Research Article

Use of a Phosphorylation Site Mutant To Identify Distinct Modes of Gene Repression by the Control of Virulence Regulator (CovR) in Streptococcus pyogenes

Nicola Horstmann, Pranoti Sahasrabhojane, Hui Yao, Xiaoping Su, Samuel A. Shelburne
Ann M. Stock, Editor
DOI: 10.1128/JB.00835-16

ABSTRACT

Control of the virulence regulator/sensor kinase (CovRS) two-component system (TCS) serves as a model for investigating the impact of signaling pathways on the pathogenesis of Gram-positive bacteria. However, the molecular mechanisms by which CovR, an OmpR/PhoB family response regulator, controls virulence gene expression are poorly defined, partly due to the labile nature of its aspartate phosphorylation site. To better understand the regulatory effect of phosphorylated CovR, we generated the phosphorylation site mutant strain 10870-CovR-D53E, which we predicted to have a constitutive CovR phosphorylation phenotype. Interestingly, this strain showed CovR activity only for a subset of the CovR regulon, which allowed for classification of CovR-influenced genes into D53E-regulated and D53E-nonregulated groups. Inspection of the promoter sequences of genes belonging to each group revealed distinct promoter architectures with respect to the location and number of putative CovR-binding sites. Electrophoretic mobility shift analysis demonstrated that recombinant CovR-D53E protein retains its ability to bind promoter DNA from both CovR-D53E-regulated and -nonregulated groups, implying that factors other than mere DNA binding are crucial for gene regulation. In fact, we found that CovR-D53E is incapable of dimerization, a process thought to be critical to OmpR/PhoB family regulator function. Thus, our global analysis of CovR-D53E indicates dimerization-dependent and dimerization-independent modes of CovR-mediated repression, thereby establishing distinct mechanisms by which this critical regulator coordinates virulence gene expression.

IMPORTANCE Streptococcus pyogenes causes a wide variety of diseases, ranging from superficial skin and throat infections to life-threatening invasive infections. To establish these various disease manifestations, Streptococcus pyogenes requires tightly coordinated production of its virulence factor repertoire. Here, the response regulator CovR plays a crucial role. As an OmpR/PhoB family member, CovR is activated by phosphorylation on a conserved aspartate residue, leading to protein dimerization and subsequent binding to operator sites. Our transcriptome analysis using the monomeric phosphorylation mimic mutant CovR-D53E broadens this general notion by revealing dimerization-independent repression of a subset of CovR-regulated genes. Combined with promoter analyses, these data suggest distinct mechanisms of CovR transcriptional control, which allow for differential expression of virulence genes in response to environmental cues.

INTRODUCTION

Bacteria causing infections in humans must closely modulate virulence factor production in response to changing environmental conditions (1–3). Two-component gene regulatory systems (TCS) are a major mechanism by which bacteria coordinate gene expression, are critical to bacterial pathogenesis, and are potential targets for novel drug development (4–7). TCS, which are absent in metazoans but particularly abundant in bacteria, generally consist of a membrane-embedded histidine kinase that determines the phosphorylation level of a cognate response regulator protein, which in turn affects regulator function (8, 9). Given their critical role in a broad array of key cellular processes, TCS have been extensively investigated; yet, knowledge regarding numerous important aspects of TCS function continues to accumulate (10, 11). For example, it has only recently been appreciated that the effect of response regulator phosphorylation on gene expression can exhibit a gradient rather than an “all or none” pattern, which in turn markedly expands the ability to fine-tune gene transcript levels in response to changing environmental stimuli, such as those encountered during infection (12, 13).

The control of virulence regulator (CovR; also known as capsule synthesis regulator [CsrR]) of group A Streptococcus (GAS) serves as a model system for studying how TCS impact Gram-positive bacterial pathogenesis (14–16). GAS, also called Streptococcus pyogenes, is a strictly human pathogen that causes a variety of disease manifestations ranging from uncomplicated pharyngitis to life-threatening invasive infections (17). Thus, GAS virulence gene expression is tightly regulated to adapt to changing environmental challenges (18). Along with its cognate histidine kinase CovS, CovR is a major transcriptional regulator controlling virulence factor production in GAS (19). CovR primarily serves to repress virulence factor gene expression by binding to promoter regions of target genes (20). Similar to other members of the large OmpR/PhoB family of response regulators, phosphorylation of a conserved aspartate in the N-terminal domain of CovR is thought to be pivotal for its regulatory activity, as it elicits interdomain surface perturbations that promote protein dimerization and subsequent increased affinity for target DNA (8, 21). Accordingly, a CovR deletion strain and a strain harboring the phosphoablative D53A substitution are hypervirulent due to relieved repression of virulence factor expression (22).

Despite being studied for greater than 15 years, new information about the critical role of the CovRS system and its mechanistic principles continues to accumulate at a high rate (23–26). It has recently been discovered that several regulatory pathways play a part in fine-tuning CovR phosphorylation status in response to different environmental cues (22–24, 27, 28). This includes phosphorylation or dephosphorylation of CovR D53 by the cognate sensor histidine kinase CovS, as well as negative regulation of CovR D53 phosphorylation through mutually exclusive phosphorylation on a second phosphorylation site, T65, by the serine/threonine kinase (Stk) (22, 23). Additionally, the putative standalone histidine kinase regulator of CovR (RocA) was also recently discovered to impact CovR D53 phosphorylation, and it was shown that a naturally occurring mutation in RocA results in low-level CovR phosphorylation and hypervirulence in serotype M3, as well as serotype M18 GAS strains (24, 27, 29).

