Respiratory syncytial virus G glycoprotein promotes Streptococcus pneumoniae-induced severe pneumonia

Respiratory syncytial virus G glycoprotein promotes Streptococcus pneumoniae-induced severe pneumonia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Respiratory syncytial virus G glycoprotein promotes Streptococcus pneumoniae-induced severe pneumonia Lu Li, Xin Long, Yushun Wan, Yuncheng Wang, Peiru Shi, Enmei Liu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5978497/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objectives Co-infection with RSV and S.pn is linked to severe, often fatal pneumonia, with unclear molecular mechanisms. This study investigates whether RSV and S.pn interaction enhances pneumococcal pathogenicity and explores the underlying molecular mechanisms. Methods We co-incubated S.pn with RSV and transnasally infected mice, using RNA-seq and proteomics to analyze bacterial pathogenesis. Airway epithelial cells were infected with RSV and subsequently challenged with S.pn . We constructed an in vitro biofilm model for further study. Confocal microscopy and Western blotting investigated the association between the RSV G glycoprotein and S.pn . Mass spectrometry and pull-down assays identified surface proteins involved in direct binding. Results After incubation with RSV, S.pn exhibited a significant increase in inflammatory response and adherence to epithelial cells, as well as enhanced virulence in a murine pneumonia model. These effects were associated with extensive changes in the proteomics of S.pn and significantly upregulated expression of peptidoglycan biosynthesis genes. Additionally, we found that the RSV G glycoprotein binds to the maltodextrin ABC transporter substrate-binding protein of S.pn , which may explain how RSV infection enhances S.pn adhesion to cells. Conclusions The direct interaction between the RSV G glycoprotein and S.pn , leading to increased bacterial pathogenicity and more severe disease outcomes, represents a novel paradigm in respiratory infections. Clinical trial number Not applicable. Respiratory syncytial virus Streptococcus pneumoniae pneumonia G glycoprotein co-infection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Lower respiratory tract infections remain the leading cause of childhood morbidity and mortality worldwide, despite advancements in healthcare systems, expanded immunization coverage, and improved nutritional standards. Pneumonia alone accounts for nearly one million deaths each year in children under the age of 5, predominantly from low- and middle-income countries ( 1 ). Among the etiological agents of pediatric pneumonia, respiratory syncytial virus (RSV) and Streptococcus pneumoniae ( S.pn ) are particularly prominent ( 2 ). S.pn is part of the normal respiratory microbiota and frequently exists in a benign, asymptomatic state in children. However, during viral respiratory infections, asymptomatic colonization can become pathogenic, with emerging evidence implicating RSV as a critical factor in triggering this transition ( 3 , 4 ). Epidemiological research has consistently highlighted a correlation between seasonal RSV activity and the incidence of pneumococcal disease in infants and older children ( 5 – 7 ). Experimental studies using mouse models have demonstrated that prior RSV infection significantly increases the risk of pneumococcal sepsis and impairs bacterial clearance from the lungs ( 8 ), emphasizing the complex interplay between viral and bacterial pathogens. Understanding the molecular mechanisms underlying these pathogenic interactions is important for advancing targeted interventions. Recent developments in RSV prophylaxis, including the approval of maternal vaccines and monoclonal antibodies for infant immunoprophylaxis ( 9 ), offer promising strategies to mitigate RSV-associated disease. The potential impact of RSV vaccination could be further enhanced by the concurrent availability of broad-spectrum pneumococcal vaccines, which may synergistically reduce the severity of RSV and pneumococcal co-infections. These combined measures have the potential to significantly decrease the burden of severe disease, reduce reliance on antibiotics, and inform healthcare strategies aimed at improving outcomes for affected populations. Our previous retrospective study revealed that S.pn colonization of the upper respiratory tract worsened clinical outcomes in RSV-infected children aged 6 months and older ( 10 ). In a prospective cohort analysis employing 16S rRNA sequencing, RSV-infected children exhibited lower alpha diversity in upper respiratory tract microbiota (0.84, interquartile range 0.35–1.17) compared to healthy controls (3.06, interquartile range 2.88–3.61) ( P < 0.001). Additionally, the bacterial composition in healthy controls was more diverse, while the RSV-infected group displayed a dominance of S.pn within their upper respiratory tract flora. This study investigated the effects of RSV on pneumococcal pathogenicity after co-incubation, with a particular focus on the molecular mechanisms enhancing bacterial adhesion to epithelial cells. Severe pneumonia development was evaluated in a murine model, revealing how RSV co-incubation influences disease progression and related molecular pathways. Further analysis showed that RSV infection enhanced S.pn adhesion and induced the expression of viral surface proteins in infected cells, highlighting specific interactions between RSV G glycoproteins and pneumococcal surface proteins. The effects of RSV on S.pn biofilm formation were also assessed, providing novel insights into the interactions between RSV and S.pn . MATERIALS AND METHODS Viral and bacterial growth conditions Respiratory syncytial virus subtype A strain A2 (RSV A2) was purchased from ATCC and inoculated into human laryngeal epidermoid carcinoma (HEp-2) cells for expansion, which were harvested once syncytium formation was evident (approximately 48–72 h). The virus was purified using sucrose gradient centrifugation, aliquoted, and stored in liquid nitrogen until use. Viral titers, expressed as plaque-forming units (PFU), were quantified using a plaque assay. The RSV A2 viral titer used in this study was 1 × 10 7 PFU/mL. The S.pn serotype 2 strain D39 ( S.pn D39) was kindly donated by Ms Xuemei Zhang’s research group at the Institute of Life Sciences, Chongqing Medical University, China. This pathogenic strain was preserved in glycerol at − 80°C and resuscitated on Columbia blood agar plates at 37°C overnight, then resuspended in Todd-Hewitt broth + Yeast powder (THY) and cultured at 37°C in 5% CO 2 for 4–6 h until the optical density at 600 nm (OD 600 ) reached 0.45–0.5. Colony-forming unit (CFU) determination was conducted using plate counting. Briefly, 1 mL of S.pn bacterial solution was serially diluted 10-fold with phosphate-buffered saline (PBS) and plated onto blood agar. Colony counts were recorded after 24 h of incubation at 37°C. The S.pn concentration used in this study was 1.5 × 10 8 CFU/mL. Viral and bacterial co-incubation A 1-mL aliquot of S.pn bacterial solution (OD 600 = 0.45–0.50) was serially diluted 10-fold with PBS. Subsequently, 1 mL of the diluted bacterial solution was centrifuged (1200rpm, 8min, 4℃), and the resulting pellet was resuspended in RSV A2 to prepare the RSV + S.pn mixture. For the control group ( S.pn only), an equal amount of S.pn was resuspended in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) containing 2% fetal bovine serum (FBS). Both suspensions were incubated on a shaker at 37°C and 200 rpm for 30 minutes. After incubation, the mixtures were centrifuged, the supernatants were discarded, and the pellets were resuspended in PBS for further use. Mouse pneumonia model Groups of 10 female BALB/c mice (6–8 weeks old) were randomly assigned to three groups: blank, S.pn , and RSV + S.pn . The mice were anaesthetized with chloral hydrate and intranasally inoculated with 1.5 × 10 6 CFU/50 µL of S.pn (treatment groups) or 50 µL of PBS (blank group), then monitored for 7 days to evaluate survival rates and extent of acute lung and extrapulmonary organ invasion. At predetermined time points, mice were euthanized via cervical dislocation. The left lung was fixed in 4% paraformaldehyde for histological analysis, while the right lung was homogenized and divided into three portions for storage at − 80°C. Total RNA from lung homogenates was extracted using the Simply P Total RNA Extraction Kit (Bioflux, U.S.). Reverse transcription was immediately performed using the PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, J.P.N.), following the manufacturer's instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using ChamQ Universal SYBR Qpvr Master Mix (Vazyme, C.H.N.). The primers for the RSV A2 N gene were as follows: forward primer 5’-AGATCAACTTCTGTCATCCAGCAA-3’, reverse primer 5’-TTCTGCACATCATAATTAGGAGTATCAAT-3’. Lung paraffin sectioning and hematoxylin and eosin (H&E) staining Fixed lung tissue was dehydrated and embedded in paraffin to create paraffin blocks. These blocks were sectioned using a paraffin slicer, and the sections were placed on slides and baked in an oven at 60°C overnight. After the paraffin was sufficiently melted, H&E staining was performed according to the following procedure: Xylene (I) 10 min, xylene (II) 5 min, followed by gradual hydration; 100% ethanol 3 min, 95% ethanol 3 min, 75% ethanol 3 min, hematoxylin staining 3 min, flowing water 1 min, hydrochloric acid-ethanol 3 s, flowing water 1 min, saturated lithium carbonate 10 s, flowing water 1 min, 95% ethanol 1 min, eosin staining 10 s, flowing water 1 min, 95% ethanol 1 min, 100% ethanol 1 min, xylene (I), and xylene (II) 2 min. Finally, the sections were mounted with neutral resin and lung lesions were examined under a light microscope, with images captured using a pathology scanner(TEKSQRAY, C.H.N.). Bronchoalveolar lavage fluid (BALF) inflammatory cell differential count assay Mice were restrained in the supine position, and the trachea was bluntly separated and fully exposed for intubation using an indwelling needle. Subsequently, 0.5 mL of pre-cooled PBS was slowly injected with a syringe along the direction of the tracheal intubation to collect bronchoalveolar lavage fluid (BALF). This process was repeated three times to ensure adequate recovery. The collected BALF was centrifuged (1500rpm, 10min, 4℃), and the resulting cell precipitate was resuspended in 1 mL of PBS. A 20-µL aliquot was used to determine total cell count using a Countstar cell counting plate. The remaining suspension was centrifuged again to remove the supernatant. The resulting cell pellet was smeared onto slides, stained with Rachel's stain, and examined under an oil microscope to differentiate neutrophils and macrophages based on their morphological characteristics. Lung cytokine measurements by enzyme-linked immunosorbent assay (ELISA) Mouse lung proteins were extracted using RIPA lysis buffer according to the manufacturer’s instructions (Sigma, G.E.R.). Protein concentrations in the samples were determined using a Bicinchoninic Acid (BCA) Protein Assay Kit (Beyotime, C.H.N.). For cytokine measurements, 45-µg aliquots of each sample were prepared and TNF-α, IL-1β, IL-10, IFN-γ, GM-CSF, MCP-1, IL-6, IL-17A, IL-1α, G-CSF, KC, and MIP-1α levels were quantified using the Bio-Plex Pro Mouse Cytokine Grp I Panel 23-Plex on the Bio-Plex MAGPIX System (Wayen Biotechnologies, C.H.N.) according to the manufacturer's instructions. Cytokine concentrations were calculated based on fluorescence values obtained from recombinant cytokine standards in 96-well plates using Bio-Plex Manager software. Lung tissue RNA sequencing (RNA-seq) Total RNA was extracted from mouse lungs using TRIzol® reagent (Invitrogen, U.S.), and genomic DNA was removed using DNase I (TaKara, U.S.). RNA quality was determined using the NanoDrop 2000 spectrophotometer (NanoDrop Technologies, U.S.) and RNA degradation was verified by agarose gel electrophoresis. Only RNA samples meeting quality standards were used for library preparation. RNA purification, reverse transcription, library construction, and sequencing were carried out by Shanghai Majorbio Bio-Pharm Technology Co., Ltd, China. Differential gene expression was analyzed using standard criteria: |log₂FC| ≥ 1, and one of the following thresholds for significance: FDR < 0.05 (DESeq2, edgeR, or Limma), FDR 0.8 (NOIseq). A gene was considered differentially expressed if both conditions were satisfied. In addition, functional enrichment analysis of DEGs was conducted using the Gene Ontology (GO) ( http://www.geneontology.org ) and Kyoto Encyclopedia of Genes and Genomes (KEGG) ( http://www.genome.jp/kegg/ ) databases. Quantitative reverse transcription PCR Bacterial RNA was extracted using a RNeasy Mini Kit (Qiagen, G.E.R.) according to the manufacturer’s instructions. Reverse transcription was immediately carried out using a Transcriptor First Strand cDNA Synthesis Kit (Roche, G.E.R.) as per the manufacturer’s instructions. qRT-PCR was performed using ChamQ Universal SYBR Qpvr Master Mix. Primers for lytA, comE, nanA, ply, ciaR, stkP, soda, nox, and gyrB are listed in S1 Table. Relative gene expression was analyzed using the 2 −△△Ct method, with gyrB used as the reference gene and S.pn exposed to DMEM alone used as the reference condition. Each analysis included six distinct biological replicates, and qRT-PCR was performed in triplicate. A P -value less than 0.05 was considered statistically significant. Protein label-free quantitative analysis by mass spectrometry Bacterial protein identification and analysis were conducted by Shanghai Majorbio Bio-Pharm Technology Co., Ltd, China. Bacterial protein supernatants were extracted by adding SDT (SDS + MDTT + Tris-HCL) lysis buffer to each sample, and protein quantification was performed using the BCA method. For each sample, 10 µg of protein was subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Blue. Proteins were then digested using the Protein Digestion Kit (Abcam, U.K.), the resulting peptides were dried and resolubilized with 0.1% TFA. Peptide concentrations were determined prior to liquid chromatography-mass spectrometry (LC-MS). Peptides were chromatographically separated using an Easy nLC 1200 chromatography system (Thermo Scientific, U.S.) with a nanoliter flow rate. The resulting LC-MS/MS RAW files were imported into the SEQUEST HT search engine in Proteome Discoverer v2.4 (Thermo Scientific, U.S.). Database searches were performed using UniProt-Human respiratory syncytial virus A (strain A2) [208893]-8928-20230803.fasta ( https://www.uniprot.org/taxonomy/12088 ) and UniProt- Streptococcus pneumoniae serotype 2 (strain D39 NCTC 7466) [373153]-1922-20230803.fasta ( https://www.uniprot.org/taxonomy/373153 ). Proteins meeting the screening criteria of the fold change in expression level greater than 1.5 (up- and down-regulated) and P -value less than 0.05 were considered as significantly differentially expressed proteins. Identified bacterial proteins were subjected to subcellular localization analysis using the GO database and functional enrichment analyses with both the GO and KEGG databases. S.pn adhesion assay Human non-small cell lung cancer cells (A549) were purchased from ATCC and cultured in DMEM, supplemented with 10% FBS, penicillin (100 IU/mL) and streptomycin (100 µg/mL) at 37°C under 5% CO 2 . When the A549 cells reached 80–90% confluence, they were infected with RSV A2 at a multiplicity of infection (MOI) of 1 or mock-infected with DMEM in 2% FBS. After 24 h of RSV infection, the cells were washed three times with sterile PBS. Subsequently, S.pn D39 was added to the cells and incubated at 37°C for 3 h. Non-adherent bacteria were removed by washing with PBS, and adherent bacteria were collected and quantified using plate counting. Immunofluorescence assay by confocal laser scanning microscopy Bacteria were fixed in 4% paraformaldehyde for 30 min, followed by incubation with 5% bovine serum albumin (BSA) in PBS for 30 min. After washing three times with PBS, all antibody incubations were performed in PBS containing 5% BSA. Rabbit anti-pneumococcal capsular polysaccharide and mouse anti-RSV antibodies were applied to the bacteria and incubated overnight at 4°C. After washing three times with PBS, bound pneumococcal polysaccharide and RSV primary antibodies were detected using Alexa Fluor 594 conjugated donkey anti-rabbit antibody and Alexa Fluor 488 donkey conjugated anti-mouse antibody, respectively (Abcam, U.K.). After washing three times with PBS, bacteria were transferred to sterile glass-bottomed petri dishes, sealed with an anti-fluorescence quenching agent, and observed using confocal laser scanning microscopy (CLSM, Nikon C2, J.P.N.). A549 cells were cultured in 24-well plates containing round coverslips. The cells were fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.5% Triton X-100 for 15 min, and blocked with 5% BSA for 30 min. Primary antibodies targeting the desired proteins were added, and the cells were incubated overnight at 4°C, washed with