ligand, or through a protein-protein interaction. Sensing of activating signals via the transmembrane domains within the membrane has also been proposed for LiaS of the LiaF/S/R system. LiaF/S/R modulates resistance to the membrane-active antimicrobial daptomycin in E. faecalis and E. faecium (29). The LiaS sensor kinase is conserved in Firmicutes and belongs to the IM-HK family of sensor kinases, with an input domain consisting of two transmembrane domains connected by a 15–20 amino acid extracellular segment with a predicted helical structure. The function of the LiaS extracellular segment is currently unknown. Unlike CroS, LiaS is associated with an accessory protein (LiaF), which is encoded in the liaFSR operon and is involved in the regulation of LiaS activation (29, 30).

Although the flexible linker mutants were able to respond to ceftriaxone-mediated stress by becoming activated to enhance phosphorylation of CroR, we also found that they promoted aberrantly high levels of CroR phosphorylation (Fig. 4) and ceftriaxone resistance (Table 1) compared with wild-type CroS, suggesting that the extracellular segment of CroS acts to modulate the amplitude of the response. To identify specific residues in the extracellular segment that are functionally important, we exploited our previous observation that the CroS/R orthologs from E. faecium functionally complement the E. faecalis ΔcroRS deletion mutant and therefore focused on a stretch of conserved residues (Fig. 5) near one end of the extracellular segment (L₇₁N₇₂T₇₃D₇₄L₇₅F₇₆W₇₇) in both of the CroS orthologs. Substitution of these residues in E. faecalis CroS with Ala (creating the CroS 7A variant) resulted in similar phenotypes as observed with the flexible linker mutants: the CroS 7A variant was able to respond to ceftriaxone-mediated stress by becoming activated to enhance phosphorylation of CroR, and also promoted aberrantly high levels of CroR phosphorylation (Fig. 6) and ceftriaxone resistance (Table 1) compared with wild-type CroS. Thus, one or more of the residues in this conserved stretch of the CroS extracellular segment appears to be important for modulating the magnitude of the CroS response to ceftriaxone-induced cell wall stress, indicating that although the extracellular segment is not responsible for binding to activating signals, it also does not act simply as a passive linker between the transmembrane domains. This is reminiscent of recent findings for GraS, an IM-HK with a short extracellular segment, in which a charged residue (D35) in the GraS extracellular segment impacted signaling through the system, suggesting a role for the GraS extracellular segment beyond acting simply as a passive linker (17).

Based on our previous work (1, 7), the observations that CroS variants with substitutions in the extracellular segment led to increased phosphorylation of CroR and enhanced CroR-dependent gene expression together indicate that the CroS extracellular variants are promoting elevated ceftriaxone resistance by driving signaling through the canonical CroS/R pathway. To explicitly test this, we created a mutant combining the CroS 7A extracellular mutation with D173A, a mutation that we previously showed specifically abrogates the kinase activity of CroS (1). Analysis of the resulting mutant revealed that ceftriaxone resistance was lost upon introduction of D173A (Table 2). Thus, elevated ceftriaxone resistance of the CroS 7A mutant (and presumably other CroS variants with substitutions in their extracellular segments) is indeed mediated by signaling through the CroS/R pathway. The substitutions in the CroS extracellular segment appear to primarily enhance CroS kinase activity (rather than impair CroS phosphatase activity) because expression of the CroS 7A D173A mutant in an E. faecalis strain carrying CisS (which can phosphorylate CroR independently of CroS (1)) revealed the lack of ceftriaxone resistance (Table 2), indicating that the phosphatase activity of CroS remained intact despite the 7A substitutions in the extracellular segment.

In summary, we propose a model in which detection of activating signals by CroS occurs independently of the CroS extracellular segment. However, the extracellular segment is not just a passive linker to connect the two transmembrane domains. Instead, the extracellular segment appears to modulate the magnitude of the CroS response. Because excessive activation of signaling through CroS/R results in a fitness cost (mutants with constitutively active CroR exhibit a substantial growth defect (1)), it seems reasonable that fine-tuning the magnitude of the CroS/R response to be commensurate with the stress encountered would be an evolutionarily beneficial strategy to optimize overall fitness. Future work will focus on understanding the mechanism of signal detection by CroS and the mechanism by which the extracellular segment modulates CroS signaling.

