The pneumococcal two-component system SirRH is linked to enhanced intracellular survival of Streptococcus pneumoniae in influenza-infected pulmonary cells

Artículo científico de investigación en acceso abierto.The virus-bacterial synergism implicated in secondary bacterial infections caused by Streptococcus pneumoniae following infection with epidemic or pandemic influenza A virus (IAV) is well documented. However, the molecular mechanisms behind such synergism remain largely ill-defined. In pneumocytes infected with influenza A virus, subsequent infection with S. pneumoniae leads to enhanced pneumococcal intracellular survival. The pneumococcal two-component system SirRH appears essential for such enhanced survival. Through comparative transcriptomic analysis between the ΔsirR and wt strains, a list of 179 differentially expressed genes was defined. Among those, the clpL protein chaperone gene and the psaB Mn+2 transporter gene, which are involved in the stress response, are important in enhancing S. pneumoniae survival in influenza-infected cells. The ΔsirR, ΔclpL and ΔpsaB deletion mutants display increased susceptibility to acidic and oxidative stress and no enhancement of intracellular survival in IAV-infected pneumocyte cells. These results suggest that the SirRH two-component system senses IAV-induced stress conditions and controls adaptive responses that allow survival of S. pneumoniae in IAV-infected pneumocytes.publishedVersionFil: Cortes, Paulo R. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina.Fil: Cian, Melina Beatriz. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina.Fil: Olivero, Nadia B. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina.Fil: Hernández-Morfa, Mirelys. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina.Fil: Piñas, Germán. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina.Fil: Badapanda, Chandan. Bioinformatics Division. Xcelris Labs Limited; Ahmedabad; India.Fil: Rathore, Ankita. Bioinformatics Division. Xcelris Labs Limited; Ahmedabad; India.Fil: Echenique, José Ricardo. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina.Fil: Reinoso-Vizcaíno, Nicolás Martín. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina.Fil: Cortes, Paulo R. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.Fil: Cian, Melina Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.Fil: Olivero, Nadia B. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.Fil: Hernández-Morfa, Mirelys. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.Fil: Piñas, Germán. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.Fil: Echenique, José Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.Fil: Reinoso-Vizcaíno, Nicolás Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.Fil: Perez, Daniel R. University of Georgia. Department of Population Health. College of Veterinary Medicine; Athens; Georgia; United States of America.Fil: Cian, Melina Beatriz. University of Oklahoma. Departament of Microbiology and Inmunology. Health Sciences Center; Oklahoma, United States of America.Fil: Piñas, Germán. University of Utah. School of Biological Sciences; Salt Lake City; Utah; United States of America

ComE is phosphorylated by StkP.

<p>(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 <i>wt</i> and <i>ΔstkP</i> 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.</p

Thr¹²⁸-phosphorylation increases the dimeric state of ComE.

<p>(A) Localization of Thr¹²⁸ residue in the ComE structure. Based on the crystal structure of ComE reported by Boudes <i>et al</i> [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007118#ppat.1007118.ref043" target="_blank">43</a>], 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 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007118#ppat.1007118.ref045" target="_blank">45</a>], 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.</p

ComE binding affinities to p<i>comC</i>.

<p>ComE binding affinities to p<i>comC</i>.</p

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

<p>The DNA-binding affinity for the promoter region of the <i>comCDE</i> operon (p<i>comC</i>) 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 p<i>comC</i>, 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 <i>Kd</i> values are indicated in each panel.</p

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

<p><b>(</b>A) The H₂O₂ production is altered in the <i>comE</i> and <i>stkP</i> 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 <i>comE and stkP</i> mutants were more susceptible to H₂O₂ than <i>wt</i>. Susceptibility to H₂O₂ is indicated as a percentage of bacterial survival at different time points. C-D) The <i>comE</i>^<i>T128A</i> mutant was more resistant to cell-wall antibiotic-induced lysis than <i>wt</i>. 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.</p

The StkP/ComE pathway modulates intracellular survival and the acid tolerance response of <i>S</i>. <i>pneumoniae</i>.

<p>(A) The Δ<i>stkP</i> and Δ<i>comE</i> mutants showed increased intracellular survival compared with <i>wt</i> 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 Δ<i>stkP</i> and Δ<i>comE</i> mutants displayed an augmented ATR compared with <i>wt</i>. 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 <i>p</i><0.01 (**) or <i>p</i><0.001 (***).</p

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

<p>(A) Gene expression scatter plot in the <i>wt</i> and <i>comE</i>^<i>T128A</i> samples, with the <i>x</i>-axis representing the gene expression values for the control condition (<i>wt</i>) and the <i>y</i>-axis representing those for the treated condition (<i>comE</i>^<i>T128A</i>). 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 <i>wt</i> vs <i>comE</i>^<i>T128A</i> samples measured by RNAseq. The <i>y</i>-axis represents the mean expression value of the log₁₀ (<i>p</i>-value), while the <i>x</i>-axis displays the log₂ fold change value. Black dots represent genes with an expression 2-fold higher in the <i>comE</i>^<i>T128A</i> mutant relative to strain <i>wt</i> with a <i>p</i>-value < 0.05, with red dots signifying genes with an expression 2-fold lower in the <i>comE</i>^<i>T128A</i> mutant, which are relative to strain <i>wt</i> with a <i>p</i> < 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 <i>comE</i>^<i>T128A</i> mutant relative to strain <i>wt</i> under acidic conditions. (D) ComE-regulated genes expressed under acidic conditions in the <i>comE</i>^<i>T128A</i> mutant relative to strain <i>wt</i>. Gene expression determined by RNAseq was confirmed by qPCR. The <i>comE</i>^<i>T128A</i> Δ<i>lytA</i> and Δ<i>lytA</i> (referred as <i>wt</i> 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 <i>gyrA</i> gene was used as the internal control. References: <b>**</b> <i>p</i> < 0.01; <b>***</b> <i>p</i> < 0.001.</p

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

<p>Proposed model for crosstalk between StkP and ComE that impacts on the acidic stress response and intracellular survival mechanisms in <i>S</i>. <i>pneumoniae</i> exposed to acidic conditions.</p

Evaluation of ASIL and <i>comE</i> expression in <i>S</i>. <i>pneumoniae</i> mutants.

<p>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: *<i>p</i>< 0.05; **<i>p</i>< 0.01; ***<i>p</i>< 0.001, these <i>p</i>-values were referred to the <i>wt</i> strain in each panels. (D) Transcription levels of the <i>comE</i> gene measured in cells exposed to pH 6.0. To avoid autolysis, all mutants were constructed in a Δ<i>lytA</i> (autolysin deficient) background. The Δ<i>lytA</i>, <i>comE</i>^<i>D58A</i> Δ<i>lytA</i>, Δ<i>stkP ΔlytA</i>, <i>comD</i>^<i>T233I</i> <i>ΔlytA and comE</i>^<i>T128A</i> <i>ΔlytA</i> 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 <i>gyrB</i> gene was used as the internal control and the reference condition was time 0 min of strain Δ<i>lytA</i>. 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: **<i>p</i>< 0.01; ***<i>p</i>< 0.001.</p