Crosstalk between the serine/threonine kinase StkP and the response regulator ComE controls the stress response and intracellular survival of Streptococcus pneumoniae

Streptococcus pneumoniae is an opportunistic human bacterial pathogen that usually colonizes the upper respiratory tract, but the invasion and survival mechanism in respiratory epithelial cells remains elusive. Previously, we described that acidic stress-induced lysis (ASIL) and intracellular survival are controlled by ComE through a yet unknown activation mechanism under acidic conditions, which is independent of the ComD histidine kinase that activates this response regulator for competence development at pH 7.8. Here, we demonstrate that the serine/threonine kinase StkP is essential for ASIL, and show that StkP phosphorylates ComE at Thr128. Molecular dynamic simulations predicted that Thr128-phosphorylation induces conformational changes on ComE’s DNA-binding domain. Using nonphosphorylatable (ComET128A) and phosphomimetic (ComET128E) proteins, we confirmed that Thr128-phosphorylation increased the DNA-binding affinity of ComE. The non-phosphorylated form of ComE interacted more strongly with StkP than the phosphomimetic form at acidic pH, suggesting that pH facilitated crosstalk. To identify the ComE-regulated genes under acidic conditions, a comparative transcriptomic analysis was performed between the comET128A and wt strains, and differential expression of 104 genes involved in different cellular processes was detected, suggesting that the StkP/ComE pathway induced global changes in response to acidic stress. In the comET128A mutant, the repression of spxB and sodA correlated with decreased H2O2 production, whereas the reduced expression of murN correlated with an increased resistance to cell wall antibiotic-induced lysis, compatible with cell wall alterations. In the comET128A mutant, ASIL was blocked and acid tolerance response was higher compared to the wt strain. These phenotypes, accompanied with low H2O2 production, are likely responsible for the increased survival in pneumocytes of the comET128A mutant. We propose that the StkP/ComE pathway controls the stress response, thus affecting the intracellular survival of S. pneumoniae in pneumocytes, one of the first barriers that this pathogen must cross to establish an infection.