Despite these advances, critical gaps remain regarding CovRS function. For example, in vitro phosphorylation of CovR on D53 can presently be achieved only with small-molecule phosphodonors, and the physiologic relevance of this phosphorylation remains unclear. Moreover, the labile nature of the aspartate phosphorylation imparts significant problems to the study of CovR D53 phosphorylation.

Phosphorylation-mimicking mutations have been widely employed to investigate transient or difficult-to-access phosphoproteins (30, 31). In this approach, the amino acid phosphorylation site is mutated to an aspartate or glutamate to introduce a negative charge, which can impact a protein in a fashion similar to phosphorylation (30, 32, 33). For example, we recently generated strain CovR-T65E, which allowed for elucidation of the impact of Stk-mediated phosphorylation of CovR T65 on GAS gene regulation and virulence (22). Herein, we sought to mimic CovR phosphorylation on its primary phosphorylation site, D53, with the goal of creating a strain harboring a constitutively active CovR in order to investigate the functional consequence of CovR D53 phosphorylation. Unexpectedly, functional analysis of this mutant revealed a subgroup of the CovR regulon that is repressed independent of CovR dimerization. This discovery is a crucial step toward elucidating distinct molecular mechanisms by which CovR coordinates virulence gene expression in GAS.

RESULTS

CovR-D53E cannot be phosphorylated on D53 while retaining Stk-mediated phosphorylation of T65.It has been established that the small-molecule phosphodonor acetyl phosphate can phosphorylate CovR on D53 in vitro (34). Thus, we first tested whether the exchange of CovR D53 with glutamate interferes with acetyl phosphate-mediated protein phosphorylation. To this end, we generated the CovR variant CovR-D53E and purified the recombinant protein using the NEB IMPACT system, as previously described (35). Circular dichroism spectroscopy confirmed that the introduced mutation at position 53 did not lead to aberrant folding of the protein (Fig. 1A). CovR-D53E, along with wild-type CovR and the nonphosphorylatable CovR mutant D53A (22) (as positive and negative controls, respectively) were incubated with acetyl phosphate, and the phosphorylation status was analyzed by 10% Phos-tag–SDS-PAGE. Whereas wild-type CovR was phosphorylated in the presence of acetyl phosphate, as indicated by retardation in the Phos-tag gel compared to the unphosphorylated species, both the exchange of D53 to alanine and glutamate completely abolished phosphorylation (Fig. 1B).

FIG 1
FIG 1

CovR-D53E cannot be phosphorylated on D53 while retaining Stk-mediated phosphorylation of T65. (A) Far-UV (200 to 260 nm) circular dichroism (CD) spectra of CovR wild type (wt; blue) and CovR-D53E (red), showing that exchange of CovR D53 to glutamate does not affect the secondary structure of the protein. (B to D) The phosphorylation status of wild-type and mutant CovR was analyzed by 10% Phos-tag-PAGE. Shown are representative gels with identical results obtained on three different occasions. (B) In vitro phosphorylation of CovR D53. Proteins were incubated in the absence (−) and presence (+) of acetyl phosphate (Ac-P) as phosphate donor. Gels were stained with Coomassie blue. (C) In vivo phosphorylation of CovR D53. MGAS10870 and 10870-CovR-D53E were grown to mid-exponential phase in THY or THY supplemented with 15 mM MgCl2 (Mg2+). Cell lysates containing 70 μg of protein were loaded per lane, respectively. Recombinant un/phosphorylated CovR served as controls. The Phos-tag-gel was blotted and probed with anti-CovR antibodies. (D) In vitro phosphorylation of CovR T65. Proteins were incubated in the absence (−) or presence (+) of either acetyl phosphate or Stk/ATP as phosphate donor. The Phos-tag gel was blotted and probed with anti-CovR antibodies.

Next, we created an isoallelic MGAS10870 variant in which CovR D53 was exchanged with glutamate. The resulting strain, 10870-CovR-D53E, and wild-type strain MGAS10870 were grown to mid-exponential phase in Todd-Hewitt broth with 0.2% yeast extract (THY) medium or THY supplemented with 15 mM MgCl2 (low- or high-Mg2+ condition, respectively), as Mg2+ supplementation significantly increases phosphorylated CovR (CsovR∼P) levels (23, 36–38). The cells were lysed, and CovR in vivo phosphorylation status (calculated as [CovR∼P]/[CovRtotal]) was analyzed by Phos-tag SDS-PAGE, followed by Western blotting using anti-CovR antibodies. Consistent with our in vitro results, CovR∼P was detectable only in strain MGAS10870, indicating that CovR-D53E cannot be phosphorylated in vivo (Fig. 1C).

