The ComE response regulator is activated under acidic conditions by a histidine-kinase independent pathway

We previously reported that ComE was required in the acidic stress induced lysis (ASIL) mechanism, which was independent of its cognate histidine kinase ComD at pH 6.0 [34]. This is in contrast to the ComD/quorum-sensing dependence on ComE activation at pH 7.8 necessary for competence development [19]. This initial observation led us to investigate whether other pneumococcal TCS-associated HKs could be activating ComE by a crosstalk mechanism, as described for other bacteria [36]. We hypothesized that if other HKs were involved in ComE activation by phosphorylation, the corresponding hk mutant should display alterations in the ASIL induction. Thus, the lytic phenotype was determined under acidic stress conditions for all the pneumococcal hk mutants that we had previously constructed in the background of the R801 strain by insertion-duplication mutagenesis (S1 Table) [33]. We observed that all the hk mutants showed the same lytic response as the parental R801strain, indicating that none of the tested HKs was involved in ASIL (S2 Table). In addition, the comD^F183X mutant was used because it lacks the HK domain due to a stop codon at residue 163 (S2 Table) and therefore it is unable to activate ComE [33]. This mutant was constructed to avoid putative alterations in the comCDE operon expression, and it showed the same ASIL phenotype than the ΔcomD mutant (S2 Table), indicating that the truncated ComD protein expressed by the comD^F183X mutant has not impact on the ASIL activation. In order to avoid a putative residual effect of comD^F183X on the ComE activation, we determined ASIL in the double comD^F183X hk mutants, which were constructed by transforming the comD^F183X mutant with individual plasmids containing the different hk mutations. Despite the low transformability of the comD^F183X mutant, all the double comD^F183X hk mutants displayed the same ASIL phenotype as those obtained for each individual hk mutant (S2 Table). Taken together, these results indicated that HKs were not responsible for activation of ComE and the resulting ASIL.

Although ASIL is controlled by ComE activation without HK participation, this finding does not exclude the potential that ComE could have been phosphorylated by another phosphodonor at the Asp⁵⁸ residue, typically the target of ComD phosphorylation [18]. Prokaryotic response regulators can be phosphorylated in vivo by acetyl-phosphate at the conserved aspartate residue of the receiver domain, resulting in similar activation to that exerted by the cognate HK [37]. In addition, phosphorylation crosstalk between HK and RR that belong to different TCSs has been reported [38]. Therefore, we first analyzed the possibility that Asp⁵⁸ phosphorylation could be required for ASIL, and tested the comE^D58A mutant that encodes for the ComE^D58A protein, in which the phosphorylatable Asp⁵⁸ residue is replaced by alanine [18,33]. The presence of this mutation was phenotypically corroborated under competence development conditions, confirming that CSP-induced competence was eliminated in the comE^D58A mutant (S1 Fig and [18]). Like the wt R801 strain, the comE^D58A mutant autolysed under acidic conditions (Fig 1A) indicating that Asp⁵⁸ phosphorylation is not necessary for ASIL. The comE^D58A phenotype is similar to the phenotype displayed by the hk mutants. Taken together, these results suggest ComE activation under acidic conditions is independent of both Asp⁵⁸ phosphorylation and HK activity.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. Evaluation of ASIL and comE expression in S. pneumoniae mutants.

Autolysis was measured as a change in OD_620nm over 6 hours. Lytic curves corresponding to specific mutants are indicated in each panel (A-C), with data being representative of at least three independent experiments. (A) ASIL is controlled by StkP but it does not require Asp⁵⁸-phosphorylation in ComE. (B) StkP does not participate in the CiaRH-regulated ASIL pathway. (C) StkP is involved in the ComE-regulated ASIL pathway. References: *p< 0.05; **p< 0.01; ***p< 0.001, these p-values were referred to the wt strain in each panels. (D) Transcription levels of the comE gene measured in cells exposed to pH 6.0. To avoid autolysis, all mutants were constructed in a ΔlytA (autolysin deficient) background. The ΔlytA, comE^D58A ΔlytA, ΔstkP ΔlytA, comD^T233I ΔlytA and comE^T128A ΔlytA cells were grown in ABM/pH 7.8 to the mid-exponential phase and resuspended in ABM/pH 6.0. Total RNA was extracted at 0 min, 10 min, and 30 min. The fold change in gene expression was measured by quantitative real-time PCR and calculated using the 2^–ΔΔCT method. The gyrB gene was used as the internal control and the reference condition was time 0 min of strain ΔlytA. Error bars indicate the standard deviation of the mean. INSTAT software was used to perform Dunnet’s statistical comparison test for each strain with its respective basal condition (time 0 min). References: **p< 0.01; ***p< 0.001.

https://doi.org/10.1371/journal.ppat.1007118.g001

The serine/threonine kinase StkP is essential for ASIL activation and participates in the ComE pathway

Since it has been shown that StkP is involved in comCDE expression during competence development at pH 7.8 [4], and that ComE is indispensable for the induction of ASIL [34], we evaluated whether StkP participated in ASIL development. Thus, the ΔstkP mutant strain did not autolyze when cultured under acidic conditions (Fig 1A), strongly suggesting that StkP is necessary for ASIL induction. To further confirm whether StkP kinase activity was required for ASIL, we also constructed the stkP^K42R mutant, which encodes for StkP with reduced enzymatic activity, as previously described [39]. This reduced kinase activity produces multiple septa, peripheral peptidoglycan biosynthesis and elongated cells [8], and these alterations were confirmed in our mutant and compared with the wt strain (S2A and S2B Fig). As expected, the stkP^K42R mutant strain showed ASIL with a degree of autolysis inferior to the wt strains but higher than the ΔstkP strain, indicating that the residual kinase activity in the stkP^K42R mutant [39] was likely responsible for ASIL induction. These observations strongly suggest that StkP kinase activity is essential for ASIL activation.

To exclude the possibility that the ASIL blockage observed in the ΔstkP mutant is a side effect due to hampered cell division and/or compromised cell wall structure [13], we constructed another mutant that presents cell division alterations, such as the ΔmapZ mutant [9]. We verified these alterations by Van-Fl staining (S2C Fig) and found that the ΔmapZ mutant showed an ASIL phenotype similar to the wt strain (S2D Fig), indicating that the ASIL effect showed by the ΔstkP mutant is independent of cell division alterations.

We have previously demonstrated that ASIL is controlled by two independent signaling pathways, CiaRH and ComE. While CiaRH plays a protective function, the ComE acts by promoting ASIL [34]. To determine whether StkP participated in the CiaRH-controlled ASIL pathway, the ΔstkP and ΔciaR mutations were backcrossed, and the lytic phenotype of the resulting double mutants was analyzed. The ΔstkP ΔciaR strain showed enhanced autolysis, similar to the ΔciaR single mutant, demonstrating that the ciaR mutation had an epistatic effect on the non-autolytic stkP phenotype and that StkP activity did not participate in the signaling events of the CiaRH-controlled ASIL pathway (Fig 1B).