Introduction
Sensing and transducing external (or internal) signals into an appropriate physiological response is part of a microorganism strategy to survive in a constantly changing environment. Signal transduction is mainly carried out by protein kinases, which autophosphorylate upon sensing stimuli and then catalyze the phosphorylation of a specific substrate that initiates an adaptive cellular response. In prokaryotes, signaling pathways are mainly mediated by twocomponent systems (TCS) consisting of sensor histidine kinases (HK) that phosphorylate response regulators (RR) on a receiver domain, thereby activating the effector domains of these regulators to induce a physiological event in bacterial cells. Generally, the RR effector domains bind regions of DNA that control gene expression in response to environmental changes [1,2]. Each particular HK presents a remarkable specificity for its cognate RR and is capable of identifying particular RRs.
Eukaryotic-like Ser/Thr protein kinases (STKs) are also present in prokaryotes, where they play key roles in several cellular processes, including the central or secondary metabolism, developmental processes, cell division and virulence [3]. The major human bacterial pathogen Streptococcus pneumoniae (S. pneumoniae or the pneumococcus) encodes a single copy of StkP, a eukaryotic-like serine/threonine protein kinase gene [4]. StkP is a membrane protein composed of an N-terminal kinase domain facing the cytoplasm, a short transmembrane region, and an extracellular C-terminal region containing four PASTA (Penicillin-binding protein and Ser/Thr protein kinase Associated) domains [4][5][6]. Comparison of the global expression profile of the wild-type and ΔstkP strains has revealed that the transcription of genes involved in the cell wall metabolism, pyrimidine biosynthesis, DNA repair, iron uptake, and oxidative stress response are controlled by StkP, which explain why stkP mutations have pleiotropic effects [7]. It has also been described that StkP phosphorylates several target proteins, mainly on threonine residues, with PASTA domains being essential for kinase activity [8][9][10]. However, phosphorylation on serine residues seems to be independent of StkP [11]. Immunofluorescence microscopy of pneumococcal cells localized StkP to the cell division apparatus [12], with phenotypic studies having demonstrated its impact on several cellular functions [13,14]. In fact, the stkP mutant displayed morphological and growth defects, cell division alterations, increased LytA-dependent autolysis (induced by either antibiotics or growth at an alkaline pH of 7.8), reduced tolerance to stress conditions (including acidic stress), and pilus-mediated adherence in endothelial cells [4,7,11,[15][16][17]. StkP is also essential for virulence, being necessary for lung infection and for invading and growing in the bloodstream of intranasally infected mice [4].
In S. pneumoniae, transient competence development (ability to take up exogenous DNA) in exponentially growing cells is considered a stress response to alkaline pH [2], with its core regulatory circuit being controlled by the TCS ComDE. In this quorum sensing system, the membrane-integrated HK ComD senses the extracellular accumulation of a 17 amino acid competence stimulating peptide (CSP) [2]. Upon activation by a critical concentration of CSP, ComD phosphorylates the response regulator ComE at Asp 58 [18], which consequently initiates the transcription of comCDE, comAB, and comX (a gene encoding an alternative sigma factor) [19,20]. ComX turns on the transcription of genes whose products are involved in DNA binding, uptake, and recombination [21]. In this sense, the competence development is considered to be a type of stress response to alkaline pH [2]. It has been reported that StkP can also regulate competence at pH 7.8. Cells lacking StkP do not develop natural competence [4] and show severely reduced CSP-induced competence [7,22], despite having increased expression of many genes of the CSP-regulated competence regulon [7].
To invade tissues, S. pneumoniae must overcome a variety of stress situations, such as acidic pH, as a consequence of host inflammatory responses against the invading pathogen [23]. This characteristic local acidosis is caused by infiltration of neutrophils and activation of inflammatory cells, which leads to increased energy and oxygen demand, accelerated glucose consumption via glycolysis and thus increased lactic acid secretion [24][25][26]. For instance, pH values obtained from pleural fluids from patients with acute bacterial pneumonia caused by S. pneumoniae showed an acidic pH close to 6.80 [27]. Interestingly, the lowest pH value that S. pneumoniae has been shown to be tolerant to is around 4.4 in phagosomal vesicles during the first few minutes after phagocytosis [28]. Although S. pneumoniae is considered a typical extracellular pathogen, a transient intracellular life was described, suggesting that it can survive inside eukaryotic cells. S. pneumoniae can cross brain microvascular endothelial cells inside vesicles derived from early and/or late endosomes [29] [30]. It is well accepted that acidification is essential to endosome/lysosome maturation, with early endosomes having a pH in the 6.8-6.1 range, late endosomes in the 6.0-4.8 range, whereas lysosomal pH values can drop to 4.5 [31]. In the case of a putative endosomal survival, S. pneumoniae must survive acidic conditions. Martin-Galiano et al [32] described that S. pneumoniae is able to induce an acid tolerance response (ATR) mechanism. Previously, we also showed that F 0 .F 1 -ATPase, a proton pump that controls intracellular pH, is relevant for ATR induction in S. pneumoniae. In addition, we demonstrated that the F 0 .F 1 -ATPase and ATR are necessary for the intracellular survival of the pneumococcus in macrophages [33].
As part of the acidic stress response, we have reported that exposure of S. pneumoniae to acidic culture conditions triggers a lytic response by the major autolysin LytA. The acidicstress induced lysis (ASIL) response is promoted by ComE and repressed by the CiaRH TCS. Despite requiring ComE, ASIL does not depend on CSP or ComD. Curiously, the comE gene is induced by acidic stress, but the competence-related ComX sigma factor, whose expression is regulated by ComE, does not participate in this signaling pathway [34]. We have also reported that ComDE and CiaRH control pneumococcal survival in pneumocytes in contrasting ways, CiaRH was essential for ATR and intracellular survival, whereas ComE repressed its activation. Moreover, ComE in a CSP-independent manner, was necessary for ASIL, whereas CiaRH protected against its induction by modulating LytA autolysin expression on the pneumococcal surface. These results suggest that both TCSs control the acidic stress response and establish either a survival or a suicidal response by independent pathways, either in acidified culture media or in pneumocyte cultures [33]. These findings indicate that ComE is activated under acidic conditions by an alternative signaling pathway that differs from the quorum sensing mechanism reported during competence development at alkaline pH. Alternatively, it was proposed that StkP is involved in competence at pH 7.8, by the fact that cells lacking StkP do not develop natural competence [4,7,22]. StkP is also essential for virulence, to establish infections in the lung and for invading and growing in the bloodstream of intranasally infected mice [4].
The main aim of this work was to elucidate whether ComE is part of a novel activation pathway used by S. pneumoniae to induce the acidic stress response and to control its intracellular survival mechanism in pneumocytes. Here, we demonstrate that StkP controls ComE activation by phosphorylation of the Thr 128 residue of the latter, increasing both its dimerization capacity and its DNA-binding affinity. Under acidic conditions, the StkP/ComE HKindependent pathway regulated 104 genes involved in different cellular processes, such as H 2 O 2 production and oxidative stress tolerance. The StkP/ComE pathway is independent of the HK-dependent ComD/ComE system, which regulates more than 180 genes at pH 7.8 [35]. The participation in HK-independent and HK-dependent stress regulatory systems places ComE as a global regulator. This newly discovered StkP/ComE signaling pathway triggered the acidic stress response by inducing ASIL and inhibiting ATR and the intracellular survival of S. pneumoniae in pneumocytes, one of the first barriers that this pathogen must overcome to establish an infection.