PBS three times, and mixed with fluorescent antibody bound to target protein primary antibodies at room temperature for 1 h. The cells were then stained with 4’,6-diamidino-2-phenylindole (DAPI) at room temperature for 10 min, and coverslips were mounted onto microscope-adherent slides using glycerol gelatin sealing solution. Imaging was performed using CLSM. Antibodies used in this study included mouse anti-RSV nucleoprotein (N), mouse anti-RSV G glycoprotein, mouse anti-RSV fusion (F) glycoprotein (Abcam, U.K.), CoraLite 488-conjugated alpha tubulin monoclonal, Alexa Fluor 594-conjugated goat anti-mouse IgG, and Alexa Fluor 647-conjugated goat anti-mouse IgG antibodies (Proteintech, C.H.N.). S.pn adherent detection by scanning electron microscopy Cells were seeded in 24-well plates containing round coverslips and infected with RSV A2 at an MOI of 1 for 48 h, then washed three times with PBS. S.pn was added and allowed to adhere for 3 h, after which non-adherent bacteria were removed by washing with PBS three times. The cells were fixed with 3% glutaraldehyde, then sent to Chengdu Li Lai Biotechnology Co., Ltd (China). for scanning electron microscopy (SEM) analysis. The fixed samples were washed three times with ultrapure water (10 min each time), followed by fixation with 1% osmic acid for 1–2 h and an additional three washes with ultrapure water for 10 min. Dehydration was performed using an alcohol gradient (30% → 50% → 70% → 90% → 100%, with three changes of 100%), with each step lasting 15 min. The coverslips were dried using a critical point dryer, mounted onto sample stages with conductive adhesive, and sputter-coated with gold. Imaging was conducted using a JEOL JSM-IT700HR (G.E.R.) scanning electron microscope. In vitro biofilm model A biofilm model was established using round coverslips in a 24-well plate. RSV A2 viral particles, purified RSV G glycoproteins, and DMEM with 2% FBS were incubated with S.pn in vitro on a shaker at 37°C for 30 min. The bacterial suspension was centrifuged, after which the supernatant was removed and the S.pn precipitates were resuspended in 500 µL of THY medium. Subsequently, 100 µL of bacterial suspension and 900 µL of sterile THY were added to each well, and the plate was incubated at 37°C for 24 h. After 24 h, the medium was replaced with fresh sterile THY, with or without antibiotics, and the plate was incubated for another 24 h to establish the biofilm model. The antibiotics used in this study included penicillin (100 µg/mL) + gentamicin (50 µg/mL), ceftazidime (100 µg/mL), azithromycin (100 µg/mL), and tetracycline (100 µg/mL) (Solarbio). SEM and CLSM observations were used for biofilm analysis. For SEM, biofilm samples were fixed and dehydrated with ethanol gradients (30%, 50%, 70%, 90%, and 100%) for 15 min each step, dried, and prepared for observation with an ion-sputtering apparatus. For CLSM, biofilm samples were stained using a LIVE/DEAD BacLight Bacterial Viability Kit (Thermo Scientific, U.S.) according to the manufacturer’s guidelines, and images were obtained by CLSM. Biofilm thickness was measured by horizontal scanning along the z-axis of a three-dimensional coordinate system. The biofilm was scanned layer by layer, with a step size of 2.05 µm. The thickness was determined as the z-axis distance between the last plane where the biofilm structure was visible and the first plane where it was not. Western blot analysis The RSV A2 strain G (UniProt ID: P03423, 298 Amino acids) and F coding sequences (UniProt ID: P03420, 574 Amino acids) were cloned into the pcDNA3.1 vector with C-terminal HA or Myc-His tags, respectively. Human embryonic kidney 293 cells (HEK-293T) were purchased from Procell (C.H.N.), and cellular proteins were extracted 48 h after transfection using polyethylenimine linear (PEI) MW40000 (YEASEN). Total cellular proteins were isolated using the Whole-Cell Lysis Assay (KeyGEN, C.H.N.). Cells were lysed with lysis buffer mixed with phosphatase inhibitor, protease inhibitor, and phenylmethylsulfonyl fluoride (PMSF) for 20 min on ice. Protein concentrations were determined using the BCA method. Equal amounts of protein were separated on 4–20% SDS-polyacrylamide gel and subsequently transferred to polyvinylidene difluoride membranes (Millipore, U.S.). The membranes were blocked with QuickBlock solution (Beyotime, C.H.N) for 30 min, then incubated with different primary antibodies overnight at 4°C, including RSV G and fusion (F) glycoprotein antibodies (Abcam, U.K.) and HA and 6×His tag antibodies (Proteintech, C.H.N). After washing three times with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse or HRP-conjugated GAPDH antibodies (Proteintech, C.H.N) for 1 h at room temperature. Enhanced chemiluminescence immunoblotting detection reagents were used to detect bound antibodies and images were captured using a ChemiDoc Imaging system (Bio-Rad, U.S.). Quantification of the blots was performed with ImageJ v1.53c (National Institutes of Health, U.S.) and normalized to the GAPDH intensity of each lane. Pull-down assays The purified G- and F-glycoproteins of RSV A2 were synthesized and purified from E.coli BL21 strain by affility chromatography though AKTA system and confirmed by coomassie blue staining. Pull-down experiments were performed using the His-Tag Protein Purification Kit with NTA-Ni magnetic agarose beads (Beyotime, C.H.N.) following the manufacturer’s protocols. G- and F-glycoproteins were incubated with NTA-Ni magnetic agarose beads for 30 min at room temperature on a rotary mixer, centrifuged, washed three times with non-denaturing wash solution, and detected by western blotting. The collected S.pn bacterial solution was centrifuged, and total proteins were extracted by adding bacterial lysis buffer containing lysozyme. The extracted proteins were stored at − 80°C for future use. Bacterial proteins were incubated with G- and/or F-glycoprotein magnetic beads overnight at 4°C on a rotator. After incubation, the beads were magnetically separated and washed three times with non-denaturing wash solution. To elute the bound proteins, 20 µL of non-denaturing eluent was added to the beads, followed by 10 min incubation. The beads were then magnetically separated, and the eluent containing bacterial proteins bound to the G- or F-glycoprotein was collected in a fresh centrifuge tube. After electrophoretic separation of proteins on SDS-polyacrylamide gel, silver staining experiments were performed using a Fast Silver Stain Kit (Beyotime, C.H.N). Proteins were identified through LC-MS/MS by Shanghai Bioprofile, and the resulting raw files were analyzed and matched using MaxQuant software v1.6.2.10 (Computational Systems Biochemistry, G.E.R.). The S.pn D39 strain ComGG and ABC coding sequences were cloned into the pGEX-6P-1 vector with C-terminal GST tags. Both ABJ53717.1-pGEX-6P-1 and ABJ55468.1-pGEX-6p-1 plasmids were constructed by Wuhan Gene Create, and E. coli BL21 (DE3) competent cells (Thermo Scientific, U.S.) were used to express S.pn late competence protein ComGG (ComGG protein) and maltodextrin ABC transporter substrate-binding protein (ABC protein). Three to four recombinant protein-expressing colonies were picked and inoculated into 5 mL of Luria-Bertani (LB) medium containing selective antibiotics. Cultures were shaken overnight at 37°C until saturation (OD 600 ≥ 2). Overnight cultures were diluted 1:50 in fresh LB medium with antibiotics and incubated for 2–3 h until the mid-log phase (OD 600 = 0.4). Protein expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 2–3 h (OD 600 = 0.5–0.6). The GST-tagged recombinant proteins were detected by western blotting using GAT-tag antibodies (Proteintech, C.H.N.). The S.pn recombinant proteins were incubated with G-glycoprotein magnetic beads overnight at 4°C, as described previously. The eluted proteins, representing bacterial recombinant proteins bound to the G-glycoprotein, were analyzed by western blotting. Protein structure prediction and bioinformatics analysis The amino acid sequences of the human respiratory syncytial virus subtype A (strain A2) G glycoprotein (protein ID: P03423), late competence protein ComGG (protein ID: A0A0H2ZQ87), and maltodextrin ABC transporter substrate-binding protein (protein ID: A0A0H2ZLL1) of the S.pn serotype 2 (strain D39) were retrieved from the UniProt database ( https://www.uniprot.org/ ). These sequences were submitted to AlphaFold 3.0 ( https://alphafoldserver.com/ ) for protein docking model construction. The resulting structures were visualized and analyzed using PyMOL v2.6 (DeLano Scientific LLC, U.S.). Statistical analysis The statistical significance was assessed by unpaired t test and one-away ANOVA as appropriate using GraphPad Prism 9.0. Data are shown as the mean with standard error of mean (SEM), and P < 0.05 is considered statistically significant. RESULTS RSV virions enhance S.pn pathogenicity in vivo To evaluate whether incubation with RSV (A2) enhances the pathogenicity of S.pn (D39), a sublethal dose of S.pn was incubated with RSV in vitro (RSV + S.pn ) and subsequently used to intranasally infect mice (Fig. 1 A). Control groups included mice infected with an equivalent dose of S.pn alone ( S.pn ) and mice inoculated with PBS (blank control). The 7-day survival rate revealed that 30% of mice in the RSV + S.pn group and 40% in the S.pn group survived, while all mice in the blank control group survived (Fig. 1 B). Histopathological analysis of lung tissue from surviving and deceased mice showed mild pathological changes in surviving mice, whereas deceased mice exhibited severe damage, including disrupted alveolar structure, markedly thickened lung septa, airway destruction, epithelial sloughing (Fig. 1 C), and neutrophil aggregation in the vessel lumen in the RSV + S.pn . Extrapulmonary organ invasion was also more severe in the RSV + S.pn group, with 37.5% of mice exhibiting liver and spleen infections, 56.25% showing heart infections, and 62.5% displaying brain infections (Fig. 1 D). To determine whether RSV remnants after co-incubation with S.pn were involved in infection, viral loads in lung tissues were assessed in the RSV + S.pn group after 3 days of S.pn infection. Viral copy numbers were less than 1 × 10 4 (PFU/mL) in both surviving and deceased mice (Fig. 1 E), indicating minimal residual viral presence. These findings demonstrate that RSV co-incubation exacerbates S.pn infections, leading to severe pneumonia, extrapulmonary infections, and increased mortality. Analysis of BALF revealed elevated neutrophils in the RSV + S.pn group, compared to the S.pn group, at 12 h and 24 h post- S.pn infection ( P < 0.05, Fig. 1 F). Macrophage levels increased in all groups by 72 h post- S.pn infection ( P < 0.05, Fig. 1 G). RNA-seq analysis of lung tissues from surviving and deceased mice after 3 days of S.pn infection identified differential gene expression. Venn diagram analysis identified 4 609 genes uniquely expressed in the RSV + S.pn group (Fig. 1 H), enriched in pathways such as neutrophil extracellular trap formation, C-type lectin receptor signaling pathway, and necroptosis (Fig. 1 I). Conversely, 6 986 genes were uniquely expressed in the S.pn group, enriched in pathways such as regulation of actin cytoskeleton and p53 signaling (Fig. 1 I). These findings highlight the predominant involvement of neutrophil- and macrophage-associated pathways in S.pn infection in deceased mice. Inflammatory factors associated with neutrophil/macrophage chemotaxis and activation in S.pn -infected mice were further examined (Supplemental Fig. 1). Results showed elevated levels of TNF-α, IL-1α, IL-1β, and IL-10 in deceased mice compared to survivors, although the differences among deceased mice were not significant. IL-6, IL-17A, and IFN-γ were higher in deceased RSV + S.pn mice than in deceased S.pn mice, although the differences were not significant. Neutrophil chemokines KC and G-CSF were elevated in all deceased mice, particularly in the RSV + S.pn group. Macrophage-associated chemokines GM-CSF, MIP-1α, and MCP-1 were also elevated and more highly expressed in the RSV + S.pn group. These findings suggest that co-incubation with RSV triggers a heightened inflammatory response, including cytokine storm, contributing to severe outcomes in S.pn infections. RSV enhances S.pn virulence and peptidoglycan biosynthesis To clarify whether the enhanced inflammatory response observed in RSV + S.pn infections is associated with increased S.pn virulence, bacterial-specific virulence gene expression was analyzed using qRT-PCR. After co-incubation of S.pn with RSV in vitro , with the same dose of S.pn as a control, the expression levels of eight virulence and adhesion-associated genes were measured (Fig. 2 A and 2 B). Results showed a 40-fold increase in sodA gene expression in RSV + S.pn , while nox , comE , and ciaR were significantly up-regulated compared to S.pn alone. To assess protein-level expression of the eight identified virulence factors, high-throughput sequencing was performed. Results showed that only the comE and ciaR proteins were significantly up-regulated in RSV + S.pn (Fig. 2 C and 2 D), while the remaining six proteins showed no significant differences with the S. pn group. These findings suggest that S.pn virulence-associated factors were initially up-regulated at the gene level following co-incubation with RSV, but not consistently translated into increased protein expression. Label-free quantitative proteomic analysis identified 243 differentially expressed proteins between the RSV + S.pn and S.pn groups, including 80 significantly up-regulated and 163 significantly down-regulated proteins. Volcano plot analysis of differential proteins between the two groups is shown in Fig. 2 E. Subcellular localization analysis revealed that, in the comparison between RSV + S.pn and S.pn , most differential proteins were located in the bacterial cell membrane (17 proteins, 48.57%) and bacterial cytoplasm (15 proteins, 42.86%) (Fig. 2 F). KEGG pathway enrichment analysis of the differential proteins identified the top 10 enriched pathways (Fig. 2 G). Among these, the peptidoglycan biosynthesis pathway was notably enriched, with an enrichment factor of 11.5 and a P -value of < 0.001, involving five enriched proteins. This pathway was second only to the pyrimidine pathway (13 enriched proteins, enrichment factor 14.27), metal pathway (72 enriched proteins, enrichment factor 13.85) and purine pathway (14 enriched proteins, enrichment factor 12.19). Butterfly plots comparing the KEGG pathway enrichment of up- and down-regulated proteins (Fig. 2 H) highlighted peptidoglycan biosynthesis as the most prominently expressed pathway among up-regulated proteins in RSV + S.pn compared to S.pn . These results suggest that co-incubation with RSV promotes S.pn peptidoglycan biosynthesis, enhancing its role as a virulence factor for S.pn inflammation. Peptidoglycan likely contributes to the increased inflammatory response observed in the mouse lung, amplifying the bacterial inflammatory capacity. RSV infection enhances S.pn adhesion to airway epithelial cells To investigate how RSV influences bacterial pathogenicity, S.pn was co-incubated with RSV virions, purified RSV G glycoprotein, F glycoprotein, RSV RNA, and A549 RNA in vitro , respectively, with S.pn alone as the control. The adhesion of S.pn to A549 cells was then assessed. Results showed a significant increase in S.pn adhesion only after co-incubation with RSV virions (Fig. 3 A). Antigen co-localization and confocal imaging confirmed the binding of RSV virions to the bacteria during co-incubation in vitro (yellow fluorescence, Fig. 3 B). Imaging also demonstrated that S.pn adhered predominantly to the cell surface, with a higher number of bacteria adhering to A549 cells in the RSV + S.pn group compared to S.pn alone (red fluorescence, Fig. 3 C). These results suggest that intact RSV virions facilitate S.pn adhesion to airway epithelial cells. The potential mechanisms underlying increased S.pn adhesion following RSV infection were further investigated using an in vitro model of RSV-infected A549 cells (Fig. 3 D). RSV infectivity was confirmed by immunofluorescence detection of the viral N-protein, with approximately 80% of A549 cells showing positive N-protein staining at 24 h post-infection (MOI of 1), indicating successful RSV infection (Fig. 3 E). S.pn was then inoculated into RSV-infected or mock-infected A549 cells, and bacterial adhesion was quantified through plate counting at different time points. Results showed that adhesion was significantly higher in RSV-infected cells compared to mock-infected cells 24 h post-RSV infection (Fig. 3 F). SEM confirmed increased S.pn adhesion to RSV-infected A549 cells compared to the control (Fig. 3 G), consistent with the blood plate count results (Fig. 3 H). These findings indicate that RSV infection enhances S.pn adhesion to airway epithelial cells, playing a critical role in bacterial pathogenicity. Co-incubation with RSV affects S.pn biofilm formation Given that co-incubation of RSV with S.pn was