We recently demonstrated that Stk phosphorylates GAS CovR at T65, thereby providing a second CovR phosphorylation site in addition to D53 (22). Presently, it is thought that phosphorylation on one CovR site (i.e., D53 or T65) antagonizes phosphorylation at the other site (22, 30). Thus, we next tested the hypothesis that recombinant CovR-D53E would be phosphorylated at a lower rate by Stk than by wild-type CovR. In the presence of Stk, ATP, and Mg2+, recombinant CovR, CovR-D53A, and CovR-D53E proteins were phosphorylated, although to a different degree (Fig. 1D). Consistent with mutual antagonism between the two phosphorylation sites, Stk-mediated phosphorylation of CovR-D53A was increased compared to the CovR wild type but lower for CovR-D53E (Fig. 1D). Thus, although the CovR-D53E protein is not phosphorylated in vitro or in vivo on position 53, it retains its ability to be phosphorylated on T65 by Stk.

CovR expression in strain 10870-CovR-D53E is similar to that of MGAS10870.It is known that CovR autoregulates its expression (39), and we have shown previously that intracellular CovR amounts can be profoundly influenced by single amino acid substitutions in the CovR protein (see strain CovR-T65A in reference 22). Hence, conclusions about the role of a specific amino acid in CovR function can be drawn only if this particular mutant is expressed at a level similar to that of the wild-type protein. Thus, we analyzed CovR transcript and protein levels in strain 10870-CovR-D53E compared to wild-type strain MGAS10870 during growth under low- and high-Mg2+ conditions using quantitative real-time PCR (qRT-PCR) and Western blot analysis, respectively. The two strains had comparable growth curves (Fig. 2A), and cells were harvested in mid-exponential phase. We found that covR transcript and CovR protein levels were not significantly affected by the exchange of D53 to glutamate (Fig. 2B and C), indicating that the D53E alteration in CovR does not significantly impact CovR production.

FIG 2
FIG 2

In vivo expression of CovR. (A) Growth curves of strains MGAS10870 and 10870-D53E in THY medium. Optical density at 600 nm (OD600) values represent averages from three biological replicates ± standard deviations. (B) qRT analysis showing in vivo transcript levels of covR in MGAS10870 (wild type, green) and 10870-CovR-D53E (purple) strains grown to mid-exponential phase under low- and high-Mg2+ conditions. Data graphed are means ± standard deviations, n = 8. Growth was done in duplicate on two different occasions. (C) Representative Western blot (n = 2) using anti-CovR antibodies to depict CovR protein levels (upper band) in cell lysates of MGAS10870 and 10870-CovR-D53E grown to mid-exponential phase. Anti-HPr antibodies were used to detect HPr protein levels (lower band) as a loading control. Recombinant CovR (rCovR) was loaded on the gel as a control. Migration of molecular weight markers in the area of interest is indicated on the right.

Strain 10870-D53E-CovR has a colony phenotype on sheep blood agar similar to that of a covR deletion strain.Given that CovR∼P represses production of the GAS hyaluronic acid capsule by binding to the promoter of hasA, the initial gene in the capsule operon, GAS strains with altered CovR∼P levels can have distinct colony morphologies (22, 23). We recently established that changing CovS T284 to alanine impairs CovS phosphatase activity, thereby increasing CovR∼P levels (23). Therefore, we compared the phenotype of strain 10870-CovR-D53E with those of strains 10870-CovS-T284A (high CovR∼P level), MGAS10870 (medium CovR∼P level), and the covR deletion strain 10870 ΔcovR (absent CovR∼P) to evaluate whether a D53E mutation in CovR functionally mimics phosphorylation of CovR D53. Surprisingly, the phenotype (colony size) of strain 10870-CovR-D53E closely resembled that of 10870 ΔcovR, suggesting that CovR-D53E does not suppress hyaluronic acid capsule production the same way as expected from a GAS strain with a D53 constitutive phosphorylation phenotype (Fig. 3).

FIG 3
FIG 3

Phenotypical characterization of CovR and CovS variants. Photographs of indicated strains following overnight growth on 5% sheep blood agar plates. Pictures show representative colony morphologies obtained on two different occasions.

Genome-wide analysis of gene expression in GAS strain 10870-CovR-D53E.To determine whether CovR-D53E functions as a CovR constitutive phosphorylation mimic, we compared the transcriptomes of the wild-type and 10870-CovR-D53E strains via RNA sequencing (RNA-seq) analysis. To this end, strains MGAS10870 and 10870-CovR-D53E were grown in quadruplicate to mid-exponential phase in THY. Principal-component analysis (PCA) revealed that under this condition, the transcriptomes of strain 10870-CovR-D53E and MGAS10780 were distinguishable and reproducibly grouped by strains (Fig. 4A). We considered a transcript level to be significant different when the mean fold change was ≥2 and the adjusted P value was <0.05 after correction for multiple comparisons. However, only 9 genes met these stringent criteria (Table 1); including genes with at least 1.5-fold difference in transcript level increased the number to 68. However, this remains far less than the 200 genes with ≥1.5-fold change in transcript levels previously observed for strain 10870 ΔcovR or 10870-CovR-D53A versus the wild type under the same conditions (22). All genes with increased transcript levels in strain 10870-CovR-D53E compared to the wild type are known to be CovR repressed, including genes encoding the virulence factors streptococcal pyrogenic exotoxin A (SpeA) and streptococcal collagen-like protein A (SclA) (37, 40). Transcription of known virulence genes hasA, prtS (also called spyCEP, encoding Streptococcus cell envelop protease), spyM3_0105, and mac-1 (encoding a cysteine protease) was increased in 10870-D53E compared to the wild type (Fig. 4B) but with slightly less than 2-fold difference (Table 1). Importantly, we observed no statistically significant differences in transcript level for such well-described CovR-repressed genes as sagA, which contains the first gene in the operon encoding the GAS streptolysin S, or slo, which encodes the pore-forming toxin streptolysin O (Fig. 4B). All genes whose transcript levels were significantly lower in strain 10870-CovR-D53E than in the wild type were phage-derived genes, previously described as being activated by phosphorylated CovR, e.g., speC and sdn, which encode Streptococcus pyrogenic exotoxin C and an actively secreted DNase, respectively (22) (Fig. 4B). Conversely, we observed no significant differences in the transcript levels of grab, which encodes a protein G-related α2-macroglobulin-binding protein, or mf, which encodes an actively secreted DNase, both of which have also been found to be activated by phosphorylated CovR (22, 41) (Fig. 4B). Thus, under low-Mg2+ conditions, for both CovR∼P-activated and -repressed genes, CovR-D53E not only fails to function as constitutively phosphorylated CovR, but for some genes, it exerts only a portion of wild-type CovR∼P activity.