It has been previously described that the comD^T233I mutation produces a hyperactive ComD HK, which in turn hyperphosphorylates ComE, activates comCDE transcription, and results in high intracellular levels of ComE [40]. Although our results suggested that ComD was not involved in ComE-mediated ASIL, we constructed the comD^T233I mutant to artificially produce high levels of ComE and competence, as described [34,40]. The comD^T233I mutant showed constitutive high levels of comE transcripts, at 1000-fold higher compared to the wt strain. This mutant displayed accelerated ASIL compared to the wt strain, however, we previously reported that ASIL was blocked in the double comD^T233I ΔcomE double mutant [34]. These results demonstrated that ASIL induction was ComE-dependent and that increase expression of the latter leads to accelerated autolysis [34]. More importantly, when the stkP gene was disrupted in the comD^T233I mutant, ASIL was also completely blocked (Fig 1C), suggesting that StkP is essential for ASIL activation despite the high ComE levels expressed in the comD^T233I mutant.

comE expression is induced in response to acidic conditions

It is known that comE is part of the comCDE operon, with the transcription of this operon initiated at the pcomC promoter (bp 2035421–2035806, S. pneumoniae R6 genome, NCBI reference: NC_003098.1) during competence development at alkaline pH [41]. To test whether pcomC was responsible for the increase in comE observed under acidic conditions, we constructed the pcomC-lacZ reporter fusion (S1 Table), which was integrated via a single crossover event upstream of comC in a bgaA mutant (deficient in β-galactosidase activity). When the bgaA mutant strain carrying the pcomC-lacZ fusion was incubated at pH 6.0 for 30 min., a 1.7-fold increase in β-galactosidase activity was observed. No such increase was detected in a ΔcomE knocked out mutant (S3 Fig). To further confirm this observation, the levels of comE transcript in the wt strain were determined by qPCR, which showed a 4-fold increase in cells exposed for 30 min at pH 6.0 (Fig 1D). These results indicate that the increased number of comE transcripts was caused by acidic stress, with this activation being dependent on ComE. Similarly, the levels of comE transcript in the comE^D58A mutant were increased 4.5-fold after 30 min incubation at pH 6.0. This result indicates that ComE^D58A was able to induce ASIL under acidic stress conditions (Fig 1D). Consequently, these results suggest that ComE is activated by an alternative signaling pathway that does not require phosphorylation of Asp⁵⁸.

We previously reported that induction of comE transcripts by acidic stress is a characteristic of the ComE-mediated pathway that controls ASIL [34]. To examine whether StkP could be involved in this pathway, we analyzed the comE transcript levels by qPCR in the ΔstkP and stkP^K42R mutants constructed in a lytA background to avoid autolysis (S1 Table). After incubation of ΔstkP cells for 30 min at acidic pH 6.0, we observed a five-fold reduction in the levels of comE transcripts in the ΔstkP mutant (Fig 1D). In contrast, the stkP^K42R mutant showed a 2-fold decrease in comE transcripts, likely due to reduced kinase activity of StkP^K42R. In addition, we observed that StkP was capable of controlling pcomC activation by acidic stress since increased β-galactosidase activity was observed from the pcomC-lacZ reporter fusion presence in the ΔstkP mutant in the bgaA background (S3 Fig). Taken together, these results indicate that StkP kinase activity was required for comE induction under acidic conditions.

In the comD^T233I ΔstkP double mutant, the levels of comE transcript increased 50 times over those in the ΔstkP strain and 10 times over those in the wt strain (Fig 1D). These results suggest that StkP kinase activity is required for full activation of ComE in order to induce ASIL under acidic conditions, regardless of the presence of high levels of ComE unnaturally induced by the ComD^T233I kinase. Such observations led us to speculate that StkP activates ComE by an alternative mechanism other than the classical ComD HK-mediated Asp⁵⁸ phosphorylation.

ComE is phosphorylated in vitro and in vivo by StkP

We hypothesized that StkP controls ComE by a crosstalk phosphorylation event. To test this hypothesis, we carried out an in vitro phosphorylation assay using purified recombinant His_x6-ComE fusion protein, in the presence or absence of purified recombinant GST-StkP. The phosphorylation reactions were examined by immunoblotting using either anti-phosphoserine or anti-phospho-threonine antibodies. No signal was detected with the anti-phosphoserine antibody, in contrast, positive reactions were detected with the anti-phosphothreonine antibody (Fig 2A), with the phosphorylation reaction occurring at a molar ratio range of GST-StkP/His_x6-ComE between 1:2 and 1:20 (Fig 2B). The GST-GFP or His_x6-GFP were included as controls of reaction specificity, and His_x6-DivIVA was used as a positive control of a StkP target, as previously described [8,11] (Fig 2A). We also evaluated the possibility that StkP could trigger ASIL by phosphorylating the major pneumococcal autolysin LytA. As overexpression of the full-length LytA protein was toxic in E. coli cells, the N-terminal region of LytA, which contains the catalytic domain was expressed instead fused to a His_x6-tag (N-LytA-His_x6)[42]). Incubation with or without GST-StkP, resulted in no evidence of N-LytA-His_x6 phosphorylation (Fig 2A), suggesting that LytA was not phosphorylated by StkP, at least under the experimental conditions described here.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. ComE is phosphorylated by StkP.

(A) ComE is phosphorylated at a threonine residue by StkP. Top: nitrocellulose membrane stained with Ponceau S as a loading control. Bottom: Immunodetection of phosphorylated proteins. Phosphorylation reactions were carried out with purified GST-StkP and substrate proteins (0.5 μg each) mixed in kinase buffer and incubated at 37°C for 1 hour. Phosphorylated proteins were detected with an anti-phosphothreonine polyclonal antibody. Lane 1: His_x6-ComE. Lane 2: His_x6-ComE + GST-StkP. Lane 3: His_x6-GFP. Lane 4: His_x6-GFP + GST-StkP. Lane 5: LytA(N)-His_x6. Lane 6: LytA(N)-His_x6 + GST-StkP. Lane 7: His_x6-DivIVA. Lane 8: His_x6-DivIVA + GST-StkP. (B) ComE phosphorylation assays with different StkP:ComE molar ratios. GST-StkP and His_x6-ComE were mixed at different molar ratios in kinase buffer and incubated at 37°C for 1 hour. Detection of phosphorylated proteins was performed as described above. (C) In vivo StkP-dependent and acid-induced ComE phosphorylation. C-terminal His-tagged ComE was purified from wt and ΔstkP strains grown in ABM (pH 7.8), and exposed to acidic stress in medium MD5, pH 6.0. Protein samples were separated by SDS-PAGE and phosphorylated or total ComE-His was detected with Pro-Q Diamond and SYPRO Ruby staining, respectively.

https://doi.org/10.1371/journal.ppat.1007118.g002

To determine whether StkP-mediated ComE phosphorylation occurs in vivo and because response regulators are usually expressed at low level in bacteria, we constructed by insertion-duplication mutagenesis wt and ΔstkP strain derivatives that express ComE fused to the His_x6-epitope tag at the C-terminus (ComE-His_6x) to improve ComE detection. Cells were incubated at either pH 7.8 or pH 6.0 and ComE-His_x6 was purified from protein lysates as described in Material and Methods and separated by SDS-PAGE. Phosphoproteins were detected by ProQ Diamond staining while total proteins were detected by SYPRO Ruby staining. We observed that phosphorylated form of ComE in wt cells grown at pH 7.8, that increases 2.3 times when cells are grown at pH 6.0 (Fig 2C). In contrast, ComE remained unphosphorylated in the ΔstkP mutant, confirming that ComE is phosphorylated by StkP in vivo.

StkP phosphorylates ComE at the Thr¹²⁸ residue to regulate ASIL

To determine the amino acid residues in ComE that are targeted for phosphorylation by StkP, we performed HPLC-MS/MS analysis of the in vitro StkP-phosphorylated ComE-His_6x recombinant protein. A single amino acid was identified as a target for StkP-mediated phosphorylation in His_6x-ComE, Thr¹²⁸, located inside the trypsin-digested ¹²¹IEQNIFYTK¹²⁹ ComE peptide (Fig 3A). To further confirm this observation, we created the non-phosphorylatable ComE^T128A-His_6x recombinant mutant protein that remained unphosphorylated in the presence StkP in vitro (Fig 3B). To evaluate the role of Thr¹²⁸ phosphorylation on ComE activity in vivo, we constructed the comE^T128A mutant, which showed significantly blocked autolysis compared to the wt (Fig 3C). Using the comE^T128A mutant, we produced the revertant comE^A128T strain, which showed an ASIL phenotype similar to the wt strain (Fig 3C). To further support the role of ComE Thr¹²⁸ phosphorylation in ASIL activation, we attempted to replace Thr¹²⁸ by Glu¹²⁸ to construct the phosphomimetic comE^T128E protein, which is typically used to mimic the phosphorylated form of Thr residues [43]. In vitro, the phosphomimetic ComE^T128E-His_x6 protein was hyper-activated, as demonstrated by EMSA assays (see next), which may explain our inability to produce a viable comE^T128E mutant. These assays confirmed that Thr¹²⁸ phosphorylation is essential for the StkP-mediated ComE activation that controls ASIL and that ComE hyper-activation is likely lethal to S. pneumoniae.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 3. StkP phosphorylates ComE on the Thr¹²⁸ residue to control ASIL.