MATERIALS AND METHODS

Bacterial strains, growth media, and chemicals

Table 3 lists all bacterial strains and plasmids used in this study. E. faecalis strains were grown in Mueller-Hinton Broth (MHB) (Difco). E. coli strains were grown in half-strength brain heart infusion (BHI) medium (Bacto). Erythromycin (Em) was used at 10 µg/mL or 100 µg/mL for E. faecalis and E. coli respectively. Chloramphenicol (Cm) was used at 10 µg/mL for E. faecalis and E. coli. All cultures were grown aerobically with shaking (225 rpm).

TABLE 3.

Strains and plasmids used in this study

Strain	Genotype or description	Source or reference
Strains
E. coli
Top10	Routine cloning host	Lab stock
E. faecalis
OG1	Wild-type laboratory strain isolate	(31)
SB105	OG1 ∆croRS ∆cisRS	(1)
SB35	OG1 ∆croRS	(1)
Plasmids
pCI3340	E. coli-E. faecalis shuttle vector (Cm^r)	(32)
pJLL170	P_croR’-lacZ in pCI3340	(33)
pJRG8	E. faecalis expression vector (Em^r)	(34)
pJLL59	P_croR -croR-croS in pJRG8	(1)
pSLK134	P_croR -croR-croS-HA in pJRG8	(33)
pSBT80	P_croR -croR-croS V59C-HA in pJRG8	This work
pSBT81	P_croR -croR-croS W61C-HA in pJRG8	This work
pSBT82	P_croR -croR-croS A66C-HA in pJRG8	This work
pSBT88	P_croR -croR-croS A57C-HA in pJRG8	This work
pSBT89	P_croR -croR-croS A68C-HA in pJRG8	This work
pSBT90	P_croR -croR-croS E70C-HA in pJRG8	This work
pSBT103	P_croR -croR-croS 7A -HA in pJRG8	This work
pSBT123	P_croR -croR-croS (G4S)₆ HA in pJRG8	This work
pSBT124	P_croR -croR-croS (G4S)₂ HA in pJRG8	This work
pSBT125	P_croR -croR-croS 7A D173A -HA in pJRG8	This work
pSLB31	P_croR -croR in pJRG8	This work
pSLK136	P_croR -croR-croS D173A -HA in pJRG8	(33)

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Plasmid construction

Plasmids were constructed using Gibson Assembly (35). All inserts in recombinant plasmids were sequenced in their entirety to confirm the absence of undesired mutations. Ectopic expression of croRS alleles (wild type and mutants) under the control of the native croRS promoter in E. faecalis OG1 ∆croRS∆cisRS and E. faecalis OG1 ∆croRS host strains was accomplished using the enterococcal expression plasmid pJRG8. The constitutive promoter found in pJRG8 was replaced with the croRS fragment during cloning to introduce croRS and its native promoter.

Antibiotic susceptibility assays

The minimal inhibitory concentration (MIC) for ceftriaxone was determined using broth microdilution as described previously (1). Bacteria from stationary-phase cultures in MHB supplemented with 10 µg/mL Em for plasmid carrying strains were inoculated into microtiter plates containing 2-fold serial dilutions of antibiotic in fresh MHB supplemented with Em (to maintain plasmids) at a normalized cell density of ~10⁵ CFU/mL. Plates were incubated at 37°C for 24 h in a Bioscreen C plate reader. The optical density at 600 nm (OD₆₀₀) was read every 15 min, with brief shaking prior to each measurement. The lowest concentration of antibiotic that prohibited bacterial growth was recorded as the MIC.

Whole cell lysate preparation

Stationary-phase cultures of E. faecalis strains were diluted to OD₆₀₀ = 0.01 in fresh MHB (supplemented with 10 µg/mL Em for maintenance of plasmids) and grown to exponential phase at 37°C and 225 rpm (OD₆₀₀ ~0.175). Where specified, ceftriaxone was added at the time of dilution to stimulate activation of CroS/R and bacterial cells were grown to exponential phase in the presence of the drug. Where indicated, cultures were treated with a final concentration of 0.5 mg/mL N-ethylmaleimide (NEM) for 5 min prior to cell collection to react with free thiols and prevent disulfide bond formation during lysate preparation. Bacteria were collected by mixing cultures with an equal volume of cold ethanol-acetone (1:1 vol/vol) to rapidly kill the bacteria and prevent any further physiological changes. Cells were harvested by centrifugation (4,200 × g for 10 min at 4°C), washed with water, and resuspended in lysozyme solution (10 mM Tris [pH 8], 50 mM NaCl, 20% sucrose) followed by normalization to an equivalent OD₆₀₀ and subsequent treatment with lysozyme (5 mg/mL lysozyme in lysozyme buffer) for 30 min at 37°C. Samples were mixed with non-reducing 5× SDS Laemmli sample buffer and where indicated treated with DTT and boiled 5 min. Cell lysates used for Phos-tag analysis were not treated with DTT or boiled.