FIG 4
FIG 4

RNA-seq analysis of strain 10870-CovR-D53E versus MGAS10870. (A) Principal-component analysis (PCA) showing that strains MGAS10870 and 10870-CovR-D53 have distinct transcriptomes. The transcriptomes of four biological replicates were analyzed for each strain. (B) Heat map of CovR-regulated genes for 10870-CovR-D53E versus MGAS10870. The heat map depicts the variance stabilizing transformed read counts of selected genes for individual samples of 10870-CovR0D53E relative to the averaged value for strain MGAS10870.

View this table:
TABLE 1

Summary of RNA-seq data, with selected genes differentially expressed in strain 10870-CovR-D53E compared to MGAS10870

Transcript levels of CovR-regulated genes in strain 10870-CovR-D53E reveal distinct CovR-regulated gene groupings.We have previously determined that the effect of CovR single-amino-acid substitutions on transcription regulation strongly depends on the Mg2+ concentration in the medium (22). Thus, to further analyze the regulatory ability of CovR-D53E, we measured the transcript levels of various known CovR-regulated virulence genes in strains 10870-CovR-D53E, 10870 ΔcovR, and 10870-CovS-T284A compared to strain MGAS10870 (Fig. 5). In concert with our transcriptome data, CovR-D53E evidenced only partial regulatory capacity, but the extent of CovR-D53E activity depended on the growth medium and on the particular gene studied. For numerous CovR-regulated genes, such as sagB (second gene of sag operon involved in biosynthesis of streptolysin S), cbp (encoding a putative collagen binding protein), and covR itself, the CovR-D53E protein retains normal regulatory activity (Fig. 5A and B and data not shown). For these genes, in both growth media, the transcript levels in strain 10870-CovR-D53E were similar to those in strains MGAS10870 and 10870-CovS-T284A, whereas strain 10870 ΔcovR had significantly higher transcript levels (Fig. 5A and B). These data indicate that for this group of CovR-repressed genes, the presence of the CovR-D53E protein is sufficient to control gene expression, and increasing the amount of phosphorylated CovR does not significantly reduce gene transcript levels.

FIG 5
FIG 5

Transcript level analysis of various CovR-regulated promoters. Transcript levels (means ± standard deviations; n = 8) of selected genes in the indicated strains relative to those in MGAS10870 as measured by TaqMan qRT-PCR: sagB (A), cbp (B), hasA (C), prtS (D), mac-1 (E), and grab (F). Strains were grown in duplicate on two different occasions to mid-exponential phase under low- and high-Mg2+conditions, as indicated.

In contrast, the transcript levels of the CovR-repressed virulence genes hasA, prtS, and mac-1 were significantly different in strain 10870-CovR-D53E from those of the wild type and were highly dependent on CovR∼P levels (Fig. 5C to E). Specifically, the transcript levels of these genes were similar between strains 10870-CovR-D53E and 10870 ΔcovR and were significantly increased in both of these strains compared to MGAS10870, in particular during growth under high-Mg2+ conditions. Consistent with being highly susceptible to alterations in CovR∼P, transcript levels of hasA, prtS, and mac-1 were significantly decreased in strain 10870-CovS-T284A compared to those in MGAS10870 (Fig. 5C to E).

The grab gene represents an exceptional case in that it is negatively regulated by CovR but activated by the kinase activity of CovS (41). Given that CovS primarily serves to increase CovR∼P levels, it appears that high CovR∼P increases the grab transcript level, whereas low CovR∼P levels decrease the grab transcript level. In concert with this theory, the grab transcript level was increased in both 10870 ΔcovR and CovS-T284A (i.e., when there is either no CovR protein or high levels of CovR∼P) compared to MGAS10780. In strain 10870-CovR-D53E, the grab transcript level was similar to that of the wild type under low-Mg2+ conditions but was lower under high-Mg2+ conditions, indicating that CovR-D53E retains partial activity for grab regulation (Fig. 5F). Taken together, these data demonstrate that CovR-D53E retains activity similar to wild-type CovR for only a subset of CovR-influenced genes, suggesting distinct mechanisms of CovR-mediated gene expression at various promoters.