(A) To identify the phosphorylation site, tryptic peptides obtained from ComE previously incubated with StkP were analyzed by nano-LC-MS/MS. The figure shows the MS/MS spectrum of the di-charged ion of m/z 618.8 corresponding to the phosphorylated sequence IEQNIFYTK. C-terminal y ions are labeled in blue, while N-terminal a or b fragment ions are labeled in red. Ions containing pT residue present the phosphorylation characteristic neutral loss of 98 Da. Thr¹²⁸ is unequivocally identified as the phosphorylated residue (Xcorr 3, 45; pRS score 148). (B) StkP phosphorylates ComE at Thr¹²⁸. In vitro phosphorylation assays were performed with purified GST-StkP and His_x6-ComE^wt or His_x6-ComE^T128A proteins mixed in kinase buffer at a StkP/ComE ratio of 1:20. Phosphorylated proteins were detected with an anti-phospho-threonine polyclonal antibody. Lane 1: His_x6-ComE. Lane 2: His_x6-ComE + GST-StkP. Lane 3: His_x6-ComE^T128A. Lane 4: His_x6-ComE^T128A + GST-StkP. (C) ASIL requires the Thr¹²⁸ residue in ComE for lysis induction. Autolysis was determined as indicated in the legend of Fig 1. Lytic curves corresponding to specific mutants are indicated, which data is representative of at least three independent experiments. References: ***p> 0.001.

https://doi.org/10.1371/journal.ppat.1007118.g003

Competence regulation is independent of Thr¹²⁸ phosphorylation in ComE

StkP is involved in competence in response to stress conditions such as pH 7.8 [2,4,7]. Since, our results indicated that a signaling pathway that involves StkP and ComE controls autolysis in response to acidic stress at pH 6, we investigated whether this crosstalk mechanism could also regulate competence development at pH 7.8. We have previously described that the stkP mutant showed no competence development at pH 7.8 [4]. In contrast, the comD^T233I mutant shows a hypercompetent phenotype, accompanied by constitutively high levels of ComE expression [34]. We observed that the competence phenotype of the comD^T233I ΔstkP double mutant was similar to the comD^T233I single mutant (S1 Fig), indicating an epistatic effect of the comD^T233I mutation on the ΔstkP mutation. We also observed that the competence of the non-phosphorylatable comE^T128A and revertant comE^A128T mutants were similar to the wt strain (S1 Fig).

These observations suggest that StkP regulates competence at an early stage, which is independent of ComE Thr¹²⁸ phosphorylation. Furthermore, StkP was not essential for competence once ComE was activated by ComD at pH 7.8. In contrast, StkP is necessary to activate ComE and to trigger autolysis under acidic conditions.

StkP-mediated Thr¹²⁸ phosphorylation increases ComE dimerization

Response regulators are composed by a conserved receiver domain, which is phosphorylated on an aspartate residue by their cognate histidine kinase, and DNA-binding domains [44]. In ComE, the receiver domain corresponds to the first 130 residues [43]. Thr¹²⁸ is located at the end of the α-5 region (Asp¹¹⁴-Ser¹³⁰) of the receiver domain of ComE, next to the α-4 region (Ala⁹⁴-Gln¹⁰¹), and near the loop between α-4 and β-5 (Val¹⁰²-Leu¹⁰⁵) that is involved in ComE dimerization and considered as a dimerization interface [43] (Fig 4A). The proximity between the dimerization interface and the two phosphorylated residues (Asp⁵⁸ and Thr¹²⁸) suggest a putative influence on the dimerization capacity of ComE, which was confirmed by in vitro dimerization assays using the phosphomimetic mutants. The ComE^D58E-His_6x, ComE^T128E-His_6x, and ComE^T128A-His_6x mutants showed dimer steady-state levels, which were 70, 72 and 2.9 times higher, respectively, than ComE^wt-His_6x (reference level). In addition, when the ComE^wt-His_6x protein was incubated with StkP, the dimerization rate increased 8.9 times, whereas ComE^T128A-His_6x showed only a 3.3-fold increase (Fig 4B). The different dimerization capabilities found for ComE^wt/StkP (8.9 times) compared to the phosphomimetic ComE^T128E-His_6x (70 times) suggest that ComE^wt-His_6x is partially phosphorylated by StkP (~12%). These observations suggest that Thr¹²⁸ phosphorylation modifies ComE in a manner that strongly affects its dimerization interface, which is a condition sine qua non for the response regulator activation.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. Thr¹²⁸-phosphorylation increases the dimeric state of ComE.

(A) Localization of Thr¹²⁸ residue in the ComE structure. Based on the crystal structure of ComE reported by Boudes et al [43], this figure reveals the localization of the Thr¹²⁸ residue, as well as the alternative phosphorylation site Asp⁵⁸. The three loops in the DNA-binding domain are also shown, which are apparently altered when ComE is phosphorylated on Thr¹²⁸. At the bottom of this image, a sequence alignment between the DNA-binding domains of ComE and AgrA is also shown. Positively charged or polar residues, which are described in AgrA to have a direct contact with DNA bases [45], are indicated in red. (B) The dimerization capacity of recombinant ComE proteins, such as the phosphomimetic ComE^D58E and ComE^T128E proteins, as well as the non-phosphorylatable ComE^T128A mutant, was analyzed and compared with ComE^wt (left panel). ComE^wt and ComE^T128A were also pre-incubated with GST-StkP (right panel). Dimerization states were assessed by native PAGE/Tris-MOPS buffer. Proteins were electroblotted onto PVDF membranes, and His_x6-ComE was detected using anti-His antibody.

https://doi.org/10.1371/journal.ppat.1007118.g004

Thr¹²⁸ phosphorylation changes conformation of the ComE DNA-binding domain

In order to determine how Thr¹²⁸ phosphorylation affected ComE's conformation, we performed molecular dynamic simulations at 40–150 ns comparing ComE^wt (PDB ID: 4CBV, [43]) and the in silico phosphomimetic ComE^T128E-His_6x protein (S4 Fig, video). The simulations clearly indicated that 3 loops spanning the DNA-binding domain (Lys²¹⁸-Asn²¹⁹-Leu²²⁰, Thr¹⁶⁴-Gly¹⁶⁵-Val¹⁶⁶-Ser¹⁶⁷-His¹⁶⁸, and Ser²⁰⁰-Pro²⁰¹-His²⁰²-Lys²⁰³) presented different dynamics in the ComE^T128 mutant compared to ComE^wt (Fig 4B and accompanying video shown in S4 Fig). ComE is a member of the AgrA/LytTR family of bacterial response regulators, which present certain structural homologies. Coincidently, two of these three loops have been described for the AgrA RR in S. aureus [45], as key residues that determine the DNA binding affinity for promoters in the phosphorylated form of AgrA (Fig 4B). ComE also revealed positively charged or polar residues (His¹⁶⁸ and Lys¹⁶⁹ in loop 1; His²⁰² and Lys²⁰³ in loop 2; Arg²¹⁷ and Lys²¹⁸ in loop 3), which are shown in AgrA to have a direct contact with DNA bases [45]. These results suggest that after Thr¹²⁸ phosphorylation ComE may undergo conformational changes. Consistent with this notion, limited proteolysis assays revealed structural differences between ComE-His_6x and ComE^T128E-His_6x (S5 Fig). Treatment with trypsin showed more contrasting proteolytic patterns than proteinase K treatment. These findings confirmed that Thr¹²⁸ phosphorylation causes evident changes in ComE conformation, likely in the DNA-binding domain of ComE, that may modify its DNA-binding affinity, and we explored this possibility with electrophoretic mobility shift assays (EMSAs).