shown to up-regulate bacterial peptidoglycan biosynthesis and enhance adhesion to host cells—processes both linked to biofilm formation (( 11 , 12 )To test this hypothesis, S.pn was co-incubated with RSV virions (RSV + S.pn ) or purified RSV G glycoprotein (G.pro + S.pn ) in vitro , with S.pn alone as the control ( S.pn ). Biofilm formation was then evaluated. SEM images showed that S.pn formed a strong, dense, and uniformly distributed surface biofilm across all groups, with only minor differences observed between them (Fig. 4 A). However, measurement of biofilm thickness using z-axis distance with CLSM revealed significant differences, with thicknesses of 25.86 µm for RSV + S.pn , 18.36 µm for S.pn , and 16.54 µm for G.pro + S.pn (Fig. 4 B), indicating that co-incubation with RSV enhanced S.pn biofilm thickness. To assess the impact of antibiotics on biofilm formation, penicillin + gentamicin, ceftazidime, azithromycin, and tetracycline were added to the cultures. Biofilm disruption was observed and quantitatively analyzed using CLSM. Mean fluorescence intensity comparisons indicated that penicillin + gentamicin resulted in the lowest intensity among the antibiotics tested. However, the overall mean fluorescence intensity of the RSV + S.pn group was slightly higher than that of the other two groups (Fig. 4 C). CLSM imaging of the S.pn biofilms after penicillin + gentamicin intervention showed a more uniform and densely distributed biofilm in the RSV + S.pn group compared to the other groups (Fig. 4 D). These results suggest that co-incubation with RSV enhances S.pn biofilm formation, which may contribute to the reduced efficacy of antimicrobial drugs. RSV enhances S.pn adhesion associated with G glycoprotein The RSV G glycoprotein has been reported to act as a receptor for S.pn on infected cells, facilitating bacterial invasion ( 13 , 14 ). To examine the association between increased S.pn adhesion and RSV infection, western blotting was used to detect changes in A549 G- and F-glycoproteins following RSV infection. Results showed that both glycoproteins were most highly expressed 24 h post-infection (Fig. 5 A and 5 B). Immunofluorescence further confirmed that G- and F-glycoproteins were predominantly localized on the cell membrane (Fig. 5 C and 5 D, white arrows with yellow fluorescence). Given the increased S.pn adhesion observed at 24 h post-infection, the role of RSV G- and F-glycoproteins in mediating this adhesion was further investigated. Notably, 293T cells were transfected with RSV G and F plasmids, resulting in the overexpression of G and F proteins, as confirmed by western blotting (Fig. 5 E and 5 F). Adhesion assays demonstrated increased S.pn adhesion to cells overexpressing G- and F-glycoproteins (Fig. 5 G), although the difference was only significant for G glycoprotein overexpression ( P < 0.05). These findings suggest that the RSV G glycoprotein plays a key role in enhancing S.pn adhesion to infected cells. RSV interacts with S.pn through viral G glycoprotein and bacterial surface proteins Given the evidence that RSV binds to S.pn and that RSV G- and F-glycoproteins are associated with S.pn adhesion, the potential interaction between these glycoproteins and S.pn was further investigated. The RSV G- and F-glycoproteins were immobilized on magnetic beads, as confirmed by western blotting (Fig. 6 A). The S.pn protein lysates were extracted and co-incubated with these glycoprotein magnetic beads, using BSA as a control. Silver staining results showed that only the G-glycoprotein interacted with S.pn (Fig. 6 B). Mass spectrometry analysis of bacterial proteins bound to G-glycoprotein magnetic beads identified eight S.pn surface proteins, with the late competence ComGG protein and maltodextrin ABC transporter substrate-binding protein displaying the highest binding scores and strongest binding abilities (Fig. 6 C, Table 1 ). Table 1 Mass spectrometry identification of pneumococcus membrane elution protein co-incubated with G glycoprotein magnetic beads. Protein IDs Protein names Mol. weight [kDa] Score Intensity ABJ53717.1; A0A0H2ZLL1 Late competence protein ComGG 16.003 0.44399 247150000 WP_000095484.1; ABJ55468.1; A0A0H2ZQ87 MULTISPECIES: maltodextrin ABC transporter substrate-binding protein 45.367 0.38272 466940000 WP_000750085.1; ABJ55268.1; A0A0H2ZRA8 Cell wall-associated serine protease PrtA(S8 family serine peptidase) 240.43 0.22085 890310 ABJ53888.1; A0A0H2ZMA6 Competence-induced protein Ccs4 58.026 0.16567 834390 ABJ55011.1; A0A0H2ZQ38 CAAX amino terminal protease family protein 26.254 0.06640 1331900 WP_001826592.1 type IV secretory system conjugative DNA transfer family protein 72.421 0.04186 1387500 WP_001224760.1; ABJ54249.1; A0A0H2ZN49 beta-lactamase(serine hydrolase) 48.13 0.03127 531140 ABJ53655.1; AAF73779.1; AAF13456.1; A0A0H2ZLS0 Choline binding protein A CbpA(PspC) 79.097 0.00089 59160000 BL21 competent cells were transfected with plasmids encoding ComGG and ABC proteins, with successful expression confirmed by western blotting (Fig. 6 D). A pull-down assay was performed to evaluate G glycoprotein binding to these proteins. Results showed that while the ComGG protein bands were not obvious, clear ABC protein bands were visible following pull-down with G-protein magnetic beads (Fig. 6 E and 6 F). Protein docking analysis using AlphaFold ( 15 ) also predicted that the G glycoprotein had two docking sites with ComGG protein (Fig. 6 G) and six docking sites with ABC protein (Fig. 6 H). These findings suggest that the RSV G glycoprotein primarily interacts with the maltodextrin ABC transporter substrate-binding protein of S.pn , facilitating bacterial adhesion and potentially enhancing pathogenicity. DISCUSSION The murine pneumonia model demonstrated that co-incubation of RSV with S.pn exacerbated bacterial virulence, increasing susceptibility to extrapulmonary organ invasion and lethal infection. These effects were closely associated with an enhanced inflammatory response and significant alterations in neutrophil dynamics within the airways. Early infection in RSV + S.pn mice showed increased neutrophil levels in the BALF, reflecting an intensified immune response. While neutrophils are critical for bacterial clearance following S.pn infection ( 16 ) ( 17 ), their release of elastase can compromise lung integrity ( 18 ), facilitating bacterial dissemination and extrapulmonary organ invasion. The severe pulmonary damage and excessive neutrophil infiltration detected in deceased RSV + S.pn mice underscored the detrimental consequences of this immune response. RNA-seq analysis of lung tissue identified distinct S.pn and RSV + S.pn activation pathways involved in invasive and lethal outcomes. Differentially expressed genes in the RSV + S.pn group were predominantly enriched in the C5 lectin pathway, known to drive neutrophil extracellular trap (NET) formation. Previous studies have linked C5 activation during bacterial sepsis to neutrophil recruitment to the lungs, resulting in an inflammatory storm ( 19 , 20 ), while neutrophils with impaired apoptosis accumulate in pulmonary microcirculation, exacerbating lung injury ( 21 ). This aligns with the elevated expression of pro-inflammatory cytokines, such as IL-17A, in deceased RSV + S.pn mice. IL-17A plays a dual role, promoting neutrophil bactericidal activity ( 22 , 23 ) while inducing the release of elastase and myeloperoxidase, key components of NETs, thus contributing to histopathological lung damage ( 24 ). RSV co-incubation fundamentally altered the interaction between S.pn and the host immune system, disrupting innate immune responses and amplifying pulmonary inflammation, as evidenced by the increased myeloperoxidase levels and NET formation observed in the RSV + S.pn group. The up-regulation of sodA , a gene associated with invasive S.pn infections ( 25 , 26 ), suggested that RSV exacerbated bacterial infections by promoting S.pn virulence factors. However, the sodA protein was not significantly expressed after co-incubation with RSV, suggesting the involvement of alternative mechanisms driving increased bacterial pathogenicity. Subsequent proteomic analysis comparing the S.pn and RSV + S.pn groups revealed significant up-regulation of the S.pn peptidoglycan synthesis pathway following co-incubation with RSV. Peptidoglycan, a key component of the bacterial wall, plays an important role in S.pn adhesion and the host inflammatory response ( 27 ). Host peptidoglycan recognition proteins or nucleotide oligomerization domain (NOD)-like receptors are known to trigger innate immune responses through the detection of bacterial cell wall peptides ( 28 ). Peptidoglycan homeostasis is essential for bacterial survival, growth, and division, and is critical for the pathogenicity of S.pn . This study provides the first evidence that RSV influences S.pn peptidoglycan biosynthesis, revealing a novel mechanism by which RSV enhances bacterial pathogenicity. Our results demonstrated that RSV, despite the detection of low viral loads in S.pn-infected mice after co-incubation, contributed to bacterial pathogenicity but was not the primary driver. In vitro bacterial adhesion assays and immunofluorescence imaging confirmed that only intact RSV virions co-incubated with S.pn significantly increased bacterial adhesion to epithelial cells. Immunofluorescence further demonstrated direct binding between RSV and S.pn after co-incubation. Additionally, RSV infection promoted S.pn adhesion to airway epithelial cells in a model of RSV-secondary S.pn infection. These findings are consistent with previous studies showing that RSV-induced airway damage increases host susceptibility to bacterial infections, such as S.pn , through non-specific mechanisms. Notably, pharyngeal cells co-infected with RSV and S.pn show an increase in the transcription of genes involved in adhesion ( psaA , pilus islet ) as well as transport and binding ( 29 ). RSV infection has also been shown to increase the expression of host mediators, such as platelet-activating factor (PAF) receptor and intercellular adhesion molecule 1 (ICAM-1), which facilitate S.pn adhesion ( 30 , 31 ). Imaging studies using fluorescence and SEM have reported a redistribution of adherent S.pn across the surface of RSV-infected epithelial cells, with dense bacterial accumulations near syncytia, suggesting viral and bacterial co-localization ( 13 ). These results indicate that RSV infection enhances S.pn adhesion to epithelial cells through multiple mechanisms, including direct viral-bacterial binding, modulation of host cell receptors, and redistribution of bacteria on epithelial surfaces. These interactions highlight the complex synergy between viral and bacterial pathogens in exacerbating respiratory infections. Our results indicated that RSV enhanced S.pn biofilm formation in vitro , a process likely related to the observed increase in bacterial adhesion and up-regulation of peptidoglycan biosynthesis. Biofilms are highly organized bacterial communities that adhere to solid surfaces and are embedded within a self-produced extracellular matrix of polysaccharides, proteins, and DNA ( 32 ). Although biofilm formation is a dynamic and continuous process, the initial adhesion phase is critical for biofilm development ( 33 ). This phase relies on specific components of the bacterial cell surface ( 34 ), including wall teichoic acid (WTA) and lipoteichoic acid (LTA), which are integral to peptidoglycan and cell membranes of Gram-positive bacteria. These structural components play pivotal roles in surface attachment and biofilm establishment ( 35 ). Biofilms pose a significant clinical challenge by conferring bacterial resistance to conventional antibiotics, resulting in persistent nosocomial infections ( 36 ). This resistance is attributed to the protective matrix of the biofilm and the development of multiple antibiotic resistance mechanisms in bacteria. These challenges were evident in the antibiotic intervention experiments, where RSV + S.pn formed the thickest biofilm and exhibited the lowest susceptibility to antibiotics. The observed reduction in antibiotic efficacy highlights the complex interplay between RSV-enhanced biofilm formation and bacterial resistance. Furthermore, our findings also indicated a potential association between RSV infection and S.pn adhesion, mediated by RSV G glycoproteins expressed on infected epithelial cells. These glycoproteins serve as adhesion receptors for S.pn , facilitating bacterial adhesion ( 37 ). Notably, this enhanced adhesion appears to be independent of the pneumococcal strain or of variability in RSV G glycoprotein sequences ( 38 ). Pull-down assays and mass spectrometry confirmed direct binding between RSV G glycoproteins and S.pn , consistent with earlier studies reporting increased bacterial adhesion and invasiveness in a mouse model due to this interaction ( 39 ). The interaction between RSV G glycoprotein and pneumococcus has also been linked to significant changes in the pneumococcal transcriptome, including the up-regulation of key virulence-related genes ( 40 ). This study identified specific S.pn membrane proteins that interacted with RSV G glycoproteins, including the late competence ComGG protein and the ABC transporter substrate-binding protein. Among these, the ABC transporter substrate-binding protein demonstrated a higher binding affinity, underscoring its potential role in mediating bacterial adhesion and virulence. The conventional view is that viral infections are self-limiting and require minimal intervention has been challenged, particularly in the context of RSV infections. The use of broad-spectrum antiviral drugs, such as interferons, remains highly debated. Although the virus itself is not typically harmful to the host, clinical evidence strongly supports a positive correlation between S.pn and RSV disease severity ( 41 , 42 ), , ( 43 ). Our study also confirmed that RSV enhanced susceptibility to S.pn infection and exacerbated disease severity. Notably, early and aggressive antiviral therapy appeared to be beneficial in mitigating secondary bacterial infections. However, the lack of specific antiviral treatments for RSV-infected individuals presents a significant clinical challenge. Furthermore, while some studies have reported that antibiotics improve outcomes in children with RSV-associated respiratory failure, highlighting the role of bacterial co-infections in aggravating viral disease severity ( 44 , 45 ), other studies have failed to identify a clear benefit of antibiotics in RSV disease management ( 46 , 47 ). Until more definitive evidence emerges, clinicians must exercise careful judgment in the use of antibiotics in these cases. Beta-lactam antibiotics remain the first choice for managing S.pn infections, particularly in co-infection scenarios. Of note, our study demonstrated that RSV co-incubation up-regulated S.pn peptidoglycan biosynthesis—a critical target for penicillin-based antimicrobials. This suggests that penicillin-based treatments, such as penicillin and amoxicillin, may be especially effective in patients co-infected with RSV and S.pn . This highlights the potential for prioritizing these drugs in clinical practice.While current RSV vaccines primarily focus on the RSV fusion (F) glycoprotein ( 48 ), our results underscore the importance of RSV G glycoproteins in the pathogenic interaction between RSV and S.pn . Given the critical role of RSV G glycoproteins in facilitating bacterial adherence and virulence, it is worth considering whether vaccine development targeting this glycoprotein could simultaneously protect against RSV and S.pn co-infections, offering a broader scope of prevention. In conclusion, our study showed that RSV infection influenced susceptibility to S.pn infections and disease severity through both host-dependent and host-independent mechanisms. Notably, RSV directly interacted with S.pn , increasing bacterial adherence, enhancing bacterial virulence, reducing neutrophil-mediated bacterial clearance, and intensifying pulmonary inflammation. Furthermore, RSV infection facilitated S.pn adhesion to airway epithelial cells, with RSV G glycoproteins playing a key role in this pathogenic process. These findings provide valuable insights for clinicians to rationally select anti-infective therapies, emphasizing the utility of penicillin-based antimicrobials in managing co-infections. Additionally, the study offers a theoretical foundation for the development of pathogen-targeted treatments, including vaccines against RSV G glycoproteins, which could reduce the burden of co-infections involving RSV and S.pn. Declarations Author Contributions Lu Li contributed to the study design, experiments operation, data analysis, data interpretation, preparation of tables/figures and writing of the report. Xin Long contributed to the study design, data acquisition, data analysis, data interpretation and preparation of figures. Yuncheng Wang and Peiru Shi contributed to the data acquisition, data analysis, and data interpretation. Yushun Wan contributed to the study design, instruction in experimental techniques, data interpretation and writing of the report. Enmei Liu conceived the idea for the study and critically reviewed the manuscript for intellectual content. Yu Deng conceived the idea as well as secured funding for the study, and critically reviewed the manuscript for intellectual content. All authors make an approval of the version to be submitted. Funding This work was funded by a National key Research and Development Program of China [2022YFC2704900]; Natural Science Foundation of Chongqing, China[cstc2019jycj-msxmX0858]; CQMU Program for Youth Innovation in Future Medicine. The funding bodies had no part in the design, conduct, analysis or interpretation of this study or the decision to submit this paper for publication. Data Availability The data that supports the findings of this study are available from the corresponding author upon reasonable request. Ethical approval All experiments involving animals were carried out in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Animal Experiments of Chongqing Medical University (Permit number: SYXK-(YU) 2012-0001). Consent for publication Not applicable. Competing interests The authors have declared that no competing interests exist. Acknowledgements We thank Prof. Yushun Wan for excellent technical support, and Christine Watts for her support in writing this article. Streptococcus pneumoniae strains D39 was kindly provided by Xuemei Zhang (Chongqing, China). References McAllister DA, Liu L, Shi T, Chu Y, Reed C, Burrows J, et al. Global, regional, and national estimates of pneumonia morbidity and mortality in children younger than 5 years between 2000 and 2015: a systematic analysis. Lancet Glob Health 2019;7(1):e47-e57. Li Y, Wang X, Blau DM, Caballero MT, Feikin DR, Gill CJ, et al. Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in children younger than 5 years in 2019: a systematic analysis. Lancet 2022;399(10340):2047-64. Shibata T, Makino A, Ogata R, Nakamura S, Ito T, Nagata K, et al. Respiratory syncytial virus infection exacerbates pneumococcal pneumonia via Gas6/Axl-mediated macrophage polarization. J Clin Invest Morpeth SC, Munywoki P, Hammitt LL, Bett A, Bottomley C, Onyango CO, et al. Impact of viral upper respiratory tract infection on the concentration of nasopharyngeal pneumococcal carriage among Kenyan children. Sci Rep 2018;8(1):11030. Ampofo K, Bender J, Sheng X, Korgenski K, Daly J, Pavia AT, et al. Seasonal invasive pneumococcal disease in children: role of preceding respiratory viral infection. Pediatrics 2008;122(2):229-37. Weinberger DM, Grant LR, Steiner CA, Weatherholtz R, Santosham M, Viboud C, et al. Seasonal drivers of pneumococcal disease incidence: impact of bacterial carriage and viral activity. Clin Infect Dis 2014;58(2):188-94. Ben-Shimol S, Greenberg D, Hazan G, Shemer-Avni Y, Givon-Lavi N, Dagan R. Seasonality of both bacteremic and nonbacteremic pneumonia coincides with viral lower respiratory tract infections in early childhood, in contrast to nonpneumonia invasive pneumococcal disease, in the pre-pneumococcal conjugate vaccine era. Clin Infect Dis 2015;60(9):1384-7. Stark JM, Stark MA, Colasurdo GN, LeVine AM. Decreased bacterial clearance from the lungs of mice following primary respiratory syncytial virus infection. J Med Virol Bourassa MH, Lands LC. Preventative therapies for respiratory Syncytial virus (RSV) in children: Where are we now? Paediatr Respir Rev 2024;49:24-7. Long X, Shi PR, Luo ZX, Luo J, Ren L, Liu EM, et al. Impact of Streptococcus pneumoniae colonization in upper airway on the clinical manifestations of children with respiratory syncytial virus infection. Zhonghua Er Ke Za Zhi 2022;60(7):694-9. Krishnamurthy A, Kyd J. The roles of epithelial cell contact, respiratory bacterial interactions and phosphorylcholine in promoting biofilm formation by Streptococcus pneumoniae and nontypeable Haemophilus influenzae. Microbes Infect 2014;16(8):640-7. Anderson EM, Sychantha D, Brewer D, Clarke AJ, Geddes-McAlister J, Khursigara CM. Peptidoglycomics reveals compositional changes in peptidoglycan between biofilm- and planktonic-derived Pseudomonas aeruginosa. J Biol Chem 2020;295(2):504-16. Hament JM, Aerts PC, Fleer A, Van Dijk H, Harmsen T, Kimpen JL, et al. Enhanced adherence of Streptococcus pneumoniae to human epithelial cells infected with respiratory syncytial virus. Pediatr Res 2004;55(6):972-8. Levine S, Klaiber-Franco R, Paradiso PR. Demonstration that glycoprotein G is the attachment protein of respiratory syncytial virus. J Gen Virol 1987;68 ( Pt 9):2521-4. Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. Highly accurate protein structure prediction with AlphaFold. Nature 2021;596(7873):583-9. Kadioglu A, Gingles NA, Grattan K, Kerr A, Mitchell TJ, Andrew PW. Host cellular immune response to pneumococcal lung infection in mice. Infect Immun 2000;68(2):492-501. Bou Ghanem EN, Clark S, Roggensack SE, McIver SR, Alcaide P, Haydon PG, et al. Extracellular Adenosine Protects against Streptococcus pneumoniae Lung Infection by Regulating Pulmonary Neutrophil Recruitment. PLoS Pathog . 2015;11(8):e1005126. Domon H, Oda M, Maekawa T, Nagai K, Takeda W, Terao Y. Streptococcus pneumoniae disrupts pulmonary immune defence via elastase release following pneumolysin-dependent neutrophil lysis. Sci Rep 2016;6:38013. Russkamp NF, Ruemmler R, Roewe J, Moore BB, Ward PA, Bosmann M. Experimental design of complement component 5a-induced acute lung injury (C5a-ALI): a role of CC-chemokine receptor type 5 during immune activation by anaphylatoxin. Faseb j 2015;29(9):3762-72. Bosmann M, Ward PA. Role of C3, C5 and anaphylatoxin receptors in acute lung injury and in sepsis. Adv Exp Med Biol 2012;946:147-59. Park I, Kim M, Choe K, Song E, Seo H, Hwang Y, et al. Neutrophils disturb pulmonary microcirculation in sepsis-induced acute lung injury. Eur Respir J 2019;53(3). Domon H, Nagai K, Maekawa T, Oda M, Yonezawa D, Takeda W, et al. Neutrophil Elastase Subverts the Immune Response by Cleaving Toll-Like Receptors and Cytokines in Pneumococcal Pneumonia. Front Immunol 2018;9:732. Lu YJ, Gross J, Bogaert D, Finn A, Bagrade L, Zhang Q, et al. Interleukin-17A mediates acquired immunity to pneumococcal colonization. PLoS Pathog 2008;4(9):e1000159. Ritchie ND, Ritchie R, Bayes HK, Mitchell TJ, Evans TJ. IL-17 can be protective or deleterious in murine pneumococcal pneumonia. PLoS Pathog 2018;14(5):e1007099. Berry AM, Paton JC. Additive attenuation of virulence of Streptococcus pneumoniae by mutation of the genes encoding pneumolysin and other putative pneumococcal virulence proteins. Infect Immun 2000;68(1):133-40. Mahdi LK, Van der Hoek MB, Ebrahimie E, Paton JC, Ogunniyi AD. Characterization of Pneumococcal Genes Involved in Bloodstream Invasion in a Mouse Model. PLoS One 2015;10(11):e0141816. Skovsted IC, Kerrn MB, Sonne-Hansen J, Sauer LE, Nielsen AK, Konradsen HB, et al. Purification and structure characterization of the active component in the pneumococcal 22F polysaccharide capsule used for adsorption in pneumococcal enzyme-linked immunosorbent assays. Vaccine 2007;25(35):6490-500. Johnson JW, Fisher JF, Mobashery S. Bacterial cell-wall recycling. Ann N Y Acad Sci 2013;1277(1):54-75. Kimaro Mlacha SZ, Peret TC, Kumar N, Romero-Steiner S, Dunning Hotopp JC, Ishmael N, et al. Transcriptional adaptation of pneumococci and human pharyngeal cells in the presence of a virus infection. BMC Genomics 2013;14:378. Avadhanula V, Rodriguez CA, Devincenzo JP, Wang Y, Webby RJ, Ulett GC, et al. Respiratory viruses augment the adhesion of bacterial pathogens to respiratory epithelium in a viral species- and cell type-dependent manner. J Virol 2006;80(4):1629-36. Yokota S, Okabayashi T, Hirakawa S, Tsutsumi H, Himi T, Fujii N. Clarithromycin suppresses human respiratory syncytial virus infection-induced Streptococcus pneumoniae adhesion and cytokine production in a pulmonary epithelial cell line. Mediators Inflamm 2012;2012:528568. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science 1999;284(5418):1318-22. Khan F, Pham DTN, Kim YM. Alternative strategies for the application of aminoglycoside antibiotics against the biofilm-forming human pathogenic bacteria. Appl Microbiol Biotechnol 2020;104(5):1955-76. Khan F, Jeong GJ, Javaid A, Thuy Nguyen Pham D, Tabassum N, Kim YM. Surface adherence and vacuolar internalization of bacterial pathogens to the Candida spp. cells: Mechanism of persistence and propagation. J Adv Res 2023;53:115-36. Jeong GJ, Khan F, Tabassum N, Cho KJ, Kim YM. Controlling biofilm and virulence properties of Gram-positive bacteria by targeting wall teichoic acid and lipoteichoic acid. Int J Antimicrob Agents 2023;62(4):106941. Yan Z, Huang M, Melander C, Kjellerup BV. Dispersal and inhibition of biofilms associated with infections. J Appl Microbiol . 2020;128(5):1279-88. Avadhanula V, Wang Y, Portner A, Adderson E. Nontypeable Haemophilus influenzae and Streptococcus pneumoniae bind respiratory syncytial virus glycoprotein. J Med Microbiol 2007;56(Pt 9):1133-7. Brealey JC, Sly PD, Young PR, Chappell KJ. Analysis of phylogenetic diversity and in vitro adherence characteristics of respiratory syncytial virus and Streptococcus pneumoniae clinical isolates obtained during pediatric respiratory co-infections. Microbiology (Reading) 2020;166(1):63-72. Hament JM, Aerts PC, Fleer A, van Dijk H, Harmsen T, Kimpen JL, et al. Direct binding of respiratory syncytial virus to pneumococci: a phenomenon that enhances both pneumococcal adherence to human epithelial cells and pneumococcal invasiveness in a murine model. Pediatr Res 2005;58(6):1198-203. Smith CM, Sandrini S, Datta S, Freestone P, Shafeeq S, Radhakrishnan P, et al. Respiratory syncytial virus increases the virulence of Streptococcus pneumoniae by binding to penicillin binding protein 1a. A new paradigm in respiratory infection. Am J Respir Crit Care Med 2014;190(2):196-207. Brealey JC, Chappell KJ, Galbraith S, Fantino E, Gaydon J, Tozer S, et al. Streptococcus pneumoniae colonization of the nasopharynx is associated with increased severity during respiratory syncytial virus infection in young children. Respirology 2018;23(2):220-7. Diaz-Diaz A, Bunsow E, Garcia-Maurino C, Moore-Clingenpeel M, Naples J, Juergensen A, et al. Nasopharyngeal Codetection of Haemophilus influenzae and Streptococcus pneumoniae Shapes Respiratory Syncytial Virus Disease Outcomes in Children. J Infect Dis 2022;225(5):912-23. Esposito S, Zampiero A, Terranova L, Ierardi V, Ascolese B, Daleno C, et al. Pneumococcal bacterial load colonization as a marker of mixed infection in children with alveolar community-acquired pneumonia and respiratory syncytial virus or rhinovirus infection. Pediatr Infect Dis J 2013;32(11):1199-204. Shein SL, Kong M, McKee B, O'Riordan M, Toltzis P, Randolph AG. Antibiotic Prescription in Young Children With Respiratory Syncytial Virus-Associated Respiratory Failure and Associated Outcomes. Pediatr Crit Care Med 2019;20(2):101-9. Juvén T, Mertsola J, Waris M, Leinonen M, Ruuskanen O. Clinical response to antibiotic therapy for community-acquired pneumonia. Eur J Pediatr 2004;163(3):140-4. Farley R, Spurling GK, Eriksson L, Del Mar CB. Antibiotics for bronchiolitis in children under two years of age. Cochrane Database Syst Rev 2014;2014(10):Cd005189. Kneyber MC, Kimpen JL. Antibiotics in RSV bronchiolitis: still no evidence of effect. Eur Respir J 2007;29(6):1285. Crank MC, Ruckwardt TJ, Chen M, Morabito KM, Phung E, Costner PJ, et al. A proof of concept for structure-based vaccine design targeting RSV in humans. Science 2019;365(6452):505-9. Additional Declarations No competing interests reported. Supplementary Files SupplementalFigure1.tiff Supplemental Figure 1. Cytokines level in the lungs of living and dead mice. Mice were challenged intranasally with 1.5*10^6 cfu Streptococcus pneumoniae D39 (co-incubated with RSV), exposed to S.pn alone or exposed to PBS alone as a control, and lung cytokines were detected 3 days post S.pn infection using the Bio-plex Pro Mouse Cytokine Grp I Panel 23-plex. Data were shown as mean ± SD of 1 biological replicates, * P<0.05, ** P<0.01, *** P<0.001. (TIFF) Supplementaryfile.docx SupplementalTable1.docx Supplemental Table 1. Primer sequences for the S.pn genes’ amplification during reverse transcription PCRs. (DOCX) Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5978497","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":415968672,"identity":"bfc37fad-14f8-44c4-8b0b-a88ea90038dc","order_by":0,"name":"Lu Li","email":"","orcid":"","institution":"Children's Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Li","suffix":""},{"id":415968673,"identity":"5eff8a52-fdb5-4fe7-bb4c-ab9bed3ecc24","order_by":1,"name":"Xin Long","email":"","orcid":"","institution":"Children's Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Long","suffix":""},{"id":415968674,"identity":"3acc1318-c324-4bd3-bf3f-27ec470b87eb","order_by":2,"name":"Yushun Wan","email":"","orcid":"","institution":"College of Basic Medicine of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yushun","middleName":"","lastName":"Wan","suffix":""},{"id":415968675,"identity":"201a6f85-7024-4189-b200-62e2ec03a90d","order_by":3,"name":"Yuncheng Wang","email":"","orcid":"","institution":"College of Basic Medicine of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuncheng","middleName":"","lastName":"Wang","suffix":""},{"id":415968676,"identity":"7c678a8b-ec8b-4a35-b402-0d7b0932014d","order_by":4,"name":"Peiru Shi","email":"","orcid":"","institution":"Children's Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Peiru","middleName":"","lastName":"Shi","suffix":""},{"id":415968677,"identity":"00dbbc9c-1d89-4dcf-9d52-1523f14c46e7","order_by":5,"name":"Enmei Liu","email":"","orcid":"","institution":"Children's Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Enmei","middleName":"","lastName":"Liu","suffix":""},{"id":415968678,"identity":"308569ab-55da-43ed-8f3c-57064b9099ae","order_by":6,"name":"Yu Deng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYDCCAzAGM/PBBx8YLHhI0MLOlmw4g0GCFC38PGbCPAwShHXwHe89/Jrnz+F8fma2NGabGgkZgxu5Bxh+VGzDqUXyzLk0a962w5Yzm5mPPc45JsFjcCMvgbHnzG2cWgxu5JgZ8zYcNjA4zJZunMMG0pJjwMzYhkfL/TdmxkCHGdgf5jGTtvhHjJYbPMaPediAtjADtTC2EaFF8kyOGePctnQDicPAQO7tk+CRPPPG4CA+v/AdP2P84c0fawP+/sMHH/z4ZmPPdzzH8MGPCtxagIBNCiX6FA4gRRYOwPzxBzJXvoGA+lEwCkbBKBhxAAD9MVai8sVyXAAAAABJRU5ErkJggg==","orcid":"","institution":"Children's Hospital of Chongqing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yu","middleName":"","lastName":"Deng","suffix":""}],"badges":[],"createdAt":"2025-02-07 06:53:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5978497/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5978497/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":76491966,"identity":"bb988d1c-6058-49fc-9c43-45b7b5e66ea0","added_by":"auto","created_at":"2025-02-17 17:04:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3100065,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCo-incubation with RSV promotes \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS.pn\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003epathogenicity in vivo.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) A simplified schematic representation of mouse pneumonia model, RSV (A2) was co-incubated with \u003cem\u003eS.pn\u003c/em\u003e(D39) on a shaker at 37°C for 30 min, mixture centrifuged, RSV discarded, \u003cem\u003eS.pn\u003c/em\u003eresuspended in PBS and intranasally infected Balb/c. (\u003cstrong\u003eB\u003c/strong\u003e) Survival curves of \u003cem\u003eS.pn\u003c/em\u003e-infected mice after co-incubation with RSV (RSV+\u003cem\u003eS.pn\u003c/em\u003e), exposure to \u003cem\u003eS.pn\u003c/em\u003e alone (\u003cem\u003eS.pn\u003c/em\u003e) or PBS alone (control). (\u003cstrong\u003eC\u003c/strong\u003e) Lung histopathology of live and dead mice infected with \u003cem\u003eS.pn\u003c/em\u003e for 3 days, the control group lacking dead lung histopathology because no mouse died. (\u003cstrong\u003eD\u003c/strong\u003e) The percentage of bacteria invading extrapulmonary organs (including liver, spleen, heart and brain) after 3 days of \u003cem\u003eS.pn\u003c/em\u003e infection in living mice. (\u003cstrong\u003eE\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eRSV \u003cem\u003eN\u003c/em\u003e gene copies from surviving and dead mice 3 days after infection with \u003cem\u003eS.pn\u003c/em\u003e (co-incubated with RSV). (\u003cstrong\u003eF\u003c/strong\u003e) Percentage of neutrophils in the BALF of mice 12, 24, 48 and 72 hours after infection with\u003cem\u003eS.pn\u003c/em\u003e. (\u003cstrong\u003eG\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003ePercentage of macrophages in the BALF of mice 12, 24, 48 and 72 hours after infection with\u003cem\u003e S.pn\u003c/em\u003e. (\u003cstrong\u003eH\u003c/strong\u003e) Enrichment of differentially expressed genes in the lungs of dead \u003cem\u003eS.pn\u003c/em\u003e infected mice after 3 days. (\u003cstrong\u003eI\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eKEGG enrichment analysis of differentially expressed genes in the lungs of dead mice after 3 days of \u003cem\u003eS.pn\u003c/em\u003einfection, where DA_alone indicates the gene specifically expressed in \u003cem\u003eS.pn \u003c/em\u003e(after co-incubation with RSV) infected mice and DS_alone indicates the gene specifically expressed in \u003cem\u003eS.pn\u003c/em\u003e alone infected mice. Data were shown as mean ± SD of 3 biological replicates, * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, nd = no significant difference (\u003cem\u003eP\u003c/em\u003e＞0.05).