CovR-D53E protein retains its ability to bind promoter DNA.Given that strain 10870-CovR-D53E possessed different CovR regulatory activity depending on the examined gene, we next performed electrophoretic mobility shift analyses (EMSA) to test the ability of recombinant CovR-D53E protein to bind DNA from selected promoters of both groups. To this end, approximately 300-bp promoter regions of covR and sagA (D53E regulated), as well as prtS and mac-1 (D53E nonregulated), were amplified by PCR and incubated with increasing amounts of recombinant CovR-D53E protein. The samples were subsequently separated by native Tris-borate-EDTA–PAGE (TBE-PAGE). We did not attempt to calculate any equilibrium dissociation constant (KD) values for CovR-DNA binding, because CovR tends to bind stepwise to promoter DNA, thereby forming complexes of increasing molecular weight (symbolized with triangle in Fig. 6), and the importance of those complexes for gene regulation has not been clarified. Therefore, DNA-binding patterns were compared instead. CovR-D53E bound to all promoters tested, and DNA binding was comparable to that of CovR∼P (reference 22, and data not shown). Surprisingly, DNA binding of CovR-D53E was not reduced for the D53E-nonregulated promoters prtS or mac-1 compared to D53E-regulated promoters, like covR and sagA (Fig. 6), indicating that factors besides CovR-DNA binding are likely to play a decisive role in CovR-mediated transcriptional regulation of this gene group.

FIG 6
FIG 6

CovR-D53E binding to promoter DNA. Binding of recombinant CovR-D53E protein to DNA of the CovR-D53E regulated promoters of covR (A) and sagA (B) and the CovR-D53E-nonregulated promoters prtS (C) and mac-1 (D). Increasing protein concentrations (in micromolar) were used as indicated. Samples were electrophoresed on a 6% TBE-PAA gel for 60 min at 120 V. The gels were stained with ethidium bromide. ns, nonspecific DNA; f, free DNA; c, protein-DNA complex. The triangle symbolizes the continuum from lower- to higher-molecular-weight complexes. Gels shown are representatives of identical results obtained on two separate occasions.

CovR-D53E is a monomeric protein.It is currently thought that phosphorylation of an OmpR/PhoB family response regulator shifts the equilibrium from a monomeric inactive protein to a dimeric active regulator via the α4-β5-α5 interface, and it has been shown that in vitro phosphorylation of CovR results in dimerization (9, 34). We therefore analyzed the oligomerization state of CovR-D53E. Recombinant CovR wild-type and CovR-D53E mutant proteins were incubated in kinase buffer in the absence and presence of acetyl phosphate and subsequently electrophoresed on a 4 to 16% blue native polyacrylamide (BN-PAGE) gel. During BN-PAGE, proteins are separated exclusively by molecular weight (theoretical molecular mass for CovR is ∼26 kDa for monomer versus 52 kDa for dimer), as opposed to a combination of molecular weight and surface charge as in conventional native PAGE. While the CovR wild type dimerized upon phosphorylation with acetyl phosphate, CovR-D53E was monomeric under both conditions (Fig. 7). Thus, the elongation of amino acid residue 53 by one methylene group with the D53E substitution is not sufficient to induce CovR dimerization.

FIG 7
FIG 7

Analysis of CovR oligomerization state. Four to 16% BN-PAGE of recombinant CovR wild type and CovR-D53E in the absence (−) and presence (+) of acetyl phosphate. MW, molecular weight standard. The gel shown is representative of identical results obtained on three separate occasions.

Strains with mutations in CovR dimerization interface have transcription pattern similar to 10870-CovR-D53E.Our results suggested that CovR might be capable of regulating transcription of D53E-regulated genes independent of its oligomerization state. To further explore this hypothesis, we generated two isoallelic GAS strains, which harbor mutations within the CovR α4-β5-α5 dimer interface: 10870-CovR-D93A and 10870-CovR-D97A. CovR aspartates 93 and 97 are highly conserved within the OmpR/PhoB family of transcription regulators. Based on structural modeling, they engage in salt bridges with arginines 114 and 119, respectively, from the other CovR protomer, thereby contributing to the dimer interface of the activated response regulator (42) (corresponding residues in PhoB are E96-K117 and D100-R119). Thus, a mutation of aspartate residue 93 or 97 to an alanine would be expected to impair CovR dimerization. The resulting strains were grown under high-Mg2+ conditions to mid-exponential phase, and transcription levels were measured for the promoters of sagB and cbp (D53E regulated), as well as prtS and hasA (nonregulated) (Fig. 8). Notably, for both strains, the regulation pattern resembled that of strain 10870-CovR-D53E. Specifically, transcript levels of sagB and cbp did not significantly differ in strains 10870-CovR-D93A and 10870-CovR-D97A compared to wild-type strain MGAS10870 (Fig. 8A). In contrast, repression of prtS and hasA transcription was remarkably relieved in these strains (Fig. 8B).