StkP-mediated Thr¹²⁸ phosphorylation in ComE increases its DNA-binding affinity in a pH-dependent manner

During competence development at alkaline pH, Asp⁵⁸ phosphorylation by ComD results in increased binding of ComE to pcomC and transcriptional activation of the comCDE operon [2,46,47]. We have observed that at acidic pH, pcomC activation and induction of comE transcription depended both on ComE and on StkP (Fig 1D and S3 Fig) suggesting that Thr¹²⁸ phosphorylation by StkP influences the binding of ComE to pcomC. Electrophoretic mobility shift assays (EMSAs) proved that the phosphomimetic ComE^T128E-His_6x protein bound pcomC 5-fold stronger than ComE^wt-His_6x (Kd 74 nM vs Kd 371 nM, respectively). The DNA binding affinity of the non-phosphorylatable ComE^T128A-His_6x mutant was unaffected (Kd 375 nM) whereas ComE^D58E-His_6x affinity for pcomC was 17- fold greater that ComE^wt-His_6x (Fig 5 and Table 1). Curiously, when ComE^wt-His_6x was pre-incubated with StkP in phosphorylation buffer at pH 7.8 no binding to pcomC was observed (S6 Fig, Table 1). As ASIL is regulated by StkP-mediated phosphorylation of ComE Thr¹²⁸ residue under acidic conditions, we tested if ComE DNA-binding affinity could be affected by pH. When ComE^wt-His_6x was previously incubated with StkP at pH 6.0, its affinity for pcomC increased (Kd 64 nM) and was similar to that shown by ComE^T128E-His_6x (S6 Fig, Table 1). To determine which of these contrasting effects actually depended on StkP phosphorylation, similar assays were performed with an inactive StkP enzyme (StkP^K42M) [8] and ComE^wt-His_6x.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 5. The phosphomimetic ComE^T128E protein shows an increased DNA-binding affinity.

The DNA-binding affinity for the promoter region of the comCDE operon (pcomC) of ComE^wt (A), the non-phosphorylatable (by StkP) ComET^128A mutant (B) and the phosphomimetic ComE^T128E (C) and ComE^D58E (D) proteins was determined by EMSA. Binding interactions were examined by incubating variable amounts of the different ComE versions with Cy5-labeled pcomC, followed by electrophoretic separation of the protein-DNA complexes. Black or white triangles are indicating the free or ComE-bound probe, respectively. Images were obtained with a fluorescence scanner as described in Materials and Methods. The Kd values are indicated in each panel.

https://doi.org/10.1371/journal.ppat.1007118.g005

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. ComE binding affinities to pcomC.

https://doi.org/10.1371/journal.ppat.1007118.t001

Binding to pcomC was comparable in ComE^wt-His_6x pre-treated with StkP^K42M at pH 6.0 and untreated ComE^wt (Kd 300 nM vs Kd 371 nM) but was still absent after incubation at pH 7.8 (S7 Fig, Table 1). These results indicate that StkP phosphorylation at pH 6.0 underlied the enhanced pcomC-binding affinity of ComE, but not the blocking of ComE-pcomC interaction observed at pH 7.8, which suggests that at a slightly alkaline pH, StkP makes a complex with ComE masking its DNA-binding sites. The DNA-binding affinity of the phosphomimetic ComE^T128E-His_6x and the non-phosphorylatable ComE^T128A-His_6x proteins were not affected when preincubated with StkP at either pH 6.0 or pH 7.8 (S7 Fig, Table 1), indicating that Thr¹²⁸ mediated the observed StkP effects on ComE: (1) at pH 6.0, Thr¹²⁸ phosphorylation by StkP increases ComE DNA binding affinity; (2) at pH 7.8 Thr¹²⁸ mediates the StkP-ComE interaction that blocks DNA binding. Thus, these experiments indicate that pH modulates the interplay between StkP and ComE.

The StkP/ComE interaction increases at acidic pH

To test for a putative protein-protein interaction between StkP and ComE, a sandwich fluorescence-linked immunosorbent assay (FLISA) was utilized, in which the StkP-coated surface of a microtiter plate was incubated with increasing amounts of His-tagged ComE at either pH 6.0 or pH 7.8. This assay clearly showed that acidic pH augmented the number of binding sites between ComE and StkP, as reflected by a 3-fold increase in F_max (maximum fluorescence when binding is saturated) at pH 6.0 compared to pH 7.8 (Table 2, S8 Fig). The interaction between ComE^wt and the StkP^K42M mutant revealed the same F_max as the ComE/StkP at different pH values, indicating that kinase activity did not alter the saturation of this protein complex. However, for the StkP^K42M mutant, the K_1/2 values were lower, indicating that StkP^K42M bound more tightly to ComE at any pH value. In comparison with ComE^wt, the interaction between non-phosphorylatable ComE^T128A mutant and StkP or StkP^K42M was 3-fold stronger and was not affected by pH. In contrast, the phosphomimetic ComE^T128E protein produced a 3-fold increment in K_1/2 at pH 7.8, which further raised to 9-fold at pH 6.0, demonstrating that the phosphorylated form of ComE had a lower affinity for StkP (Table 2, S8 Fig).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 2. Estimates of ComE binding affinities for StkP and StkP^K42M.

https://doi.org/10.1371/journal.ppat.1007118.t002

These data confirm that the StkP/ComE interactions are mediated by ComE residue Thr¹²⁸ and favored by acidic conditions, which may facilitate ComE phosphorylation.

ComE induces global changes in the transcriptome of S. pneumoniae

To understand the effect of the StkP/ComE signaling pathway on pneumococcal physiology, we compared the transcriptomes of the comE^T128A mutant and wt by RNAseq analysis. Three replicates of each strain strains were grown in ABM (pH 6.0) for 1 hr at the exponential growth phase (OD_620nm 0.3) and analyzed. In total, the differential expression of 104 genes was detected, 51 were down-regulated genes and 53 were up-regulated, considering relevant genes to be those with expressions higher than 2 fold and p values <0.05 (Fig 6A). The full list of these genes is shown (S3 Table). Based on this differential gene expression analysis, we observed that the StkP/ComE pathway affected, directly or indirectly, the expression of genes involved in oxidative stress, and the purine/pyrimidine, amino-acid and central metabolisms, as well as the ribosomal and translation structures, metabolite transport, molecular chaperones and cell wall biosynthesis, among others (Fig 6B). The list of genes regulated by Thr¹²⁸-phosphorylated ComE indicated that this new signaling pathway induces global changes in the pneumococcal transcriptome, such as the physiological response to acidic stress. Bioinformatic analysis of the promoter regions (240 bp upstream of the start codon) of 22 ComE-regulated genes obtained from RNAseq assays predicted a putative DNA binding motif (S9A Fig). Martin et al [47] described a potential ComE^D58~P binding site (CEbs, 32 bp) in the comC promoter constituted by two repeats (DR1 and DR2) separated by 12 bp. In this report, we established that the putative ComE^T128~P binding site (26 bp) only partially overlaps with theses repeats suggesting a different consensus binding sequence to that described for ComE^D58~P (S9B Fig).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 6. ComE is a global regulator that controls gene expression during the stress response.

