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

Abstract

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.

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.

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.

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.

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.

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.

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.

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.

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 (***).

Fig 9. Proposed model for crosstalk between StkP and ComE that impacts on the acidic stress response and intracellular survival mechanisms in S. pneumoniae exposed to acidic conditions.