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 Asp 58 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 Asp 58 phosphorylation could be required for ASIL, and tested the comE D58A mutant that encodes for the ComE D58A protein, in which the phosphorylatable Asp 58 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 Asp 58 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 Asp 58 phosphorylation and HK activity.

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 Asp 58 .
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 Asp 58 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 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 acidinduced 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 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.
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 insertionduplication mutagenesis wt and ΔstkP strain derivatives that express ComE fused to the His x6epitope 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 Thr 128 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, Thr 128 , located inside the trypsin-digested 121 IEQNIFYTK 129 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 Thr 128 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 Thr 128 phosphorylation in ASIL activation, we attempted to replace Thr 128 by Glu 128 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 Thr 128 phosphorylation is essential for the StkP-mediated ComE activation that controls ASIL and that ComE hyper-activation is likely lethal to S. pneumoniae.

Competence regulation is independent of Thr 128 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 nonphosphorylatable 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 Thr 128 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 Thr 128 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]. Thr 128 is located at the end of the α-5 region (Asp 114 -Ser 130 ) of the receiver domain of ComE, next to the α-4 region (Ala 94 -Gln 101 ), and near the loop between α-4 and β-5 (Val 102 -Leu 105 ) 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 (Asp 58 and Thr 128 ) 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 Thr 128 phosphorylation modifies ComE in a manner that strongly affects its dimerization interface, which is a condition sine qua non for the response regulator activation.