(A) Gene expression scatter plot in the wt and comE^T128A samples, with the x-axis representing the gene expression values for the control condition (wt) and the y-axis representing those for the treated condition (comE^T128A). Each black dot represents a significant single transcript, with the vertical position of each gene representing its expression level in the experimental conditions and the horizontal one representing its control strength. Thus, genes that fall above the diagonal are over-expressed whereas genes that fall below the diagonal are underexpressed as compared to their median expression levels in the experimental groups. (B) Volcano plot of gene expression in wt vs comE^T128A samples measured by RNAseq. The y-axis represents the mean expression value of the log₁₀ (p-value), while the x-axis displays the log₂ fold change value. Black dots represent genes with an expression 2-fold higher in the comE^T128A mutant relative to strain wt with a p-value < 0.05, with red dots signifying genes with an expression 2-fold lower in the comE^T128A mutant, which are relative to strain wt with a p < 0.05. (C) Categories of ComE-regulated genes obtained from an RNAseq analysis. An RNAseq generated distribution in functional categories of genes that are regulated in the comE^T128A mutant relative to strain wt under acidic conditions. (D) ComE-regulated genes expressed under acidic conditions in the comE^T128A mutant relative to strain wt. Gene expression determined by RNAseq was confirmed by qPCR. The comE^T128A ΔlytA and ΔlytA (referred as wt for this assay) strains were grown in ABM/pH 6.0 to the mid-exponential phase in triplicate, with the fold change in gene expression measured by RT-qPCR and calculated using the 2^–ΔΔCT method. The gyrA gene was used as the internal control. References: ** p < 0.01; *** p < 0.001.

https://doi.org/10.1371/journal.ppat.1007118.g006

The StkP/ComE pathway controls H₂O₂ production and oxidative stress response

Using RT-qPCR we confirmed decreased expression of oxidative stress genes spxB, sodA [48] and tpxD [49] in comE^T128A mutant compared to wt (Fig 6C). The spxB gene encodes the pyruvate oxidase that produces H₂O₂ from O₂, sodA encodes the superoxide dismutase that produces H₂O₂ from superoxide, and tpxD encodes the thiol peroxidase that catalyzes the H₂O₂ oxidation and contributes to the oxidative stress response. In the comE^T128A mutant, we found that the spxB, sodA, and tpxD transcripts were downregulated 18, 2.8 and 2.7 times, respectively (Fig 6C). These findings were also corroborated by H₂O₂ production and H₂O₂ susceptibility assays. The comE^T128A, ΔcomE, and ΔstkP mutants showed a 4-fold decrease in their H₂O₂ production compared to the wt and comE^A128Tstrains (Fig 7A), which is likely caused by the reduced expression of spxB and sodA protein products. In addition, we observed a 10-fold reduction in the susceptibility to H₂O₂ by the comE^T128A, ΔcomE, and ΔstkP mutants compared to the wt strain (Fig 7B), likely due to reduced expression of the TpxD peroxidase. These findings support the notion that the StkP/ComE pathway is essential for the control of H₂O₂ production and for H₂O₂ tolerance.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 7. The StkP/ComE pathway controls oxidative stress and cell wall biosynthesis.

(A) The H₂O₂ production is altered in the comE and stkP mutants. Cells were grown in BHI at 37°C to an OD_620nm of 0.3, then diluted in either ABM (pH 6.0) and incubated at 37°C to an OD_620nm of 0.3. The H₂O₂ concentration was determined by the peroxidase test as described in Material & Methods. Values were calculated as the H₂O₂ concentration in mM and normalized against the number of viable cells. (B) The comE and stkP mutants were more susceptible to H₂O₂ than wt. Susceptibility to H₂O₂ is indicated as a percentage of bacterial survival at different time points. C-D) The comE^T128A mutant was more resistant to cell-wall antibiotic-induced lysis than wt. Cells were grown in BHI/pH 7.2 at 37°C to an OD_620nm of 0.3, and fosfomycin (C) and vancomycin (D) were added in independent cultures at final concentrations of 50 μg/ml and 0.4 μg/ml, respectively. Cell lysis of bacterial cultures was determined by turbidimetry at OD_620nm for more than 3 h. References: *** p < 0.001.

https://doi.org/10.1371/journal.ppat.1007118.g007

The StkP/ComE pathway regulates murN expression and modulates susceptibility to antibiotic-induced lysis

Although RNAseq analysis showed that the murN gene was overexpressed in the comE^T128A mutant, its expression by qPCR was actually found to be 4-fold lower than in wt in three independent assays (Fig 6C), suggesting a typical case of false positive that is commonly found in RNAseq studies. Regarding the physiological impact of the murN mutation, Filipe et al [50] described that a murMN mutant had cell wall alterations and presented increased susceptibility to lysis when exposed to cell wall antibiotics. To test whether an altered murN expression in the comE^T128A mutant could modify the susceptibility to cell wall antibiotics, we determined the MIC values of the comE^T128A and wt strains in the presence of either fosfomycin, vancomycin, penicillin, cefotaxime, cefazolin, or piperacillin. The fosfomycin MIC in the comE^T128A (170 μg/ml) was higher than the wt strain (50 μg/ml), whereas the MICs for vancomycin, penicillin, cefotaxime, cefazolin, and piperacillin were similar between these strains. The typical lytic effect of fosfomycin (50 μg/ml, 1xMIC; Fig 7C) and vancomycin (0.4 μg/ml, 1xMIC; Fig 7D) on the wt strain was inhibited in the comE^T128A strain. The diminished susceptibility to cell wall antibiotics in the comE^T128A strain suggests cell wall alterations consistent with the ASIL repression showed by this mutant.

Pneumococcal survival in pneumocytes is controlled by the StkP/ComE pathway

We previously described that ComE is involved in the acidic stress response and in the pneumococcal intracellular survival mechanism in pneumocytes [33]. Here, we demonstrate that StkP phosphorylates ComE, and in order to determine whether this crosstalk affects the pneumococcal survival, we measured the intracellular survival capacities in A549 pneumocyte cells of the ΔstkP, stkP^K42R (reduced kinase activity), ΔcomE, comE^T128A and comE^A128T (revertant) strains compared to the wt in A549 pneumocyte cells [33]. Mutations in either the stkP or comE genes conferred increased survival compared to comE^A128T or wt (Fig 8A), indicating that the StkP/ComE pathway controlled pneumococcal survival in pneumocytes. To discriminate whether this phenotype could result from increased ATR or decreased ASIL, we tested the ΔlytA mutant, which lacks autolysin and presented a blocked ASIL [33], but its intracellular survival was similar to the wt strain (Fig 8A). Thus, a blocked ASIL is not enough to increase intracellular survival of S. pneumoniae in pneumocytes. Consequently, the increased survival showed by either the ΔstkP, ΔcomE, or comE^T128A mutants (Fig 8A) is likely due to higher ATR capacity. To test this hypothesis, we determined the ATR phenotype of the comE^T128A mutant, but in a lytA deficient background in order to discard residual autolysis. As expected, ATR of the ΔlytA strain increased 2-fold at pH 6.0 compared with cells cultured at pH 7.8, whereas the comE^T128A ΔlytA cells showed a 20-fold increase under the same conditions (Fig 8B), supporting the notion that increased ATR explains the increased survival rate displayed by the comE^T128A mutant in pneumocytes.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 8. The StkP/ComE pathway modulates intracellular survival and the acid tolerance response of S. pneumoniae.