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5978497/v1/ffa82c04b9a4fab346e81124.png"},{"id":76491055,"identity":"39f82401-9843-4ebf-9f65-c46d72bea567","added_by":"auto","created_at":"2025-02-17 16:48:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2096744,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRSV increases \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS.pn\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e virulence and peptidoglycan biosynthesis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) and (\u003cstrong\u003eB\u003c/strong\u003e)\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eS.pn\u003c/em\u003e (with RSV co-incubation) toxicity-associated gene expression, \u003cem\u003eS.pn\u003c/em\u003e alone as a control, \u003cem\u003eGyrB\u003c/em\u003e was the internal control gene and the fold changes in gene expression were calculated by 2^\u003csup\u003e-△△Ct\u003c/sup\u003e. (\u003cstrong\u003eC\u003c/strong\u003e) and (\u003cstrong\u003eD\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eS.pn\u003c/em\u003e (with RSV co-incubation) toxicity-associated protein expression, \u003cem\u003eS.pn\u003c/em\u003e alone as a control, the relative abundance was measured by Label-free quantitative proteomics. (\u003cstrong\u003eE\u003c/strong\u003e) Volcano plot of differentially expressed proteins in \u003cem\u003eS.pn\u003c/em\u003e co-incubated with RSV and \u003cem\u003eS.pn\u003c/em\u003e alone, red dots were upregulated proteins, blue dots were downregulated proteins and grey dots were no differentially expressed proteins. (\u003cstrong\u003eF\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eSubcellular localization of differentially expressed proteins, including cytoplasm, extracellular region, membrane and ribosomes. (\u003cstrong\u003eG\u003c/strong\u003e) KEGG enrichment analysis in \u003cem\u003eS.pn\u003c/em\u003e differential expression proteins (top10), circle size represented the count and different colors indicated each pathway. (\u003cstrong\u003eH\u003c/strong\u003e) KEGG enrichment pathway of up- and down-regulated proteins (top12), the blue columns indicated the pathways to down-regulated proteins and the red columns indicated the pathways to up-regulated proteins. Data were expressed as mean ± SD with triplicate experiments, * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, *** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5978497/v1/51e9e3ef5d561db9de3bef3e.png"},{"id":76491060,"identity":"4214a553-745d-4ffa-b042-1346064554b5","added_by":"auto","created_at":"2025-02-17 16:48:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2484317,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRSV infection enhanced \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS.pn\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e adhesion on A549.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eAfter co-incubation of \u003cem\u003eS.pn \u003c/em\u003ewith RSV virons, G glycoprotein, F glycoprotein, RSV RNA and A549 RNA for 30 minutes, the adherence of \u003cem\u003eS.pn\u003c/em\u003eto epithelial cells (A549) was measured after 3 hours. (\u003cstrong\u003eB\u003c/strong\u003e) Confocal image of \u003cem\u003eS.pn\u003c/em\u003e co-incubated with RSV, bacterial and viral preparations stained with anti-pneumococcal capsular antibody (green) and RSV-specific antibody (red). (\u003cstrong\u003eC\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eConfocal images of \u003cem\u003eS.pn\u003c/em\u003e co-incubated with RSV adhering to A549, cells were stained with an antibody specific for α-tubulin (green) and an anti-pneumococcal antibody (red). (\u003cstrong\u003eD\u003c/strong\u003e) A simplified scheme of RSV infection and subsequent bacterial adherence: RSV (A2) infected or mock infected (Mock) A549 cells at MOI 1 for 3, 6, 12 and 24 hours. (\u003cstrong\u003eE\u003c/strong\u003e) RSV infectivity to A549 confirmed by N-protein immunofluorescence staining at 3, 6, 12 and 24h post infection. (\u003cstrong\u003eF\u003c/strong\u003e) After 3h, 6h, 12h and 24h of RSV infection, adherent bacteria of \u003cem\u003eS.pn\u003c/em\u003e with A549 were counted by plate counting method and three fields of view were analyzed in each group. (\u003cstrong\u003eG\u003c/strong\u003e) Representative SEM images of \u003cem\u003eS.pn\u003c/em\u003e adhering to A549, infected with RSV for 24h, followed by adhesion assay with \u003cem\u003eS.pn\u003c/em\u003e at 1 × 10^7 CFU. (\u003cstrong\u003eH\u003c/strong\u003e) Statistical analysis of \u003cem\u003eS.pn\u003c/em\u003e adherent to A549 after RSV infection for 24h, numbers of bacteria adherent to A549 were counted. The data represented the mean ± SD of 3 independent experiments, * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5978497/v1/5408d94e4f662d86e09ededa.png"},{"id":76491070,"identity":"d5bbb18b-34db-4363-915b-9ce0b40a4137","added_by":"auto","created_at":"2025-02-17 16:48:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3189175,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of RSV on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eStreptococcus pneumoniae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e biofilm.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) The morphology of the \u003cem\u003epneumococcus\u003c/em\u003e D39 biofilm by scanning electron microscope (SEM), the biofilm model was constructed by S.pn co-incubated with RSV virons or G glycoprotein, \u003cem\u003eS.pn\u003c/em\u003e alone as control. (\u003cstrong\u003eB\u003c/strong\u003e) Confocal laser scanning microscope (CLSM) measured \u003cem\u003eS.pn\u003c/em\u003e biofilm thickness, the Z-axis distance was the biofilm thickness. (\u003cstrong\u003eC\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eROI Mean Intensity (Syto9) of \u003cem\u003eS.pn\u003c/em\u003e biofilm with/without antibiotic treatment (including Penicillin + Gentamicin, Ceftizoxime, Azithromycin, and Tetracycline), the bacteria were stained using a live/dead kit with Syto9 (green: live) and PI (red: dead). (\u003cstrong\u003eD\u003c/strong\u003e) CLSM images of \u003cem\u003eS.pn\u003c/em\u003e biofilms with/without antibiotic (Penicillin + Gentamicin) treatment. Data were expressed as mean ± SD with triplicate experiments.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5978497/v1/045d570402704bf4645101e7.png"},{"id":76491726,"identity":"044ea45c-a7c2-49bd-8428-de643f446f6a","added_by":"auto","created_at":"2025-02-17 16:56:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2543842,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRSV enhances \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS.pn\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e adhesion was associated with G glycoproteins upregulation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) and(\u003cstrong\u003eB\u003c/strong\u003e) G-glycoprotein (\u003cstrong\u003eA\u003c/strong\u003e) and F-glycoprotein (\u003cstrong\u003eB\u003c/strong\u003e) levels measured by Western blotting assay in A549 infected with RSV for 3h, 6h, 12h and 24h, respectively. (\u003cstrong\u003eC\u003c/strong\u003e) and (\u003cstrong\u003eD\u003c/strong\u003e) Localization of G-glycoprotein (\u003cstrong\u003eC\u003c/strong\u003e) and F-glycoprotein (\u003cstrong\u003eD\u003c/strong\u003e) in A549 cells infected with RSV for 24 h confirmed by immunofluorescence staining, cells were stained with an antibody specific for α-tubulin (green) and an anti-G-glycoprotein or anti-F-glycoprotein antibody (red), areas of antigen colocalisation are shown in yellow (and indicated by arrows). (\u003cstrong\u003eE\u003c/strong\u003e) and (\u003cstrong\u003eF\u003c/strong\u003e) Overexpression of G-glycoprotein (\u003cstrong\u003eE\u003c/strong\u003e) and F-glycoprotein (\u003cstrong\u003eF\u003c/strong\u003e) with G- or F-plasmid transfected 293T cells and detected by Western blotting. (\u003cstrong\u003eG\u003c/strong\u003e) Increased \u003cem\u003eS.pn\u003c/em\u003eadhesion to 293T cells overexpressing G glycoprotein, \u003cem\u003eS.pn\u003c/em\u003e adhesion measured by plate counting methods. Data were shown as the mean ± SD of 3 independent experiments, * P\u0026lt;0.05, ** P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5978497/v1/50d861b42f2dab77a5b7247d.png"},{"id":76491724,"identity":"589a1714-81c4-4450-9b21-f251f99b1647","added_by":"auto","created_at":"2025-02-17 16:56:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2538565,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRSV G glycoproteins bind directly to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eStreptococcus pneumoniae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Ni bead incubated with purified RSV G and F glycoproteins, G protein-bead and F protein-bead measured by Western blotting assay. (\u003cstrong\u003eB\u003c/strong\u003e) Silver staining assay of total pneumococcal protein extract incubated with G protein bead or F protein bead, and bovine serum albumin (BSA) as a control. (\u003cstrong\u003eC\u003c/strong\u003e) Mass spectrometry identification of pneumococcal protein, the elution protein of pneumococcal total protein extract incubated with G protein-bead was identified by mass spectrometry, BSA incubated with G protein-bead was control. (\u003cstrong\u003eD\u003c/strong\u003e) Overexpression of late competence protein ComGG (ComGG) and maltodextrin ABC transporter substrate-binding protein (ABC) with ComGG plasmid and ABC plasmid transfected into BL21(DE3) competent cells, respectively, and detected by Western blotting. (\u003cstrong\u003eE\u003c/strong\u003e) and (\u003cstrong\u003eF\u003c/strong\u003e) Pull-down assay of G protein-bead with ComGG (\u003cstrong\u003eE\u003c/strong\u003e) and ABC (\u003cstrong\u003eF\u003c/strong\u003e), G protein-beads were incubated with BL21 total protein extract of overexpressing ComGG or ABC, and the elution protein after incubation were detected by Western blotting. G protein was detected by a monoclonal antibody, ComGG and ABC were detected both by a Gst tag monoclonal antibody. (\u003cstrong\u003eG\u003c/strong\u003e) and (\u003cstrong\u003eH\u003c/strong\u003e), Protein docking of G glycoprotein with ComGG (G) and ABC (H) in AlphaFold, respectively.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5978497/v1/75d7c01453bec9973300088d.png"},{"id":84310192,"identity":"afcd38a2-9248-447d-8daa-a462b2617b93","added_by":"auto","created_at":"2025-06-10 12:17:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15655091,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5978497/v1/3fd3fbc6-066a-4a43-969c-bcec79419b02.pdf"},{"id":76491053,"identity":"ab38a214-5f68-4b93-866a-131cef9be372","added_by":"auto","created_at":"2025-02-17 16:48:56","extension":"tiff","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1512214,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Figure 1. Cytokines level in the lungs of living and dead mice. \u003c/strong\u003eMice were challenged intranasally with 1.5*10^6 cfu \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003eD39 (co-incubated with RSV), exposed to S.pn alone or exposed to PBS alone as a control, and lung cytokines were detected 3 days post S.pn infection using the Bio-plex Pro Mouse Cytokine Grp I Panel 23-plex. Data were shown as mean ± SD of 1 biological replicates, * P\u0026lt;0.05, ** P\u0026lt;0.01, *** P\u0026lt;0.001. (TIFF)\u003c/p\u003e","description":"","filename":"SupplementalFigure1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-5978497/v1/978ddcd228c6f60389f37219.tiff"},{"id":76491072,"identity":"2e89098c-7f4b-4e1e-aeff-50c3c0fef6c6","added_by":"auto","created_at":"2025-02-17 16:48:58","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":7631255,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-5978497/v1/08a3ffdb701ce2210927432b.docx"},{"id":76491068,"identity":"df1e0abe-801a-435d-8b13-64e421d6cc7f","added_by":"auto","created_at":"2025-02-17 16:48:57","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":17210,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Table 1. Primer sequences for the S.pn genes’ amplification during reverse transcription PCRs. (DOCX)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"SupplementalTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5978497/v1/421f1b54c61d72a6e24c4a03.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Respiratory syncytial virus G glycoprotein promotes Streptococcus pneumoniae-induced severe pneumonia","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eLower respiratory tract infections remain the leading cause of childhood morbidity and mortality worldwide, despite advancements in healthcare systems, expanded immunization coverage, and improved nutritional standards. Pneumonia alone accounts for nearly one million deaths each year in children under the age of 5, predominantly from low- and middle-income countries (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Among the etiological agents of pediatric pneumonia, respiratory syncytial virus (RSV) and \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e (\u003cem\u003eS.pn\u003c/em\u003e) are particularly prominent (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). \u003cem\u003eS.pn\u003c/em\u003e is part of the normal respiratory microbiota and frequently exists in a benign, asymptomatic state in children. However, during viral respiratory infections, asymptomatic colonization can become pathogenic, with emerging evidence implicating RSV as a critical factor in triggering this transition (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEpidemiological research has consistently highlighted a correlation between seasonal RSV activity and the incidence of pneumococcal disease in infants and older children (\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Experimental studies using mouse models have demonstrated that prior RSV infection significantly increases the risk of pneumococcal sepsis and impairs bacterial clearance from the lungs (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), emphasizing the complex interplay between viral and bacterial pathogens. Understanding the molecular mechanisms underlying these pathogenic interactions is important for advancing targeted interventions.\u003c/p\u003e \u003cp\u003eRecent developments in RSV prophylaxis, including the approval of maternal vaccines and monoclonal antibodies for infant immunoprophylaxis (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), offer promising strategies to mitigate RSV-associated disease. The potential impact of RSV vaccination could be further enhanced by the concurrent availability of broad-spectrum pneumococcal vaccines, which may synergistically reduce the severity of RSV and pneumococcal co-infections. These combined measures have the potential to significantly decrease the burden of severe disease, reduce reliance on antibiotics, and inform healthcare strategies aimed at improving outcomes for affected populations.\u003c/p\u003e \u003cp\u003eOur previous retrospective study revealed that \u003cem\u003eS.pn\u003c/em\u003e colonization of the upper respiratory tract worsened clinical outcomes in RSV-infected children aged 6 months and older (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). In a prospective cohort analysis employing 16S rRNA sequencing, RSV-infected children exhibited lower alpha diversity in upper respiratory tract microbiota (0.84, interquartile range 0.35\u0026ndash;1.17) compared to healthy controls (3.06, interquartile range 2.88\u0026ndash;3.61) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Additionally, the bacterial composition in healthy controls was more diverse, while the RSV-infected group displayed a dominance of \u003cem\u003eS.pn\u003c/em\u003e within their upper respiratory tract flora.\u003c/p\u003e \u003cp\u003eThis study investigated the effects of RSV on pneumococcal pathogenicity after co-incubation, with a particular focus on the molecular mechanisms enhancing bacterial adhesion to epithelial cells. Severe pneumonia development was evaluated in a murine model, revealing how RSV co-incubation influences disease progression and related molecular pathways. Further analysis showed that RSV infection enhanced \u003cem\u003eS.pn\u003c/em\u003e adhesion and induced the expression of viral surface proteins in infected cells, highlighting specific interactions between RSV G glycoproteins and pneumococcal surface proteins. The effects of RSV on \u003cem\u003eS.pn\u003c/em\u003e biofilm formation were also assessed, providing novel insights into the interactions between RSV and \u003cem\u003eS.pn\u003c/em\u003e.