Thr 128 phosphorylation changes conformation of the ComE DNA-binding domain
In order to determine how Thr 128 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). 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 (His 168 and Lys 169 in loop 1; His 202 and Lys 203 in loop 2; Arg 217 and Lys 218 in loop 3), which are shown in AgrA to have a direct contact with DNA bases [45]. These results suggest that after Thr 128 phosphorylation ComE may undergo conformational changes. Consistent with this notion, limited proteolysis assays revealed structural differences between ComE-His 6x and ComE T128E [43], this figure reveals the localization of the Thr 128 residue, as well as the alternative phosphorylation site Asp 58 . The three loops in the DNA-binding domain are also shown, which are apparently altered when ComE is phosphorylated on Thr 128 . 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 showed more contrasting proteolytic patterns than proteinase K treatment. These findings confirmed that Thr 128 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 Thr 128 phosphorylation in ComE increases its DNAbinding affinity in a pH-dependent manner
During competence development at alkaline pH, Asp 58 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 Thr 128 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 Thr 128 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 .
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 Thr 128 mediated the observed StkP effects on ComE: (1) at pH 6.0, Thr 128 phosphorylation by StkP increases ComE DNA binding affinity; (2) at pH 7.8 Thr 128 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 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 StkP/ComE crosstalk controls stress response and intracellular survival 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).
These data confirm that the StkP/ComE interactions are mediated by ComE residue Thr 128 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 StkP/ComE pathway controls H 2 O 2 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 H 2 O 2 from O 2 , sodA encodes the superoxide dismutase that produces H 2 O 2 from superoxide, and tpxD encodes the thiol peroxidase that catalyzes the H 2 O 2 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 H 2 O 2 production and H 2 O 2 susceptibility assays. The comE T128A , ΔcomE, and ΔstkP mutants showed a 4-fold decrease in their H 2 O 2 production compared to the wt and comE A128T strains (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 H 2 O 2 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 StkP/ComE crosstalk controls stress response and intracellular survival

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 log 2 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) ComEregulated 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: ÃÃ p < 0.01; ÃÃÃ p < 0.001. https://doi.org/10.1371/journal.ppat.1007118.g006

Fig 7. The StkP/ComE pathway controls oxidative stress and cell wall biosynthesis. (A)
The H 2 O 2 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. (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.