(A) The ΔstkP and ΔcomE mutants showed increased intracellular survival compared with wt in A549 pneumocytes. Bacteria cells were initially incubated for 3 h in monolayers of A549 pneumocytes, and survival progression of different strains was monitored using a typical protection assay. Survival percentages were calculated by considering the total amount of internalized bacteria after 30 min of extracellular antibiotic treatment as representing 100% for each strain. After antibiotic treatment, samples were taken at 0 (white bars), 3 (grey bars) and 7 (black bars) hours, and pneumocytes were lysed to release pneumococci. Samples were diluted in BHI spread on BHI-blood-agar plates and incubated at 37°C for 16 h. (B) The ΔstkP and ΔcomE mutants displayed an augmented ATR compared with wt. To determine the survival percentage of bacterial strains, the non-induced cells (white bars) were directly exposed for 2 h at pH 4.4 (lethal pH) in THYE medium, with the acid-induced cells (grey bars) being previously incubated for 2 h at pH 6.0 (sub-lethal pH) in THYE medium. After exposition to lethal pH, pneumococcal survival was determined by spreading dilutions in BHI-blood-agar plates and incubating these at 37°C for 16 h. For both panels, data are representative of at least three independent experiments and statistically significant differences are indicated as p<0.01 (**) or p<0.001 (***).

https://doi.org/10.1371/journal.ppat.1007118.g008

Bacterial strains, plasmids, cell lines, and growth conditions

All strains, plasmids, and oligonucleotides used in this study, as well as cloning and mutagenesis procedures, are listed in the supplementary material (S1 Table). The growth conditions and stock preparation for the pneumococcal and Escherichia coli strains have been reported elsewhere [34], and the transformation assays have also been previously described [76,77].

ASIL and ATR assays

ASIL was performed as described previously [33]. Firstly, bacterial cells were grown in Todd-Hewitt/yeast extract medium. When cultures reached OD_600nm ~0.3, cells were centrifuged at 10,000 g for 5 min, the pellet was resuspended in ABM pH 6.0 and cultures were re-incubated at 37°C. Autolysis was measured as a change in OD_600nm at different time points over 6 h.

ATR was performed as described previously [24]. For non-acid-induced conditions, bacterial cells were first grown in THYE (pH 7.8) at 37°C, and when cultures reached OD_600nm~ 0.3, 100 μl aliquots were taken and added to 900 μl of THYE (pH 4.4) and incubated for 2 h at 37°C. Then, serial dilutions were made in THYE (pH 7.8) and plated onto 5% of sheep blood tryptic-soy agar (TSA) plates. After 24 h of incubation at 37°C, colonies were counted to determine the number of survivors, with the total CFU being obtained by plating serial dilutions of cells grown THYE pH 7.8 onto 5% sheep blood TSA, made just before cells were switched to pH 4.4. In parallel, to determine survival under acidic-induced conditions, bacterial cells were grown in THYE (pH 7.8) until OD_600nm ~ 0.3, centrifuged at 10,000 g for 5 min, resuspended in THYE (pH 6.0) and incubated for 2 h at 37°C. Culture aliquots were taken and serially diluted in THYE pH 7.8 for total cell counting, while other aliquots were diluted ten times in THYE (pH 4.4) and incubated for 2 h at 37°C to determine the survival percentage as described above. For both assays (acid-induced and non acid-induced conditions), this was calculated by dividing the number of survivors at pH 4.4 by the number of total cells at time zero (before incubation at pH 4.4). For the ASIL and ATR assays, data were expressed as the mean percentage ± standard deviation (SD) of independent experiments performed in triplicate.

Cell lines and culture conditions

The A549 cell line (human lung epithelial carcinoma, pneumocytes type II; ATCC CCL-185) was cultured at 37°C, 5% CO₂ in Dulbecco’s modified Eagle medium (DMEM) with 4.5 g/l of glucose and 10% of heat-inactivated fetal bovine serum (FBS)(Gibco BRL, Gaithersburg, Md.). Fully confluent A549 cells were split once every two or three days via trypsin/EDTA treatment and diluted in fresh media before being cultivated in Filter cap cell flasks of 75 cm² (Greiner Bio-one no. 658175) until passage 6.

In vitro phosphorylation assays

In vitro phosphorylation was carried out with 0.5 μg of purified recombinant substrate protein and 0.5 μg of purified GST-StkP in 30 μl of kinase buffer (50 mM Tris-HCl, 5 mM MgCl₂, 100 μM ATP, 1mM DTT, pH 7.5). The reaction was started by the addition of ATP and stopped after 60 min of incubation at 37°C by the addition of 5x Laemmli SDS sample buffer. Samples were separated by standard Tris-glycine-SDS polyacrylamide gel electrophoresis (PAGE) gels and electroblotted onto a nitrocellulose membrane. Phosphorylated proteins were detected with an anti-phosphothreonine polyclonal antibody (1∶1,000; Cell Signaling) and a goat anti-rabbit immunoglobulin G secondary antibody conjugated to horseradish peroxidase (1∶2,500; Invitrogen). Detection was performed with an enhanced chemiluminescence substrate (SuperSignal West Pico Chemiluminescent Substrate; Pierce) and Hyperfilm CL film (GE) using exposures of between 1 and 10 min. The pRSET-divIVA_spn plasmid was generously provided by Dr. Orietta Massidda (Università degli studi di Cagliari, Italy) [78].

In vivo phosphorylation assays

The RC838 (comE-his) and RC839 (ΔstkP comE-his) strains (S1 Table) were grown in 2 l of ABM (pH 7.8) at 37°C until OD_600nm 0.3. Half of the cultures (1 l) were centrifuged for 10 min at 5,000 x g, snap-frozen in liquid-air and stored at -80°C. The remaining 1 l was centrifuged as before, resuspended in ABM (pH 6.0), incubated at 37°C for 10 min, and finally centrifuged for 10 min at 5,000 x g, snap-frozen in liquid-air and stored at -80°C. Cell pellets were thawed in ice and resuspended in 10 ml of L8 buffer (100 mM NaH₂PO₄/10 mM Tris·HCl, pH 8.0/150 mM NaCl/20 mM imidazole/20mM PMSF/8 M urea) supplemented with MS-SAFE protease/phosphatase inhibitor cocktail (Sigma-Aldrich) and lysed by stirring for 1 h followed by sonication. The lysate was cleared by centrifugation at 15,000 g for 20 min, and the supernatant was added to a column packed with to 0.5 ml of Ni-NTA resin (Qiagen) equilibrated in L8 buffer. The column was washed sequentially with 10 ml of LX buffer (L buffer with X = 8, 6, 4, 2, and 0 M urea), and bound ComE-His_6x protein was eluted with 2 ml of L0 buffer containing 500 mM imidazole. Protein samples (approximately 0.5 μg ComE-His) were separated by SDS-PAGE, and the gels were stained with ProQ Diamond (Invitrogen) to detect phosphorylated ComE-His, followed by SYPRO Ruby (Invitrogen) total protein staining. Gels were imaged under fluorescence mode in a Typhoon FLA 9500 scanner (GE) and protein bands were quantified using ImageQuant software (GE).

Identification of phosphorylated residues by nano-LC-MS/MS analysis

Identification of the phosphorylation site was carried out by nano-LC-MS/MS analysis as previously described [79]. Protein bands were in-gel-digested overnight with sequencing grade trypsin (Promega) at 37°C, desalted using micro-reverse phase columns (C18 Omix tips, Varian), vacuum dried and resuspended in 0.1% formic acid (v/v) in water. Tryptic peptides were injected into a nano-HPLC system (Proxeon Easy nLC, Thermo) fitted with a trap column (Easy-column C18 2 cm x 100 um ID). Posteriorly, the samples were separated on a reverse phase nano-column (Easy-Column C18 10 cm x 75 um ID; Thermo) using a linear gradient of acetonitrile 0.1% formic acid (0–45% in 70 min) at a flow rate of 400 nL/min. Mass analysis was performed using a linear ion trap mass spectrometer (LTQ Velos, Thermo) in a data-dependent mode (full scan followed by MS/MS of the top 5 peaks)[79]. Raw data was analyzed using the Proteome Discoverer software package (v.1.3.0.339, Thermo), and Sequest search engine, with the following parameters: enzyme: trypsin; maximum missed cleavage: 2; precursor mass tolerance: 1 Da; fragment mass tolerance: 0.8 Da; Ser/Thr/Tyr phosphorylation and methionine oxidation as dynamic modifications. Searches were performed using a Streptococcus pneumoniae database downloaded from UniProt (17/5/2017) and including the His tag-ComE sequence. For phosphosite localization, the phosphoRS algorithm was used and the spectra of phosphorylated peptides were manually inspected to corroborate the phosphosite assignment [80].