FIG 8
FIG 8

Influence of CovR dimerization on the regulation of various CovR-regulated promoters. Transcript levels (means ± standard deviations; n = 8) of selected genes in the indicated strains relative to those in MGAS10870, as measured by TaqMan qRT-PCR: sagB and cbp (A) and prtS and hasA (B). Strains were grown in duplicate on two different occasions to mid-exponential phase under high-Mg2+ conditions.

Genes responsive and nonresponsive to CovR-D53E repression reveal distinct promoter architectures.The ability of CovR-D53E to influence the expression of a subset of CovR-regulated genes despite being unable to dimerize suggests distinct modes of CovR-mediated gene expression at various promoters. Thus, we next inspected the promoter region of several genes that either were or were not regulated in strain 10870-CovR-D53E (Fig. 9). We searched 200 bp up- and downstream of the respective transcription start site for possible CovR-binding sites, in particular for the established CovR consensus sequence ATTARA (20) and an expanding motif (TATTTTAAT) identified in group B Streptococcus, which has a CovR protein highly similar to that in GAS (43). We found that promoters from CovR-D53E-regulated and -nonregulated genes have distinct architectures with respect to the number and location of predicted CovR-binding sites. The promoters of hasA, prtS, mac-1, and spyM3_0105 exemplifying the group of CovR-D53E-nonregulated genes generally contain more potential CovR-binding sites, which are conspicuously clustered around the −35 and −10 promoter elements and the transcription start point (Fig. 9). Moreover, the occurrence of several consecutive or overlapping sites in both strands provides opportunities for CovR dimerization at these promoters. In contrast, the promoters of CovR-D53E-regulated genes, like cbp, sagA, or covR, contain a limited number of putative CovR-binding sites which are nonoverlapping and less concentrated around the promoter elements and transcription start site than CovR-D53E-nonregulated genes. Rather, putative CovR-binding motifs in the promoters of CovR-D53E-regulated genes were often 100 bp or greater in the 5′ direction to the transcription start or located in the coding area or the untranslated sequence in the 3′ direction to the transcription start site, a configuration that we did not observe for CovR-D53E-nonregulated genes. In conjunction with the transcript level and CovR-D53E dimerization assays, these data suggest that variance in promoter architecture of CovR-regulated genes dictates distinct modes of CovR-mediated gene regulation.

FIG 9
FIG 9

Distinct promoter architectures of CovR-regulated gene groupings. Scheme of promoter architectures (within 200 bp upstream of and downstream from transcription start) of selected genes exemplifying the CovR-D53Eregulated (A) and CovR-D53E-nonregulated (B) gene groups. The name of the gene is indicated on the right. The −35 and −10 promoter regions are labeled in red. The transcription start is marked by a black arrow and labeled +1. Pink arrows indicate ATTARA CovR-binding sites. Blue arrows depict GBS CovR consensus motifs (TATTTTAAT), with dark blue allowing maximally one mismatch and light blue allowing two mismatches. Note that actual role of all of these binding sites in vivo has not yet been verified.

DISCUSSION

As the key regulatory system of GAS, the CovRS TCS serves as a prototype for investigating the impact of TCS on bacterial pathogenesis, yet an understanding of the mechanisms by which CovRS regulates GAS virulence gene expression remains incomplete. A major hurdle in studying the structural and functional consequences of CovRS phosphorylation is the labile nature of the CovR D53 phosphoanhydride bond. Thus, herein we sought to investigate the impact of CovR D53 phosphorylation via creation of a phosphorylation mimic strain, 10870-CovR-D53E, which we predicted to have a constitutive phosphorylation phenotype.

Contrary to our expectations, strain 10870-CovR-D53E exhibited only partial CovR activity. Fractional activity has been observed previously for other TCS response regulators in which the regulatory aspartate residue had been mutated to a glutamate (44–46). In these examples, although being constitutive (independent of cognate histidine kinase signaling), the activity of the mutant response regulator was reduced compared to the phosphorylated wild type and therefore contingent on copy number, i.e., amount of active protein in the cell (44–46). In contrast, in the case of CovR, the repressor activity was promoter dependent, indicating that CovR might apply more diverse regulatory mechanisms to differentially adjust gene expression of its large regulon.

On this note, a key finding of our work was that the CovR D53E substitution impeded CovR dimerization, yet this inability to dimerize only abolished CovR regulation of a subset of CovR-controlled genes. The current model of OmpR/PhoB family regulator function predicates that response regulator dimerization is the driving force for phosphorylation-mediated gene regulation through binding to tandem DNA-binding sites (47–49). Our CovR-D53E transcription data suggest that CovR may employ additional regulatory mechanisms beyond that of typical OmpR/PhoB family members, the more so as in contrast to CovR many of them mainly function as transcriptional activators (50, 51). Given that strains 10870-CovR-D53E, 10870-CovR-D93A, and 10870-CovR-D97A repressed gene expression from certain promoters (e.g., sagA and cpb) similarly to strains MGAS10870 and CovS-T284A, our data suggest that CovR is in principle capable of functioning as a monomeric protein. Our findings resemble those reported for CtrA from Caulobacter crescentus, in which an analogous CtrA-D51E isoform lacked the ability to bind DNA from the origin of replication in a congruent fashion to CtrA∼P, yet the mutant strain retained activity for a subset of CtrA functions (46, 52, 53). Interestingly, subsequent studies have demonstrated that CtrA promoter DNA binding is achieved by different interaction modes in terms of protein oligomerization and with different effects of phosphorylation depending on spatial distinct arrangements of DNA-binding consensus motifs in the promoters of CtrA-controlled genes (54). This finding is strikingly similar to the distinct promoter architectures we identified in CovR-D53E-regulated and -nonregulated genes, suggesting that diverse modes of response regulator function may be more common in OmpR/PhoB family members than is currently appreciated. Thus, our genome-wide analysis of CovR modes of gene expression extend the findings of potentially different modes of CovR gene regulation dependent upon promoter architecture, as noted by Churchward (19).