Discussion
Two-component systems (TCSs) represent one of the most important mechanisms of gene regulation in bacteria. Alternatively, eukaryotic-like serine-threonine kinases (STKs) constitute another signaling mechanism that bacteria utilize to regulate different cellular functions, such as stress response and pathogenesis. STKs are more promiscuous than the TCS-associated kinases and can phosphorylate different protein substrates producing pleiotropic effects [16,51,52]. STKs are also able to interact with TCSs by direct phosphorylation of RRs, as reviewed in [3,53]. STK-mediated RR activation takes place on either serine or threonine residues, instead of aspartate, which is the typical residue target for HK phosphorylation. STKmediated phosphorylation on DNA-binding domains of RR have been reported, as described for GraR in S. aureus [54], YvcK in Listeria monocytogenes [55] and RitR in S. pneumoniae [56]. STKs may also phosphorylate on receiver domains, as observed for CovR in S. pyogenes [57], WalR in B. subtillis [58], DosR in M. tuberculosis [59], or in both domains, as demonstrated for VraR in S. aureus [60]. ComE is the most studied RR in S. pneumoniae and belongs to the AlgR/AgrA/LytTR transcription factor subfamily, showing a typical receiver domain and a DNA-binding (or LytTR) domain. When phosphorylated by the ComD histidine kinase at the Asp 58 residue, ComE undergoes conformational changes that increase their DNA affinity and modify transcription regulation of competence genes by binding to their promoter regions [2,47,61]. In the present work, we show that S. pneumoniae utilizes an alternative signal transduction pathway to control acidic stress response (ASIL and ATR), oxidative stress, cell wall biosynthesis, and intracellular survival in pneumocytes. Under acidic conditions, a phosphorylation crosstalk between StkP and ComE involving phosphorylation at Thr 128 in the receiver domain resulted in activation of this RR.
Using the crystal structure of ComE [43], a molecular dynamic simulation of ComE permitted a comparison with the phosphomimetic ComE T128E protein, predicting that the Thr 128 phosphorylation produces structural changes in the DNA-binding domain. These putative conformational changes were confirmed by limited proteolysis assays that revealed differences between ComE and ComE T128E . In this sense, we observed that phosphorylation at either Asp 58 or Thr 128 increases the dimerization and DNA-binding capacity of ComE. These results were coincident with the activation model proposed by Boudes et al [43], where the most plausible activation mechanism of ComE is first a phosphorylation reaction to induce its dimerization, which occurs at the canonical receiver domain of ComE, followed by binding to DNA via the LytTR domain. It remains to be elucidated how phosphorylation at alternative sites in the receiver domain (Asp 58 or Thr128) modifies the DNA-binding domain of ComE.
Because ComE Thr 128 activation is independent of CSP/ComD activation by a quorum sensing mechanism, this RR requires alternative factors to act as a sensor and/or an environmental signal to trigger an adaptive stress response. We propose that StkP senses an alternative environmental signal, acidic pH. Our results are consistent with such notion: the level of comE transcripts induced under acidic conditions is the indicator of ComE activation due to the comCDE operon is autoregulated. 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 ( ÃÃ ) or p<0.001 ( ÃÃÃ ). https://doi.org/10.1371/journal.ppat.1007118.g008 An additional aspect to consider in the StkP/ComE crosstalk phosphorylation is the effect of pH on protein-protein interactions. We observed that the non-phosphorylated forms of both proteins show strong interaction at acidic pH. Once StkP auto-phosphorylates, it becomes metastable complex until it dissociates from the phosphorylated ComE form. Such cycle is favored at pH 6.0 and evidence for the outcome of such cycle is shown by the fact that phosphorylation of ComE at Thr 128 by StkP prevents further interaction between these two proteins. We propose the following cycle for StkP/ComE interaction: Pneumococcal H 2 O 2 production is one of the most significant among bacterial pathogens, and S. pneumoniae utilizes this intermediate metabolite to compete with the respiratory tract microbiota and to produce cytotoxic effects on the host tissues [62][63][64]. In this investigation, the transcriptome analysis of the comE T128A mutant revealed a marked decrease in the expression of spxB and sodA when cells were grown under acidic conditions. These results correlated with very low H 2 O 2 production by the comE T128A mutant associated with low expression of SpxB and SodA. Considering that H 2 O 2 is toxic for eukaryotic cells, we propose that reduction H 2 O 2 production in the comE T128A mutant facilitates its intracellular survival in pneumocytes. S. pneumoniae generates hyper-virulent mutants with defective spxB during infection [65], supporting the hypothesis that the H 2 O 2 levels are controlled during pneumococcal pathogenesis.
Because S. pneumoniae lacks catalase, and H 2 O 2 overproduction must be controlled for this pathogen to survive, this pathogen induces an oxidative stress resistance that is induced by endogenous H 2 O 2 [66]. In this sense, the transcriptome analyses of the comE T128A mutant revealed a decreased expression of tpxD, which encodes the thiol peroxidase. These findings correlate with the increased H 2 O 2 susceptibility by the comE T128A mutant. Similarly, a previous study showed that a tpxD (or psaD) mutant eliminated the H 2 O 2 -mediated response to high H 2 O 2 levels [67]. Previously, a microarray analysis of the stkP mutant revealed that tpxD expression is repressed, which is correlated with a low H 2 O 2 resistance of this mutant, but the putative regulatory mechanism was not mentioned [7]. In the present study, we have shown for the first time that the StkP/ComE pathway controls oxidative stress resistance and H 2 O 2 production under acidic conditions, which are probably responsible for the intracellular survival of S. pneumoniae in pneumocytes.