EMSA

The promoter region of comCDE (255 bp; pcomC) was PCR-amplified using the 5’-Cy5 labeled oligonucleotides NGEP516 and NGEP517 (IDT) and purified using the QIAquick PCR Purification Kit (QIAGEN). DNA-binding assays were performed in a total volume of 10 μl containing 50 mM NaCl, 50 mM Tris/HCl pH 7.5, 5% (v/v) glycerol, 7.5 nM Cy5-labeled PCR fragments, 1 mM MgCl₂, 0.15 mg Poly(dI-dC) (as the non-specific competitor), and varying concentrations of untreated or StkP-treated ComE proteins. In the latter case, phosphorylation of ComE-His_x6 by StkP was carried out with an equimolar amount of StkP in kinase buffer (50 mM Bis-Tris propane, 5 mM MgCl_2, 1mM DTT, 0.1 μM ATP, pH 7.8 or pH 6.0) for 60 min at 37°C. Protein-DNA binding reactions were incubated at room temperature for 30 min, and “frozen” with an equal volume of 2X Stop Solution [40% (v/v) Triethylene Glycol, 10 mM Tris, pH 7.5]. DNA-protein complexes were resolved by electrophoresis in native Tris-Borate-EDTA polyacrylamide gels [10% (w/v)] containing 30% Triethylene Glycol. Gels were run at 4°C for 120 min at constant voltage (25 V cm^-1) in a 0.5X TBE buffer and scanned in a Typhoon FLA 9500 biomolecular imager (GE) under fluorescence mode. Free and protein-bound DNA were quantified using ImageQuant (GE). The fraction of DNA bound (FB) at each ComE concentration was fit with a standard binding isotherm using Kaleidagraph (Synergy Software), according to the equation: FB = [ComE]/(K_d +[ComE]), where K_d is the apparent equilibrium dissociation constant and reflects the protein concentration required to shift 50% of the labeled DNA fragment.

Molecular dynamics simulation

MD simulations were carried out with the NAMD program [81], using the CHARMM27 force field [82]. Spherical boundary conditions and a non-bonded cut-off of 12.0 Å with a switching function of 10.0 Å were used. All systems were submitted to structural minimization in vacuum, and then embed in a water sphere for the MD. The temperature was set to 310 K by a Langevin thermostat. MD simulations were run for 40 ns with an integration step of 2 fs. Analysis of the trajectories was performed using VMD software [83]. Crystal structure images were analyzed using PyMOL [84].

StkP and ComE interaction by FLISA

StkP and ComE binding interactions were assessed by a sandwich fluorescence-linked immunosorbent assay (FLISA) in black 96-well high binding capacity microplates with clear flat-bottoms (Corning #3601). Each well was filled with 50 μL of 10 μg/ml GST-StkP (500 ng of GST-StkP) dissolved in a 0.1 M coating buffer (0.1 M NaHCO₃/Na₂CO₃, pH 9.4) and incubated overnight at 4°C to allow protein adsorption. Wells were rinsed five times with Tris-buffered saline, 0.05% Tween 20, pH 7.4 (TBS-T) and the reactive sites were blocked with 2% w/v bovine serum albumin dissolved in TBS-T for 2 h at room temperature. Wells were washed three times with TBS-T. Different amounts of ComE-His_x6 (200–1200 ng) were dissolved in 50 μL of 50 mM Bis-Tris-Propane-HCl, 1 mM MgCl₂, pH 7.8, or in the same buffer but at pH 6.0, and added to StkP-coated wells and incubated for 1h at 37°C. Wells were washed 5 times with TBS-T and incubated for 1h at RT with 50 μL of a Dylight 650-conjugated anti-6X His antibody (Invitrogen MA1-21315-D650) diluted 100-fold in TBS-T. After 5 washes with TBS-T, plates were read in a Typhoon FLA 9500 scanner (GE) under fluorescence mode. Fluorescence (F) was fit using a Kaleidagraph to a standard binding isotherm with the form F = F_max [ng ComE/(K_1/2 + ng ComE)], where F_max is the maximum fluorescence at binding saturation and reflects the maximum binding capacity (B_max), and K_1/2 is the amount of ComE (ng) required to reach half F_max. The inverse of K_1/2 represents an estimate of ComE affinity for the StkP binding sites.

Protein expression and purification

The comE gene was amplified from R801 genomic DNA with the primer pair FhkE/RhkE and cloned into the BamHI/EcoRI sites of the pRSET-A expression plasmid (Invitrogen), yielding pRSET-ComE. stkP and stkP-KD (kinase domain, amino acids 1–282) were amplified with primer pairs FstkP-ex/Rstk-ex and FstkP-ex/ Rstk-kd, respectively, and cloned into BamHI/EcoRI sites of pGEX-4T1 expression plasmid (GE) to generate pGEX-StkP and pGEX-StkP-KD. Plasmid pTrc-LytA(N) expressing the amino-terminal region of LytA (1–206) was obtained by cloning a lytA fragment generated by PCR amplification with primers F_lytA1/Rl_ytA2 into the BamHI/EcoRI sites of pTrcHis2A (Invitrogen). Mutations were introduced in pRSET-ComE by Quickchange site-directed mutagenesis (Agilent), employing primer pairs NGEP514/NGEP515, NGEP75/NGEP76 and NGEP77/NGEP78 to obtain pRSET-ComE(D58E), pRSET-ComE(T128A), and pRSET-ComE(T128E), respectively. In the same way, the K42M mutation was introduced in pGEX-StkP with primers NGEP770/771 to give pGEX-StkP(K42M).

Soluble His_6X-tagged LytA(N) and ComE-His_x6 proteins were purified from the E. coli BL21(DE3) strain co-transformed with either pTrc-LytA(N) or pRSet-ComE derivatives and chaperone expression plasmids pBB540 and pBB550 [2,85]. E. coli cells were grown on 800 ml of Terrific broth and induced with 100 μM IPTG according to de Marco [85]. His-tagged proteins were purified from protein lysates obtained by sonication using an NTA-Ni²⁺ resin (Qiagen) following the manufacturer’s protocol. Eluted protein was further purified by gel filtration using a HiPrep 16/60 Sephacryl S-200 HR column mounted in a ÄKTA purifier system (GE). ComE containing fractions were pooled, concentrated with an Amicon Ultra-4 centrifugal filter (Millipore), and dialyzed against the storage buffer [50 mM Tris, 200 mM NaCl, 1mM DTT, 50% v/v glycerol, pH 7.5). Samples were snap frozen and stored at -80°C until use. Following exactly the same protocol as above, GST-tagged StkP proteins were purified from the soluble protein fraction of BL21(DE3) cells bearing pGEX-StkP derivatives and plasmids pBB540 and pBB50. In this case, a Glutathione Sepharose resin (GE) was used to retain GST-tagged StkP. DivIVA was purified from BL21 (DE3) cells transformed with pRSET-divIVA according to Fadda et al. [78]. Purified recombinant Aequorea victoria His-tagged GFP protein was purchased from SIGMA.