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eViral and bacterial growth conditions\u003c/h2\u003e \u003cp\u003eRespiratory syncytial virus subtype A strain A2 (RSV A2) was purchased from ATCC and inoculated into human laryngeal epidermoid carcinoma (HEp-2) cells for expansion, which were harvested once syncytium formation was evident (approximately 48\u0026ndash;72 h). The virus was purified using sucrose gradient centrifugation, aliquoted, and stored in liquid nitrogen until use. Viral titers, expressed as plaque-forming units (PFU), were quantified using a plaque assay. The RSV A2 viral titer used in this study was 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e PFU/mL.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eS.pn\u003c/em\u003e serotype 2 strain D39 (\u003cem\u003eS.pn\u003c/em\u003e D39) was kindly donated by Ms Xuemei Zhang\u0026rsquo;s research group at the Institute of Life Sciences, Chongqing Medical University, China. This pathogenic strain was preserved in glycerol at \u0026minus;\u0026thinsp;80\u0026deg;C and resuscitated on Columbia blood agar plates at 37\u0026deg;C overnight, then resuspended in Todd-Hewitt broth\u0026thinsp;+\u0026thinsp;Yeast powder (THY) and cultured at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e for 4\u0026ndash;6 h until the optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) reached 0.45\u0026ndash;0.5. Colony-forming unit (CFU) determination was conducted using plate counting. Briefly, 1 mL of \u003cem\u003eS.pn\u003c/em\u003e bacterial solution was serially diluted 10-fold with phosphate-buffered saline (PBS) and plated onto blood agar. Colony counts were recorded after 24 h of incubation at 37\u0026deg;C. The \u003cem\u003eS.pn\u003c/em\u003e concentration used in this study was 1.5 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU/mL.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eViral and bacterial co-incubation\u003c/h3\u003e\n\u003cp\u003eA 1-mL aliquot of \u003cem\u003eS.pn\u003c/em\u003e bacterial solution (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.45\u0026ndash;0.50) was serially diluted 10-fold with PBS. Subsequently, 1 mL of the diluted bacterial solution was centrifuged (1200rpm, 8min, 4℃), and the resulting pellet was resuspended in RSV A2 to prepare the RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e mixture. For the control group (\u003cem\u003eS.pn\u003c/em\u003e only), an equal amount of \u003cem\u003eS.pn\u003c/em\u003e was resuspended in high-glucose Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) containing 2% fetal bovine serum (FBS). Both suspensions were incubated on a shaker at 37\u0026deg;C and 200 rpm for 30 minutes. After incubation, the mixtures were centrifuged, the supernatants were discarded, and the pellets were resuspended in PBS for further use.\u003c/p\u003e\n\u003ch3\u003eMouse pneumonia model\u003c/h3\u003e\n\u003cp\u003eGroups of 10 female BALB/c mice (6\u0026ndash;8 weeks old) were randomly assigned to three groups: blank, \u003cem\u003eS.pn\u003c/em\u003e, and RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e. The mice were anaesthetized with chloral hydrate and intranasally inoculated with 1.5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/50 \u0026micro;L of \u003cem\u003eS.pn\u003c/em\u003e (treatment groups) or 50 \u0026micro;L of PBS (blank group), then monitored for 7 days to evaluate survival rates and extent of acute lung and extrapulmonary organ invasion. At predetermined time points, mice were euthanized via cervical dislocation. The left lung was fixed in 4% paraformaldehyde for histological analysis, while the right lung was homogenized and divided into three portions for storage at \u0026minus;\u0026thinsp;80\u0026deg;C. Total RNA from lung homogenates was extracted using the Simply P Total RNA Extraction Kit (Bioflux, U.S.). Reverse transcription was immediately performed using the PrimeScript\u0026trade; RT Reagent Kit with gDNA Eraser (TaKaRa, J.P.N.), following the manufacturer's instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using ChamQ Universal SYBR Qpvr Master Mix (Vazyme, C.H.N.). The primers for the RSV A2 N gene were as follows: forward primer 5\u0026rsquo;-AGATCAACTTCTGTCATCCAGCAA-3\u0026rsquo;, reverse primer 5\u0026rsquo;-TTCTGCACATCATAATTAGGAGTATCAAT-3\u0026rsquo;.\u003c/p\u003e\n\u003ch3\u003eLung paraffin sectioning and hematoxylin and eosin (H\u0026E) staining\u003c/h3\u003e\n\u003cp\u003eFixed lung tissue was dehydrated and embedded in paraffin to create paraffin blocks. These blocks were sectioned using a paraffin slicer, and the sections were placed on slides and baked in an oven at 60\u0026deg;C overnight. After the paraffin was sufficiently melted, H\u0026amp;E staining was performed according to the following procedure: Xylene (I) 10 min, xylene (II) 5 min, followed by gradual hydration; 100% ethanol 3 min, 95% ethanol 3 min, 75% ethanol 3 min, hematoxylin staining 3 min, flowing water 1 min, hydrochloric acid-ethanol 3 s, flowing water 1 min, saturated lithium carbonate 10 s, flowing water 1 min, 95% ethanol 1 min, eosin staining 10 s, flowing water 1 min, 95% ethanol 1 min, 100% ethanol 1 min, xylene (I), and xylene (II) 2 min. Finally, the sections were mounted with neutral resin and lung lesions were examined under a light microscope, with images captured using a pathology scanner(TEKSQRAY, C.H.N.).\u003c/p\u003e\n\u003ch3\u003eBronchoalveolar lavage fluid (BALF) inflammatory cell differential count assay\u003c/h3\u003e\n\u003cp\u003eMice were restrained in the supine position, and the trachea was bluntly separated and fully exposed for intubation using an indwelling needle. Subsequently, 0.5 mL of pre-cooled PBS was slowly injected with a syringe along the direction of the tracheal intubation to collect bronchoalveolar lavage fluid (BALF). This process was repeated three times to ensure adequate recovery. The collected BALF was centrifuged (1500rpm, 10min, 4℃), and the resulting cell precipitate was resuspended in 1 mL of PBS. A 20-\u0026micro;L aliquot was used to determine total cell count using a Countstar cell counting plate. The remaining suspension was centrifuged again to remove the supernatant. The resulting cell pellet was smeared onto slides, stained with Rachel's stain, and examined under an oil microscope to differentiate neutrophils and macrophages based on their morphological characteristics.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLung cytokine measurements by enzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e \u003cp\u003eMouse lung proteins were extracted using RIPA lysis buffer according to the manufacturer\u0026rsquo;s instructions (Sigma, G.E.R.). Protein concentrations in the samples were determined using a Bicinchoninic Acid (BCA) Protein Assay Kit (Beyotime, C.H.N.). For cytokine measurements, 45-\u0026micro;g aliquots of each sample were prepared and TNF-α, IL-1β, IL-10, IFN-γ, GM-CSF, MCP-1, IL-6, IL-17A, IL-1α, G-CSF, KC, and MIP-1α levels were quantified using the Bio-Plex Pro Mouse Cytokine Grp I Panel 23-Plex on the Bio-Plex MAGPIX System (Wayen Biotechnologies, C.H.N.) according to the manufacturer's instructions. Cytokine concentrations were calculated based on fluorescence values obtained from recombinant cytokine standards in 96-well plates using Bio-Plex Manager software.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLung tissue RNA sequencing (RNA-seq)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from mouse lungs using TRIzol\u0026reg; reagent (Invitrogen, U.S.), and genomic DNA was removed using DNase I (TaKara, U.S.). RNA quality was determined using the NanoDrop 2000 spectrophotometer (NanoDrop Technologies, U.S.) and RNA degradation was verified by agarose gel electrophoresis. Only RNA samples meeting quality standards were used for library preparation. RNA purification, reverse transcription, library construction, and sequencing were carried out by Shanghai Majorbio Bio-Pharm Technology Co., Ltd, China. Differential gene expression was analyzed using standard criteria: |log₂FC| \u0026ge; 1, and one of the following thresholds for significance: FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (DESeq2, edgeR, or Limma), FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.001 (DEGseq), or Prob\u0026thinsp;\u0026gt;\u0026thinsp;0.8 (NOIseq). A gene was considered differentially expressed if both conditions were satisfied. In addition, functional enrichment analysis of DEGs was conducted using the Gene Ontology (GO) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.geneontology.org\u003c/span\u003e\u003cspan address=\"http://www.geneontology.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.genome.jp/kegg/\u003c/span\u003e\u003cspan address=\"http://www.genome.jp/kegg/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) databases.\u003c/p\u003e\n\u003ch3\u003eQuantitative reverse transcription PCR\u003c/h3\u003e\n\u003cp\u003eBacterial RNA was extracted using a RNeasy Mini Kit (Qiagen, G.E.R.) according to the manufacturer\u0026rsquo;s instructions. Reverse transcription was immediately carried out using a Transcriptor First Strand cDNA Synthesis Kit (Roche, G.E.R.) as per the manufacturer\u0026rsquo;s instructions. qRT-PCR was performed using ChamQ Universal SYBR Qpvr Master Mix. Primers for lytA, comE, nanA, ply, ciaR, stkP, soda, nox, and gyrB are listed in S1 Table. Relative gene expression was analyzed using the 2\u003csup\u003e\u0026minus;△△Ct\u003c/sup\u003e method, with \u003cem\u003egyrB\u003c/em\u003e used as the reference gene and \u003cem\u003eS.pn\u003c/em\u003e exposed to DMEM alone used as the reference condition. Each analysis included six distinct biological replicates, and qRT-PCR was performed in triplicate. A \u003cem\u003eP\u003c/em\u003e-value less than 0.05 was considered statistically significant.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eProtein label-free quantitative analysis by mass spectrometry\u003c/h2\u003e \u003cp\u003eBacterial protein identification and analysis were conducted by Shanghai Majorbio Bio-Pharm Technology Co., Ltd, China. Bacterial protein supernatants were extracted by adding SDT (SDS\u0026thinsp;+\u0026thinsp;MDTT\u0026thinsp;+\u0026thinsp;Tris-HCL) lysis buffer to each sample, and protein quantification was performed using the BCA method. For each sample, 10 \u0026micro;g of protein was subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Blue. Proteins were then digested using the Protein Digestion Kit (Abcam, U.K.), the resulting peptides were dried and resolubilized with 0.1% TFA. Peptide concentrations were determined prior to liquid chromatography-mass spectrometry (LC-MS). Peptides were chromatographically separated using an Easy nLC 1200 chromatography system (Thermo Scientific, U.S.) with a nanoliter flow rate. The resulting LC-MS/MS RAW files were imported into the SEQUEST HT search engine in Proteome Discoverer v2.4 (Thermo Scientific, U.S.). Database searches were performed using UniProt-Human respiratory syncytial virus A (strain A2) [208893]-8928-20230803.fasta (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/taxonomy/12088\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/taxonomy/12088\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and UniProt-\u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e serotype 2 (strain D39 NCTC 7466) [373153]-1922-20230803.fasta (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/taxonomy/373153\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/taxonomy/373153\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Proteins meeting the screening criteria of the fold change in expression level greater than 1.5 (up- and down-regulated) and \u003cem\u003eP\u003c/em\u003e-value less than 0.05 were considered as significantly differentially expressed proteins. Identified bacterial proteins were subjected to subcellular localization analysis using the GO database and functional enrichment analyses with both the GO and KEGG databases.\u003c/p\u003e \u003cp\u003e \u003cb\u003eS.pn\u003c/b\u003e \u003cb\u003eadhesion assay\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHuman non-small cell lung cancer cells (A549) were purchased from ATCC and cultured in DMEM, supplemented with 10% FBS, penicillin (100 IU/mL) and streptomycin (100 \u0026micro;g/mL) at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e. When the A549 cells reached 80\u0026ndash;90% confluence, they were infected with RSV A2 at a multiplicity of infection (MOI) of 1 or mock-infected with DMEM in 2% FBS. After 24 h of RSV infection, the cells were washed three times with sterile PBS. Subsequently, \u003cem\u003eS.pn\u003c/em\u003e D39 was added to the cells and incubated at 37\u0026deg;C for 3 h. Non-adherent bacteria were removed by washing with PBS, and adherent bacteria were collected and quantified using plate counting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence assay by confocal laser scanning microscopy\u003c/h2\u003e \u003cp\u003eBacteria were fixed in 4% paraformaldehyde for 30 min, followed by incubation with 5% bovine serum albumin (BSA) in PBS for 30 min. After washing three times with PBS, all antibody incubations were performed in PBS containing 5% BSA. Rabbit anti-pneumococcal capsular polysaccharide and mouse anti-RSV antibodies were applied to the bacteria and incubated overnight at 4\u0026deg;C. After washing three times with PBS, bound pneumococcal polysaccharide and RSV primary antibodies were detected using Alexa Fluor 594 conjugated donkey anti-rabbit antibody and Alexa Fluor 488 donkey conjugated anti-mouse antibody, respectively (Abcam, U.K.). After washing three times with PBS, bacteria were transferred to sterile glass-bottomed petri dishes, sealed with an anti-fluorescence quenching agent, and observed using confocal laser scanning microscopy (CLSM, Nikon C2, J.P.N.).\u003c/p\u003e \u003cp\u003eA549 cells were cultured in 24-well plates containing round coverslips. The cells were fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.5% Triton X-100 for 15 min, and blocked with 5% BSA for 30 min. Primary antibodies targeting the desired proteins were added, and the cells were incubated overnight at 4\u0026deg;C, washed with PBS three times, and mixed with fluorescent antibody bound to target protein primary antibodies at room temperature for 1 h. The cells were then stained with 4\u0026rsquo;,6-diamidino-2-phenylindole (DAPI) at room temperature for 10 min, and coverslips were mounted onto microscope-adherent slides using glycerol gelatin sealing solution. Imaging was performed using CLSM. Antibodies used in this study included mouse anti-RSV nucleoprotein (N), mouse anti-RSV G glycoprotein, mouse anti-RSV fusion (F) glycoprotein (Abcam, U.K.), CoraLite 488-conjugated alpha tubulin monoclonal, Alexa Fluor 594-conjugated goat anti-mouse IgG, and Alexa Fluor 647-conjugated goat anti-mouse IgG antibodies (Proteintech, C.H.N.).\u003c/p\u003e \u003cp\u003e \u003cb\u003eS.pn\u003c/b\u003e \u003cb\u003eadherent detection by scanning electron microscopy\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCells were seeded in 24-well plates containing round coverslips and infected with RSV A2 at an MOI of 1 for 48 h, then washed three times with PBS. \u003cem\u003eS.pn\u003c/em\u003e was added and allowed to adhere for 3 h, after which non-adherent bacteria were removed by washing with PBS three times. The cells were fixed with 3% glutaraldehyde, then sent to Chengdu Li Lai Biotechnology Co., Ltd (China). for scanning electron microscopy (SEM) analysis. The fixed samples were washed three times with ultrapure water (10 min each time), followed by fixation with 1% osmic acid for 1\u0026ndash;2 h and an additional three washes with ultrapure water for 10 min. Dehydration was performed using an alcohol gradient (30% \u0026rarr; 50% \u0026rarr; 70% \u0026rarr; 90% \u0026rarr; 100%, with three changes of 100%), with each step lasting 15 min. The coverslips were dried using a critical point dryer, mounted onto sample stages with conductive adhesive, and sputter-coated with gold. Imaging was conducted using a JEOL JSM-IT700HR (G.E.R.) scanning electron microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro biofilm model\u003c/h2\u003e \u003cp\u003eA biofilm model was established using round coverslips in a 24-well plate. RSV A2 viral particles, purified RSV G glycoproteins, and DMEM with 2% FBS were incubated with \u003cem\u003eS.pn in vitro\u003c/em\u003e on a shaker at 37\u0026deg;C for 30 min. The bacterial suspension was centrifuged, after which the supernatant was removed and the \u003cem\u003eS.pn\u003c/em\u003e precipitates were resuspended in 500 \u0026micro;L of THY medium. Subsequently, 100 \u0026micro;L of bacterial suspension and 900 \u0026micro;L of sterile THY were added to each well, and the plate was incubated at 37\u0026deg;C for 24 h. After 24 h, the medium was replaced with fresh sterile THY, with or without antibiotics, and the plate was incubated for another 24 h to establish the biofilm model. The antibiotics used in this study included penicillin (100 \u0026micro;g/mL)\u0026thinsp;+\u0026thinsp;gentamicin (50 \u0026micro;g/mL), ceftazidime (100 \u0026micro;g/mL), azithromycin (100 \u0026micro;g/mL), and tetracycline (100 \u0026micro;g/mL) (Solarbio). SEM and CLSM observations were used for biofilm analysis.