In a previous study, we proposed that ASIL may be activated under acidic conditions by a translocation of LytA from an intracellular to an extracellular compartment probably due to cell wall alterations by an unknown ComE-dependent mechanism [68]. Here, the comE T128A mutant displayed a decreased expression of murN, which encodes one of the first enzymes involved in the cell wall biosynthesis of S. pneumoniae [69]. Filipe et al [70] described that the murMN mutant showed an increased susceptibility to lysis when murMN cells were exposed to cell wall antibiotics, such as fosfomycin and vancomycin, which are involved in the inhibition of both the early and late stages of cell wall biosynthesis, respectively. Following this line of thinking, an altered expression of murN in the comE T128A mutant should cause a misbalance in the peptidoglycan biosynthesis and modify susceptibility to cell wall antibiotics. Accordingly, we observed that this mutant had an increased MIC of fosfomycin compared with wt, as well as a greater tolerance to autolysis induced by fosfomycin or vancomycin. The putative cell wall alterations indicated by antibiotic susceptibility tests may explain the autolysis inhibition shown by the comE T128A mutant under acidic stress, which probably interfered with LytA activation. Work is in progress to try to determine the nature of such cell wall alterations.
We also demonstrated that Thr 128 phosphorylation is not involved in competence. Regarding this topic, Guiral et al [71] described a phenomenon of lysis of non-competent cells triggered by competent cells, named allolysis, which involves bacteriocins and the autolysins LytA, LytC, and CbpD. Allolysis is considered to be a competence-induced mechanism of predation of non-competent cells that contributes to virulence by releasing pneumolysin [72]. Here, we show that the StkP/ComE signaling pathway can also trigger autolysis of noncompetent cells in acidic biological niches, such as inflammatory foci or endosomal compartments. This phenomenon occurred without the activation of a quorum sensing mechanism, a situation that allows bacterial cells to lyse even under low population density conditions. StkP and ComE have already been shown to be involved in pneumococcal pathogenesis in different studies using animal models, with StkP appearing to be involved in bacterial survival in vivo [4,73]. On the other hand, ComE-mediated competence for DNA transformation has been also associated with virulence [74,75]. As mentioned above, pneumolysin release by competence-mediated autolysis was considered to be essential for pneumococcal pathogenesis [71]. Concerning the impact of StkP/ComE pathway regulation on pneumococcal pathogenesis, we propose that two different scenarios should be considered. In extracellular niches, a subpopulation of pneumococci exposed to acidic stress may cause tissue damage by overproduction of H 2 O 2 and induction of ASIL to release pneumolysin, with this suicidal situation being promoted by StkP-mediated phosphorylation of ComE. On the other hand, the Thr 128nonphosphorylated form of ComE might facilitate pneumococcal survival at either the extracellular or the intracellular level in host tissues. We focused our attention on the first barrier that this pathogen must cross to establish an infection, and we hypothesized that intracellular survival in pneumocytes should be important for S. pneumoniae. The ΔstkP, ΔcomE, and comE T128A mutants were tested in the pneumococcal infection model in A549 pneumocytes, and they revealed an increased survival compared with wt. Thus, we conclude that this survival could have been caused by increasing their capacity of ATR, decreasing the H 2 O 2 production and modifying the cell wall biosynthesis to repress ASIL (Fig 9). Establishing how the balance between H 2 O 2 resistance mechanism and H 2 O 2 production affects intracellular survival is beyond the scope of this report, but this work is in progress. Finally, we propose that the StkP/ ComE pathway is relevant in the genetic regulation of physiological adaptation to environmental stress, which is necessary for pneumococcal survival in pneumocytes. This is one of the first steps in the pathogenic process that S. pneumoniae must overcome to produce infection.

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 600nm0 .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% CO 2 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 cm 2 (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 MgCl 2 , 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 NaH 2 PO 4 /10 mM TrisÁHCl, 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 MgCl 2 , 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 (

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 NaHCO 3 /Na 2 CO 3 , pH 9.4) and incubated overnight at 4˚C to allow protein adsorption. Wells were rinsed five times with Trisbuffered 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 MgCl 2 , 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.
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-Ni 2+ 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 Ä KTA 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 GSTtagged 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 × 10 5 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 H 2 O 2 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 H 2 O 2 released by 10 6 cells.

Hydrogen peroxide susceptibility assays
Bacterial strains were grown until OD 600nm in BHI and aliquots were treated with H 2 O 2 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 H 2 O 2 by the CFU of the control tube without H 2 O 2 . 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 CaCl 2 , 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.