Dimeric state of ComE proteins

Native PAGE was used to assess the ComE monomer/dimer ratio. Purified ComE-His_x6 proteins were diluted in 2x Laemmli sample buffer without 2-mercaptoethanol and SDS and loaded in a 4–20% gradient Bis-Tris precast polyacrylamide gel (GenScript). Electrophoresis was performed at 4°C using Tris-MOPS running buffer without SDS (GenScript) at a constant electric field of 15 V cm^-1. Proteins were electroblotted onto a PVDF membrane and probed with Dylight 650-conjugated anti-6X His antibody to detect His-tagged ComE. The membranes were imaged under fluorescence mode in a Typhoon FLA 9500 scanner (GE Healthcare), and bands were quantified with ImageQuant software (GE Healthcare).

RNAseq analysis

Cells were initially grown in THYE medium at pH 7.8 until OD_600nm ~0.3 (log phase), centrifuged at 14,000 g for 10 min at 4°C, resuspended in the same volume in ABM at pH 6.0 (Piñas et al, 2008) and incubated a 37°C for 1h. Then, cells were centrifuged at 14,000 x g for 10 min at 4°C, resuspended in a 1/10 vol of lysis buffer (DOC 1% in 0.9% Na Cl) and incubated 3 min a 37°C until complete lysis. Total RNA was purified by TRIzol reagent according to the manufacturer's instructions (Fisher Scientific) from three biological replicates for wt and the comE^T128A mutant. Posteriorly, we used the Ribopure Bacterial RNA Purification Kit (Ambion) following the manufacturer's protocol, with the contaminant DNA being removed using the provided Dnase. rRNA was depleted from 8μg of total RNA using the MICROBExpress Bacterial mRNA Enrichment Kit (Ambion), and then the transcriptome libraries were prepared with TruSeq Stranded RNA Library Preparation Kit (Illumina) following the manufacturer's instructions. Briefly, enriched mRNA was fragmented using reagents provided with the kit, and this was followed by first-strand cDNA synthesis and second-strand generation. The libraries were tagged with unique indexes and amplified for a limited number of PCR cycles followed by quantification and qualification using the DNA High Sensitivity Assay Kit. Samples were sequenced using PE150bp chemistry and the Illumina HiSeq. Reads were trimmed by Trimmomatic 0.36 [86] to generate high-quality reads. Subsequently, these reads of wt and the comE^T128A samples were separately aligned to the Streptococcus pneumoniae R6 genome using BWA -version 0.7.12-r1039 (bio-bwa.sourceforge.net) at default parameters. The software package SAMtools (http://samtools.sourceforge.net/) was used to convert the sequence alignment/map (SAM) file to a sorted binary alignment/map (BAM) file. The mapped reads ratio (MRR) to the reference in each dataset was calculated by applying the flagstat command of SAMtools software to the BAM file.

Differential gene expression

The aligned reads were assembled by Cufflinks (version-2.2.1), and then the differentially expressed genes were detected and quantified by Cuffdiff, which is included in the Cufflinks package, using a rigorous sophisticated statistical analysis. The expression of the genes was calculated in terms of FPKM (Fragment per kilobase per million mapped reads). Differential gene expression analysis was carried out between wt and the comE^T128A samples.

qRT-PCR

cDNA was synthesized from 2 μg RNA using the ProtoScript II First Strand cDNA Synthesis Kit (NEB) following the manufacture's protocol. cDNA was cleaned using the QIAquick PCR Purification Kit (Qiagen). Genes were amplified using the oligos listed in the S2 Table and FastStart Essential DNA Green Master Mix (Roche) following the manufacturer's protocol. Expression was determined relative to AU0158 normalized by gyrA (spr1099) expression using the ΔΔCt method [87]. The gyrA had a similar expression by RNA-Seq for wt and the comE^T128A mutant, and this had been used to normalize the expression in S. pneumoniae in other studies [88].

Intracellular survival assays

The assays to determine the intracellular survival of pneumococci were performed as reported previously [33], but with modifications. Briefly, 3.0 × 10⁵ of A549 cells per well were seeded in 6 well plates and cultured in DMEM supplemented with 10% of fetal bovine serum (FBS) and incubated for 12 h. Pneumococci were grown in THYE to the mid-log phase (OD_600nm 0.3) and resuspended in DMEM (with 10% FBS). Infection of cell monolayers was carried out using a multiplicity of infection (MOI) 20:1. Bacterial internalization after incubation and washes with extracellular antibiotics was approximately 1%, and the occurrence of apoptosis/necrosis caused by pneumococcal infection quantified by flow cytometry (Annexin V/propidium iodide labeling kit; Invitrogen) was approximately 5–10% for all time points analyzed. A549 cells were incubated 3 h with pneumococcal strains and cells were washed three times with phosphate-buffered saline (PBS) and fresh DMEM (without FBS) containing 150 μg/ml potassium penicillin G (Sigma P7794) and 900 μg/ml gentamicin sulfate (US Biological G2030). After a 20 min rest period, cells were washed three times with PBS. The eukaryotic cells were lysed by centrifugation for 5 min at 10,000 rpm and the bacterial pellet was resuspended in THYE medium. The number of internalized bacteria at different time points was quantified after serial dilutions and plating on BHI 5% sheep blood agar plates with incubation for 16 h at 37°C. The time scale referred to the time after elimination of the extracellular bacteria by antibiotic treatment. A 100% survival was defined after 20 min of antibiotic treatment (S1 Fig), and all the samples were referred to this point to calculate the respective percentages.

Hydrogen peroxide determination

For the detection of H₂O₂ released by bacterial cells, the phenol red oxidation microassay was used. Briefly, cells were grown in BHI to the mid-log phase (OD_600nm 0.3). Posteriorly, cells were centrifuged at 10,000 x g for 5 min, resuspended in Todd Hewitt broth THB (pH 6.0) and incubated by 1 h at 37°C. Aliquots were taken and serially diluted to determine viable cells by plating in BHI-blood agar. Other aliquots were centrifuged at 10,000 x g for 5 min, and 100 μl of supernatants were transferred to multiwell plates and mixed with the same volume of PRS buffer (NaCl 140 mM, dextrose 5.5 mM, phenol red 280 μM, and horseradish peroxidase 8.5 U/ml in phosphate-buffered saline, pH7.0). Reactions were incubated for 90 min at 37°C and the reaction was stopped with 10 μl of 1 N NaOH, and the reactive wells were read in a microplate reader (Bio-Rad) with a 595-nm filter. Assays were performed in triplicate and results are expressed as mmoles of H₂O₂ released by 10⁶ cells.

Hydrogen peroxide susceptibility assays

Bacterial strains were grown until OD_600nm in BHI and aliquots were treated with H₂O₂ 20 mM (final concentration). Every 30 min, aliquots were taken and serially diluted to determine viable cells by plating in BHI-blood agar. The percent survival was calculated by dividing the CFU of cultures after exposure to H₂O₂ by the CFU of the control tube without H₂O₂. Assays were performed in triplicate and results are shown survival percentage at different time points.

Limited proteolysis assays

Limited proteolysis with proteinase K was carried out in a 30 μl reaction volume with 3 μg of ComE or ComE^T128E and 6 ng of the proteinase K in 10 mM Tris, 1 mM CaCl₂, pH 7.5, for 30 min at room temperature. Reactions were stopped with 5 mM PMSF, 5 mM EDTA and 1X Laemmli loading buffer, and immediately boiled for 5 min. Digestions with trypsin were performed under the same conditions as before but in 100 mM Tris, pH 8.5. Trypsinized samples were boiled immediately for 5 min after stopping the reactions with 5 mM PMSF and 1X Laemmli loading buffer. The extension of protein digestion was verified by 12% SDS-PAGE followed by SYPRO Ruby staining.

Accession numbers

The RNA-seq data generated from this study are deposited at the NCBI SRA under the accession numbers SAMN08473835 and SAMN08473836.