\u003c/p\u003e \u003cp\u003eFor SEM, biofilm samples were fixed and dehydrated with ethanol gradients (30%, 50%, 70%, 90%, and 100%) for 15 min each step, dried, and prepared for observation with an ion-sputtering apparatus. For CLSM, biofilm samples were stained using a LIVE/DEAD BacLight Bacterial Viability Kit (Thermo Scientific, U.S.) according to the manufacturer\u0026rsquo;s guidelines, and images were obtained by CLSM. Biofilm thickness was measured by horizontal scanning along the z-axis of a three-dimensional coordinate system. The biofilm was scanned layer by layer, with a step size of 2.05 \u0026micro;m. The thickness was determined as the z-axis distance between the last plane where the biofilm structure was visible and the first plane where it was not.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eThe RSV A2 strain G (UniProt ID: P03423, 298 Amino acids) and F coding sequences (UniProt ID: P03420, 574 Amino acids) were cloned into the pcDNA3.1 vector with C-terminal HA or Myc-His tags, respectively. Human embryonic kidney 293 cells (HEK-293T) were purchased from Procell (C.H.N.), and cellular proteins were extracted 48 h after transfection using polyethylenimine linear (PEI) MW40000 (YEASEN). Total cellular proteins were isolated using the Whole-Cell Lysis Assay (KeyGEN, C.H.N.). Cells were lysed with lysis buffer mixed with phosphatase inhibitor, protease inhibitor, and phenylmethylsulfonyl fluoride (PMSF) for 20 min on ice. Protein concentrations were determined using the BCA method. Equal amounts of protein were separated on 4\u0026ndash;20% SDS-polyacrylamide gel and subsequently transferred to polyvinylidene difluoride membranes (Millipore, U.S.). The membranes were blocked with QuickBlock solution (Beyotime, C.H.N) for 30 min, then incubated with different primary antibodies overnight at 4\u0026deg;C, including RSV G and fusion (F) glycoprotein antibodies (Abcam, U.K.) and HA and 6\u0026times;His tag antibodies (Proteintech, C.H.N). After washing three times with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse or HRP-conjugated GAPDH antibodies (Proteintech, C.H.N) for 1 h at room temperature. Enhanced chemiluminescence immunoblotting detection reagents were used to detect bound antibodies and images were captured using a ChemiDoc Imaging system (Bio-Rad, U.S.). Quantification of the blots was performed with ImageJ v1.53c (National Institutes of Health, U.S.) and normalized to the GAPDH intensity of each lane.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePull-down assays\u003c/h2\u003e \u003cp\u003eThe purified G- and F-glycoproteins of RSV A2 were synthesized and purified from \u003cem\u003eE.coli\u003c/em\u003e BL21 strain by affility chromatography though AKTA system and confirmed by coomassie blue staining. Pull-down experiments were performed using the His-Tag Protein Purification Kit with NTA-Ni magnetic agarose beads (Beyotime, C.H.N.) following the manufacturer\u0026rsquo;s protocols. G- and F-glycoproteins were incubated with NTA-Ni magnetic agarose beads for 30 min at room temperature on a rotary mixer, centrifuged, washed three times with non-denaturing wash solution, and detected by western blotting. The collected \u003cem\u003eS.pn\u003c/em\u003e bacterial solution was centrifuged, and total proteins were extracted by adding bacterial lysis buffer containing lysozyme. The extracted proteins were stored at \u0026minus;\u0026thinsp;80\u0026deg;C for future use. Bacterial proteins were incubated with G- and/or F-glycoprotein magnetic beads overnight at 4\u0026deg;C on a rotator. After incubation, the beads were magnetically separated and washed three times with non-denaturing wash solution. To elute the bound proteins, 20 \u0026micro;L of non-denaturing eluent was added to the beads, followed by 10 min incubation. The beads were then magnetically separated, and the eluent containing bacterial proteins bound to the G- or F-glycoprotein was collected in a fresh centrifuge tube. After electrophoretic separation of proteins on SDS-polyacrylamide gel, silver staining experiments were performed using a Fast Silver Stain Kit (Beyotime, C.H.N). Proteins were identified through LC-MS/MS by Shanghai Bioprofile, and the resulting raw files were analyzed and matched using MaxQuant software v1.6.2.10 (Computational Systems Biochemistry, G.E.R.).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eS.pn\u003c/em\u003e D39 strain ComGG and ABC coding sequences were cloned into the pGEX-6P-1 vector with C-terminal GST tags. Both ABJ53717.1-pGEX-6P-1 and ABJ55468.1-pGEX-6p-1 plasmids were constructed by Wuhan Gene Create, and \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) competent cells (Thermo Scientific, U.S.) were used to express \u003cem\u003eS.pn\u003c/em\u003e late competence protein ComGG (ComGG protein) and maltodextrin ABC transporter substrate-binding protein (ABC protein). Three to four recombinant protein-expressing colonies were picked and inoculated into 5 mL of Luria-Bertani (LB) medium containing selective antibiotics. Cultures were shaken overnight at 37\u0026deg;C until saturation (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;\u0026ge;\u0026thinsp;2). Overnight cultures were diluted 1:50 in fresh LB medium with antibiotics and incubated for 2\u0026ndash;3 h until the mid-log phase (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.4). Protein expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 2\u0026ndash;3 h (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.5\u0026ndash;0.6). The GST-tagged recombinant proteins were detected by western blotting using GAT-tag antibodies (Proteintech, C.H.N.). The \u003cem\u003eS.pn\u003c/em\u003e recombinant proteins were incubated with G-glycoprotein magnetic beads overnight at 4\u0026deg;C, as described previously. The eluted proteins, representing bacterial recombinant proteins bound to the G-glycoprotein, were analyzed by western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eProtein structure prediction and bioinformatics analysis\u003c/h2\u003e \u003cp\u003eThe amino acid sequences of the human respiratory syncytial virus subtype A (strain A2) G glycoprotein (protein ID: P03423), late competence protein ComGG (protein ID: A0A0H2ZQ87), and maltodextrin ABC transporter substrate-binding protein (protein ID: A0A0H2ZLL1) of the \u003cem\u003eS.pn\u003c/em\u003e serotype 2 (strain D39) were retrieved from the UniProt database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). These sequences were submitted to AlphaFold 3.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://alphafoldserver.com/\u003c/span\u003e\u003cspan address=\"https://alphafoldserver.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for protein docking model construction. The resulting structures were visualized and analyzed using PyMOL v2.6 (DeLano Scientific LLC, U.S.).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical significance was assessed by unpaired \u003cem\u003et\u003c/em\u003etest and one-away ANOVA as appropriate using GraphPad Prism 9.0. Data are shown as the mean with standard error of mean (SEM), and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 is considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eRSV virions enhance S.pn pathogenicity in vivo\u003c/h2\u003e \u003cp\u003eTo evaluate whether incubation with RSV (A2) enhances the pathogenicity of \u003cem\u003eS.pn\u003c/em\u003e (D39), a sublethal dose of \u003cem\u003eS.pn\u003c/em\u003e was incubated with RSV \u003cem\u003ein vitro\u003c/em\u003e (RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e) and subsequently used to intranasally infect mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Control groups included mice infected with an equivalent dose of \u003cem\u003eS.pn\u003c/em\u003e alone (\u003cem\u003eS.pn\u003c/em\u003e) and mice inoculated with PBS (blank control). The 7-day survival rate revealed that 30% of mice in the RSV\u0026thinsp;+\u0026thinsp;S.pn group and 40% in the S.pn group survived, while all mice in the blank control group survived (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Histopathological analysis of lung tissue from surviving and deceased mice showed mild pathological changes in surviving mice, whereas deceased mice exhibited severe damage, including disrupted alveolar structure, markedly thickened lung septa, airway destruction, epithelial sloughing (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), and neutrophil aggregation in the vessel lumen in the RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e. Extrapulmonary organ invasion was also more severe in the RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e group, with 37.5% of mice exhibiting liver and spleen infections, 56.25% showing heart infections, and 62.5% displaying brain infections (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). To determine whether RSV remnants after co-incubation with \u003cem\u003eS.pn\u003c/em\u003e were involved in infection, viral loads in lung tissues were assessed in the RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e group after 3 days of \u003cem\u003eS.pn\u003c/em\u003e infection. Viral copy numbers were less than 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e (PFU/mL) in both surviving and deceased mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), indicating minimal residual viral presence. These findings demonstrate that RSV co-incubation exacerbates \u003cem\u003eS.pn\u003c/em\u003e infections, leading to severe pneumonia, extrapulmonary infections, and increased mortality.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalysis of BALF revealed elevated neutrophils in the RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e group, compared to the \u003cem\u003eS.pn\u003c/em\u003e group, at 12 h and 24 h post-\u003cem\u003eS.pn\u003c/em\u003e infection (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Macrophage levels increased in all groups by 72 h post-\u003cem\u003eS.pn\u003c/em\u003e infection (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). RNA-seq analysis of lung tissues from surviving and deceased mice after 3 days of \u003cem\u003eS.pn\u003c/em\u003e infection identified differential gene expression. Venn diagram analysis identified 4 609 genes uniquely expressed in the RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH), enriched in pathways such as neutrophil extracellular trap formation, C-type lectin receptor signaling pathway, and necroptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). Conversely, 6 986 genes were uniquely expressed in the \u003cem\u003eS.pn\u003c/em\u003e group, enriched in pathways such as regulation of actin cytoskeleton and p53 signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). These findings highlight the predominant involvement of neutrophil- and macrophage-associated pathways in \u003cem\u003eS.pn\u003c/em\u003e infection in deceased mice.\u003c/p\u003e \u003cp\u003eInflammatory factors associated with neutrophil/macrophage chemotaxis and activation in \u003cem\u003eS.pn\u003c/em\u003e-infected mice were further examined (Supplemental Fig.\u0026nbsp;1). Results showed elevated levels of TNF-α, IL-1α, IL-1β, and IL-10 in deceased mice compared to survivors, although the differences among deceased mice were not significant. IL-6, IL-17A, and IFN-γ were higher in deceased RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e mice than in deceased \u003cem\u003eS.pn\u003c/em\u003e mice, although the differences were not significant. Neutrophil chemokines KC and G-CSF were elevated in all deceased mice, particularly in the RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e group. Macrophage-associated chemokines GM-CSF, MIP-1α, and MCP-1 were also elevated and more highly expressed in the RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e group. These findings suggest that co-incubation with RSV triggers a heightened inflammatory response, including cytokine storm, contributing to severe outcomes in \u003cem\u003eS.pn\u003c/em\u003e infections.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eRSV enhances S.pn virulence and peptidoglycan biosynthesis\u003c/h2\u003e \u003cp\u003eTo clarify whether the enhanced inflammatory response observed in RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e infections is associated with increased \u003cem\u003eS.pn\u003c/em\u003e virulence, bacterial-specific virulence gene expression was analyzed using qRT-PCR. After co-incubation of \u003cem\u003eS.pn\u003c/em\u003e with RSV \u003cem\u003ein vitro\u003c/em\u003e, with the same dose of \u003cem\u003eS.pn\u003c/em\u003e as a control, the expression levels of eight virulence and adhesion-associated genes were measured (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Results showed a 40-fold increase in \u003cem\u003esodA\u003c/em\u003e gene expression in RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e, while \u003cem\u003enox\u003c/em\u003e, \u003cem\u003ecomE\u003c/em\u003e, and \u003cem\u003eciaR\u003c/em\u003e were significantly up-regulated compared to \u003cem\u003eS.pn\u003c/em\u003e alone.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess protein-level expression of the eight identified virulence factors, high-throughput sequencing was performed. Results showed that only the comE and ciaR proteins were significantly up-regulated in RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), while the remaining six proteins showed no significant differences with the \u003cem\u003eS.\u003c/em\u003epn group. These findings suggest that \u003cem\u003eS.pn\u003c/em\u003e virulence-associated factors were initially up-regulated at the gene level following co-incubation with RSV, but not consistently translated into increased protein expression.\u003c/p\u003e \u003cp\u003eLabel-free quantitative proteomic analysis identified 243 differentially expressed proteins between the RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e and \u003cem\u003eS.pn\u003c/em\u003e groups, including 80 significantly up-regulated and 163 significantly down-regulated proteins. Volcano plot analysis of differential proteins between the two groups is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE. Subcellular localization analysis revealed that, in the comparison between RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e and \u003cem\u003eS.pn\u003c/em\u003e, most differential proteins were located in the bacterial cell membrane (17 proteins, 48.57%) and bacterial cytoplasm (15 proteins, 42.86%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). KEGG pathway enrichment analysis of the differential proteins identified the top 10 enriched pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Among these, the peptidoglycan biosynthesis pathway was notably enriched, with an enrichment factor of 11.5 and a \u003cem\u003eP\u003c/em\u003e-value of \u0026lt;\u0026thinsp;0.001, involving five enriched proteins. This pathway was second only to the pyrimidine pathway (13 enriched proteins, enrichment factor 14.27), metal pathway (72 enriched proteins, enrichment factor 13.85) and purine pathway (14 enriched proteins, enrichment factor 12.19). Butterfly plots comparing the KEGG pathway enrichment of up- and down-regulated proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH) highlighted peptidoglycan biosynthesis as the most prominently expressed pathway among up-regulated proteins in RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e compared to \u003cem\u003eS.pn\u003c/em\u003e. These results suggest that co-incubation with RSV promotes \u003cem\u003eS.pn\u003c/em\u003e peptidoglycan biosynthesis, enhancing its role as a virulence factor for \u003cem\u003eS.pn\u003c/em\u003e inflammation. Peptidoglycan likely contributes to the increased inflammatory response observed in the mouse lung, amplifying the bacterial inflammatory capacity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eRSV infection enhances S.pn adhesion to airway epithelial cells\u003c/h2\u003e \u003cp\u003eTo investigate how RSV influences bacterial pathogenicity, \u003cem\u003eS.pn\u003c/em\u003e was co-incubated with RSV virions, purified RSV G glycoprotein, F glycoprotein, RSV RNA, and A549 RNA \u003cem\u003ein vitro\u003c/em\u003e, respectively, with \u003cem\u003eS.pn\u003c/em\u003e alone as the control. The adhesion of \u003cem\u003eS.pn\u003c/em\u003e to A549 cells was then assessed. Results showed a significant increase in \u003cem\u003eS.pn\u003c/em\u003e adhesion only after co-incubation with RSV virions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Antigen