In support of the concept of distinct modes of CovR-mediated gene regulation, the groups of CovR-D53E-regulated and -nonregulated genes closely mimic those previously identified as Mg2+-responsive and Mg2+-nonresponsive CovR-repressed genes (22, 37). We recently showed that Mg2+ in the growth medium increases CovR phosphorylation (23), indicating that altering CovR∼P levels has discriminative effects on subsets of CovR-regulated genes. The ability of CovR to influence gene expression via separate mechanisms adds another layer of complexity to the intricate network of GAS virulence gene regulation. Via CovS-mediated changes in CovR∼P levels, GAS is able to respond to external stimuli by altering the production of specific virulence factors in a temporal manner during the infection process. For instance, virulence factors critical to interaction with the innate human system, such as the hyaluronic acid capsule and the interleukin-8 (IL-8)-degrading enzyme SpyCEP, not only display a wide range of CovR-influenced expression levels, but their expression is also highly susceptible to alterations in CovR∼P levels. Yet, the production of many other CovR-regulated virulence factors, such as the pilus protein Cbp and streptolysin S, does not vary in response to CovS signaling. Instead, streptolysin S production has been shown to be under the control of several other transcriptional regulators, thereby allowing for fine-tuning by signals from other pathways (55–58). We like to emphasize that regulation of the promoters exemplified here represents extreme cases, while the expression of many genes is probably regulated by mixtures of several mechanisms in differing combinations. Hence, the full CovR-regulated spectrum likely manifests a continuum, with some genes regulated primarily by phosphorylation and others primarily by nondimerization mechanisms.

It has been shown previously that recombinant CovR-D53A protein, similar to unphosphorylated CovR, binds DNA from a broad variety of CovR-regulated genes, with only 2- to 4-fold lower affinity than phosphorylated CovR in vitro (20, 22, 34, 39). However, strain 10870-CovR-D53A exhibits a global expression profile very similar to that of a covR deletion strain (22). Likewise, our CovR-D53E–DNA-binding assays revealed that monomeric CovR-D53E is capable of interacting with promoter DNA from CovR-controlled genes, irrespective of its ability to regulate transcription. Thus, these data show that in vitro DNA-binding affinity is not reflective of CovR activity in vivo. Further, phosphorylation-mediated CovR dimerization per se is evidently not crucial for CovR to bind DNA but instead is critical for mechanisms of CovR-mediated gene repression at particular promoters. In this regard, it is notable that CovR∼P was found to be significantly more effective than nonphosphorylated CovR in repressing hasA gene expression because of its ability to interact with, and presumably interfere with, RNA polymerase (34). Thus, dimerization via the α4-β5-α5 interface of the CovR regulatory domain is likely necessary for interaction with RNA polymerase. Moreover, the location of CovR operator sites around the −35 and −10 promoter elements in the D53E-nonregulated group supports a model in which transcription repression is achieved by trapping RNA polymerase via repressor oligomerization, as proposed for heat-stable nucleoid structuring protein (H-NS) (59, 60). Importantly, RNA polymerase-mediated recruitment of CovR∼P does not occur at sagA or covR promoters (34). Instead, it was previously shown that phosphorylation increased CovR-binding affinity for sagA and covR promoter DNA in vitro and that CovR∼P could polymerize along broad stretches of DNA, consistent with cooperative binding among CovR monomers (39, 61). Thus, we hypothesize that conformational changes in CovR induced by the D53E exchange are sufficient to facilitate CovR∼P-like interactions at promoters where dimerization and subsequent interaction with RNA polymerase are not necessary for CovR-mediated control of gene expression.

In conclusion, although strain 10870-CovR-D53E proved to not fully mimic phosphorylated CovR, it provided valuable global information about the importance of CovR phosphorylation and oligomerization in regulating different promoter groups. These findings allow for further unraveling of the distinct molecular mechanisms by which this critical virulence regulator impacts GAS pathogenesis and augment understanding of the functionality of OmpR/PhoB family members.

MATERIALS AND METHODS

Protein expression and purification.To generate the CovR variant D53E, a single nucleotide exchange was introduced into pTXB1-covR via QuikChange mutagenesis (Stratagene) using the respective primer pair (primer sequences are available upon request). Wild-type and mutant CovR proteins were overexpressed in E. coli BL21/pLysS at 18°C overnight and purified to ≥95% homogeneity using the IMPACT protein purification system (New England BioLabs), as described previously (35). CovR proteins were extensively buffer exchanged to 50 mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) (pH 10.0) and 100 mM NaCl.