Standard and Phos-tag SDS PAGE immunoblot analysis

Proteins in undiluted lysates were separated by standard (10% SDS-PAGE) or phos-tag SDS-PAGE (10% SDS-PAGE supplemented with 20 µM Phos-tag and 40 µM MnCl₂). Electrophoresis was performed at 200 V for 45 min using Laemmli buffer system at room temperature for standard SDS-PAGE or at 4°C for Phos-tag SDS-PAGE. After electrophoresis, all Phos-tag gels were treated with 5 mM ethylenediaminetetraacetic acid (EDTA) twice for 10 min each at room temperature. Proteins were transferred to polyvinylidene difluoride (PVDF) membrane using a Bio-Rad TurboBlot apparatus (25 V for 7 min). Membranes were incubated with No-Stain Protein Labeling Reagent (Invitrogen) to assess total protein on membranes prior to incubation with 5% milk and successive incubation with primary antibody polyclonal antisera. After incubation with Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (Invitrogen), total protein was imaged using Amersham Typhoon Imager (GE Life Sciences). Membranes were then coated with chemiluminescent HRP substrate (ThermoScientific) and imaged using Chemidoc Imager (BioRad).

Beta-galactosidase activity assays

As previously described, CroR-dependent transcription was monitored using a P_croR-lacZ fusion reporter plasmid (Table 3) (7, 33, 36, 37). Stationary-phase cultures of plasmid-bearing strains were diluted to an OD₆₀₀ of 0.01 in MHB supplemented with Cm and Em for plasmid maintenance. Cells were grown to exponential phase (OD₆₀₀ ~0.175) in the absence or presence of ceftriaxone at 37°C and 225 rpm. Cells were pelleted (4,200 x g for 10 min at 4°C) and resuspended in Z buffer (60 mM Na₂HPO₄-7H₂O, 40 mM NaH₂PO₄- H₂O, 10 mM KCl, 1 mM MgSO₄-7H₂O, 50 mM β-ME). Cells were permeabilized with SDS and chloroform (10 min at RT) prior to incubation with ortho-nitrophenyl-β-galactoside (ONPG) (Sigma) as the substrate for β-galactosidase. Cellular debris was removed by centrifugation, and β-galactosidase activity was monitored by measuring sample absorbance at 420 and 550 nm; samples were normalized for cell density (OD₆₀₀). Absorbance readings (420, 550, and 600 nm) were used to calculate Miller Units. Experiments were performed in triplicate and samples were analyzed in triplicate.

ACKNOWLEDGMENTS

This study was supported in part by grants AI150895 and AI153391 from the National Institutes of Health (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.

Contributor Information

Christopher J. Kristich, Email: ckristich@mcw.edu.

Michael J. Federle, University of Illinois Chicago, Chicago, Illinois, USA

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[B1] 1. 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 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B2] 2. Comenge Y, Quintiliani R, Li L, Dubost L, Brouard JP, Hugonnet JE, Arthur M. 2003. The CroRS two-component regulatory system is required for intrinsic β-lactam resistance in Enterococcus faecalis. J Bacteriol 185:7184–7192. doi: 10.1128/JB.185.24.7184-7192.2003 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B3] 3. Muller C, Le Breton Y, Morin T, Benachour A, Auffray Y, Rincé A. 2006. The response regulator CroR modulates expression of the secreted stress-induced SalB protein in Enterococcus faecalis. J Bacteriol 188:2636–2645. doi: 10.1128/JB.188.7.2636-2645.2006 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4. Le Breton Y, Muller C, Auffray Y, Rincé A. 2007. New insights into the Enterococcus faecalis CroRS two-component system obtained using a differential-display random arbitrarily primed PCR approach. Appl Environ Microbiol 73:3738–3741. doi: 10.1128/AEM.00390-07 [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

The extracellular segment of CroS is not required for sensing but fine-tunes the magnitude of CroS signaling to regulate cephalosporin resistance in Enterococcus faecalis

Sarah B Timmler

Christopher J Kristich

Roles