In vitro phosphorylation of CovR and detection of CovR phosphorylation status.Wild-type and variant CovR proteins were phosphorylated on D53 for 2 h at 37°C in phosphorylation buffer containing 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 3 mM dithiothreitol (DTT), and 32 μM acetyl phosphate (Sigma), as previously described (61). Phosphorylation on T65 was achieved by incubating the proteins for 30 min at 37°C in phosphorylation buffer containing 100 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, and 10 mM ATP, with a 3-fold excess of recombinant Stk kinase domain (22). The phosphorylation status was detected using 10% Zn2+–Phos-tag–SDS gel electrophoresis at neutral pH, as described previously (22, 23). The polyclonal anti-CovR antibody utilized in this study has been raised in rabbits against purified untagged CovR protein and has been used and tested for specificity, as described in two studies by Horstmann et al. (22, 23).

Circular dichroism spectroscopy.Far-UV (200 to 260 nm) spectra of 0.4 mg/ml recombinant wild-type CovR and CovR-D53E were recorded on a Jasco J-810 spectropolarimeter at 37°C and a scan speed of 20 nm/min, as described previously (35).

EMSA.Promoter regions (∼300 bp) of selected CovR-regulated genes were amplified by PCR from MGAS10870 genomic DNA (primer sequences available on request). The purified PCR products (0.4 μg) were incubated with increasing amounts of recombinant CovR-D53E at 37°C for 15 min in TBE buffer [89 mM Tris, 89 mM borate, 1 mM EDTA, 5% glycerol, and 10 μg/ml poly(dI-dC) (Sigma)]. Samples were then separated on a 6% TBE-phosphonoacetic acid (TBE-PAA) gel for 70 min at 120 V and stained with ethidium bromide.

Analysis of CovR oligomerization state.The oligomerization states of recombinant wild-type CovR and CovR-D53E were analyzed by blue-native electrophoresis. Five milligrams of phosphorylated and unphosphorylated proteins was run on a 4 to 16% NativePAGE Novex Bis-Tris gel (Thermo Fisher Scientific), according to the manufacturer's instructions. NativeMark unstained protein standard (Thermo Fisher Scientific) was used as a molecular weight marker.

Bacterial strains and culture medium.The strains and plasmids used in this study are listed in Table 2. GAS strains were grown to mid-exponential phase (optical density [OD], 0.6) in standard laboratory medium (Todd-Hewitt broth with 0.2% yeast extract [THY], supplemented with 15 mM MgCl2 when indicated) at 37°C in 5% CO2. The MGAS10870 derivatives 10870-CovR-D53E, 10870-CovR-D93A, and 10870-CovR-D97A, which differ from the parental strain only by a single amino acid change, were generated using the chloramphenicol-resistant, temperature-sensitive plasmid pJL1055 (gift of D. Kasper), as described previously (62). E. coli strains were grown under agitation in Luria broth (LB) with 100 μg/ml ampicillin and 25 μg/ml chloramphenicol when needed.

View this table:
TABLE 2

Bacterial strains and plasmids used in this study

RNA isolation and transcript level analysis.RNA was purified in duplicate from the indicated GAS strains using the RNeasy minikit (Qiagen). The hundred nanograms of RNA per sample was transcribed to cDNA using a high-capacity reverse transcription kit (Applied Biosystems) and diluted 10-fold in H2O. The primers and probes used for TaqMan quantitative real-time PCR (qRT-PCR) are available on request. Transcript levels of sagB were measured instead of sagA to avoid difficulties in TaqMan qRT analysis due the short nature of the sagA transcript. Similarly, we did not measure transcript levels for speA or sclA due to problems in designing suitable primer/probes (sclA has a stop codon in M3 GAS strains). qRT-PCR was performed using an Applied Biosystems StepOnePlus system, as described previously (63). All samples were prepared at least in duplicate from cells grown on two separate occasions and analyzed in duplicate (n = 8). A two-sample t test (unequal variance) was applied to test for statistically relevant changes between strains (P < 0.05, and a mean transcript level of ≥2.0-fold change to be considered statistically significant). For transcriptome analyses, strains MGAS10870 and 10870-CovR-D53E were grown in quadruplicate to mid-exponential phase in THY. RNA was isolated as for qRT-PCR and processed and analyzed as previously described (22). Eighty-nine out of 1,853 genes were removed due to low expression levels. A transcript level difference of at least 2.0-fold and an adjusted P value of <0.05 after applying the Benjamini-Hochberg method (64) to control for false discovery was considered significantly different. A hierarchical clustering analysis was performed using the Pearson correlation coefficient as the distance metrics and the Ward's linkage rule. Principal-component analysis (PCA) was also applied to discover the multigene structure.

ACKNOWLEDGMENTS

We declare no conflicts of interest with the contents of this article.

The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

FOOTNOTES

    • Received 6 December 2016.
    • Accepted 3 March 2017.
    • Accepted manuscript posted online 13 March 2017.

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KEYWORDS

Bacterial Proteins
Gene Expression Regulation, Bacterial
Mutant Proteins
Protein Processing, Post-Translational
Repressor Proteins
Streptococcus pyogenes
Streptococcus pyogenes
dimerization
pathogenesis
regulation of gene expression
transcriptional regulation
two-component regulatory systems
virulence regulation

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