co-localization and confocal imaging confirmed the binding of RSV virions to the bacteria during co-incubation \u003cem\u003ein vitro\u003c/em\u003e (yellow fluorescence, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Imaging also demonstrated that \u003cem\u003eS.pn\u003c/em\u003e adhered predominantly to the cell surface, with a higher number of bacteria adhering to A549 cells in the RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e group compared to \u003cem\u003eS.pn\u003c/em\u003e alone (red fluorescence, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These results suggest that intact RSV virions facilitate \u003cem\u003eS.pn\u003c/em\u003e adhesion to airway epithelial cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe potential mechanisms underlying increased \u003cem\u003eS.pn\u003c/em\u003e adhesion following RSV infection were further investigated using an \u003cem\u003ein vitro\u003c/em\u003e model of RSV-infected A549 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). RSV infectivity was confirmed by immunofluorescence detection of the viral N-protein, with approximately 80% of A549 cells showing positive N-protein staining at 24 h post-infection (MOI of 1), indicating successful RSV infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). \u003cem\u003eS.pn\u003c/em\u003e was then inoculated into RSV-infected or mock-infected A549 cells, and bacterial adhesion was quantified through plate counting at different time points. Results showed that adhesion was significantly higher in RSV-infected cells compared to mock-infected cells 24 h post-RSV infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). SEM confirmed increased \u003cem\u003eS.pn\u003c/em\u003e adhesion to RSV-infected A549 cells compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), consistent with the blood plate count results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). These findings indicate that RSV infection enhances \u003cem\u003eS.pn\u003c/em\u003e adhesion to airway epithelial cells, playing a critical role in bacterial pathogenicity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eCo-incubation with RSV affects S.pn biofilm formation\u003c/h2\u003e \u003cp\u003eGiven that co-incubation of RSV with S.pn was shown to up-regulate bacterial peptidoglycan biosynthesis and enhance adhesion to host cells\u0026mdash;processes both linked to biofilm formation ((\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e)To test this hypothesis, \u003cem\u003eS.pn\u003c/em\u003e was co-incubated with RSV virions (RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e) or purified RSV G glycoprotein (G.pro\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e) \u003cem\u003ein vitro\u003c/em\u003e, with \u003cem\u003eS.pn\u003c/em\u003e alone as the control (\u003cem\u003eS.pn\u003c/em\u003e). Biofilm formation was then evaluated. SEM images showed that \u003cem\u003eS.pn\u003c/em\u003e formed a strong, dense, and uniformly distributed surface biofilm across all groups, with only minor differences observed between them (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). However, measurement of biofilm thickness using z-axis distance with CLSM revealed significant differences, with thicknesses of 25.86 \u0026micro;m for RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e, 18.36 \u0026micro;m for \u003cem\u003eS.pn\u003c/em\u003e, and 16.54 \u0026micro;m for G.pro\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), indicating that co-incubation with RSV enhanced \u003cem\u003eS.pn\u003c/em\u003e biofilm thickness.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess the impact of antibiotics on biofilm formation, penicillin\u0026thinsp;+\u0026thinsp;gentamicin, ceftazidime, azithromycin, and tetracycline were added to the cultures. Biofilm disruption was observed and quantitatively analyzed using CLSM. Mean fluorescence intensity comparisons indicated that penicillin\u0026thinsp;+\u0026thinsp;gentamicin resulted in the lowest intensity among the antibiotics tested. However, the overall mean fluorescence intensity of the RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e group was slightly higher than that of the other two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). CLSM imaging of the \u003cem\u003eS.pn\u003c/em\u003e biofilms after penicillin\u0026thinsp;+\u0026thinsp;gentamicin intervention showed a more uniform and densely distributed biofilm in the RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e group compared to the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results suggest that co-incubation with RSV enhances \u003cem\u003eS.pn\u003c/em\u003e biofilm formation, which may contribute to the reduced efficacy of antimicrobial drugs.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eRSV enhances S.pn adhesion associated with G glycoprotein\u003c/h2\u003e \u003cp\u003eThe RSV G glycoprotein has been reported to act as a receptor for \u003cem\u003eS.pn\u003c/em\u003e on infected cells, facilitating bacterial invasion (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). To examine the association between increased \u003cem\u003eS.pn\u003c/em\u003e adhesion and RSV infection, western blotting was used to detect changes in A549 G- and F-glycoproteins following RSV infection. Results showed that both glycoproteins were most highly expressed 24 h post-infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Immunofluorescence further confirmed that G- and F-glycoproteins were predominantly localized on the cell membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, white arrows with yellow fluorescence).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven the increased \u003cem\u003eS.pn\u003c/em\u003e adhesion observed at 24 h post-infection, the role of RSV G- and F-glycoproteins in mediating this adhesion was further investigated. Notably, 293T cells were transfected with RSV G and F plasmids, resulting in the overexpression of G and F proteins, as confirmed by western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Adhesion assays demonstrated increased \u003cem\u003eS.pn\u003c/em\u003e adhesion to cells overexpressing G- and F-glycoproteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG), although the difference was only significant for G glycoprotein overexpression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings suggest that the RSV G glycoprotein plays a key role in enhancing S.pn adhesion to infected cells.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eRSV interacts with S.pn through viral G glycoprotein and bacterial surface proteins\u003c/h2\u003e \u003cp\u003eGiven the evidence that RSV binds to \u003cem\u003eS.pn\u003c/em\u003e and that RSV G- and F-glycoproteins are associated with \u003cem\u003eS.pn\u003c/em\u003e adhesion, the potential interaction between these glycoproteins and S.pn was further investigated. The RSV G- and F-glycoproteins were immobilized on magnetic beads, as confirmed by western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The \u003cem\u003eS.pn\u003c/em\u003e protein lysates were extracted and co-incubated with these glycoprotein magnetic beads, using BSA as a control. Silver staining results showed that only the G-glycoprotein interacted with \u003cem\u003eS.pn\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Mass spectrometry analysis of bacterial proteins bound to G-glycoprotein magnetic beads identified eight \u003cem\u003eS.pn\u003c/em\u003e surface proteins, with the late competence ComGG protein and maltodextrin ABC transporter substrate-binding protein displaying the highest binding scores and strongest binding abilities (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMass spectrometry identification of pneumococcus membrane elution protein co-incubated with G glycoprotein magnetic beads.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtein IDs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtein names\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMol. weight [kDa]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eScore\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIntensity\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eABJ53717.1; A0A0H2ZLL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLate competence protein ComGG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.44399\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e247150000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWP_000095484.1; ABJ55468.1;\u003c/p\u003e \u003cp\u003eA0A0H2ZQ87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMULTISPECIES: maltodextrin ABC transporter substrate-binding protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e45.367\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.38272\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e466940000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWP_000750085.1; ABJ55268.1;\u003c/p\u003e \u003cp\u003eA0A0H2ZRA8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell wall-associated serine protease PrtA(S8 family serine peptidase)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e240.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.22085\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e890310\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eABJ53888.1; A0A0H2ZMA6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCompetence-induced protein Ccs4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e58.026\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.16567\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e834390\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eABJ55011.1; A0A0H2ZQ38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCAAX amino terminal protease family protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e26.254\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.06640\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1331900\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWP_001826592.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etype IV secretory system conjugative DNA transfer family protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e72.421\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.04186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1387500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWP_001224760.1; ABJ54249.1;\u003c/p\u003e \u003cp\u003eA0A0H2ZN49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ebeta-lactamase(serine hydrolase)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.03127\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e531140\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eABJ53655.1; AAF73779.1;\u003c/p\u003e \u003cp\u003eAAF13456.1; A0A0H2ZLS0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCholine binding protein A CbpA(PspC)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e79.097\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.00089\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e59160000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBL21 competent cells were transfected with plasmids encoding ComGG and ABC proteins, with successful expression confirmed by western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). A pull-down assay was performed to evaluate G glycoprotein binding to these proteins. Results showed that while the ComGG protein bands were not obvious, clear ABC protein bands were visible following pull-down with G-protein magnetic beads (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eProtein docking analysis using AlphaFold (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) also predicted that the G glycoprotein had two docking sites with ComGG protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG) and six docking sites with ABC protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). These findings suggest that the RSV G glycoprotein primarily interacts with the maltodextrin ABC transporter substrate-binding protein of \u003cem\u003eS.pn\u003c/em\u003e, facilitating bacterial adhesion and potentially enhancing pathogenicity.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe murine pneumonia model demonstrated that co-incubation of RSV with \u003cem\u003eS.pn\u003c/em\u003e exacerbated bacterial virulence, increasing susceptibility to extrapulmonary organ invasion and lethal infection. These effects were closely associated with an enhanced inflammatory response and significant alterations in neutrophil dynamics within the airways. Early infection in RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e mice showed increased neutrophil levels in the BALF, reflecting an intensified immune response. While neutrophils are critical for bacterial clearance following \u003cem\u003eS.pn\u003c/em\u003e infection (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), their release of elastase can compromise lung integrity (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), facilitating bacterial dissemination and extrapulmonary organ invasion. The severe pulmonary damage and excessive neutrophil infiltration detected in deceased RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e mice underscored the detrimental consequences of this immune response. RNA-seq analysis of lung tissue identified distinct \u003cem\u003eS.pn\u003c/em\u003e and RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e activation pathways involved in invasive and lethal outcomes. Differentially expressed genes in the RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e group were predominantly enriched in the C5 lectin pathway, known to drive neutrophil extracellular trap (NET) formation. Previous studies have linked C5 activation during bacterial sepsis to neutrophil recruitment to the lungs, resulting in an inflammatory storm (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), while neutrophils with impaired apoptosis accumulate in pulmonary microcirculation, exacerbating lung injury (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). This aligns with the elevated expression of pro-inflammatory cytokines, such as IL-17A, in deceased RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e mice. IL-17A plays a dual role, promoting neutrophil bactericidal activity (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) while inducing the release of elastase and myeloperoxidase, key components of NETs, thus contributing to histopathological lung damage (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). RSV co-incubation fundamentally altered the interaction between \u003cem\u003eS.pn\u003c/em\u003e and the host immune system, disrupting innate immune responses and amplifying pulmonary inflammation, as evidenced by the increased myeloperoxidase levels and NET formation observed in the RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e group.\u003c/p\u003e \u003cp\u003eThe up-regulation of \u003cem\u003esodA\u003c/em\u003e, a gene associated with invasive \u003cem\u003eS.pn\u003c/em\u003e infections (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), suggested that RSV exacerbated bacterial infections by promoting \u003cem\u003eS.pn\u003c/em\u003e virulence factors. However, the sodA protein was not significantly expressed after co-incubation with RSV, suggesting the involvement of alternative mechanisms driving increased bacterial pathogenicity. Subsequent proteomic analysis comparing the \u003cem\u003eS.pn\u003c/em\u003e and RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e groups revealed significant up-regulation of the \u003cem\u003eS.pn\u003c/em\u003e peptidoglycan synthesis pathway following co-incubation with RSV. Peptidoglycan, a key component of the bacterial wall, plays an important role in \u003cem\u003eS.pn\u003c/em\u003e adhesion and the host inflammatory response (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Host peptidoglycan recognition proteins or nucleotide oligomerization domain (NOD)-like receptors are known to trigger innate immune responses through the detection of bacterial cell wall peptides (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Peptidoglycan homeostasis is essential for bacterial survival, growth, and division, and is critical for the pathogenicity of \u003cem\u003eS.pn\u003c/em\u003e. This study provides the first evidence that RSV influences \u003cem\u003eS.pn\u003c/em\u003e peptidoglycan biosynthesis, revealing a novel mechanism by which RSV enhances bacterial pathogenicity.\u003c/p\u003e \u003cp\u003eOur results demonstrated that RSV, despite the detection of low viral loads in S.pn-infected mice after co-incubation, contributed to bacterial pathogenicity but was not the primary driver. \u003cem\u003eIn vitro\u003c/em\u003e bacterial adhesion assays and immunofluorescence imaging confirmed that only intact RSV virions co-incubated with \u003cem\u003eS.pn\u003c/em\u003e significantly increased bacterial adhesion to epithelial cells. Immunofluorescence further demonstrated direct binding between RSV and \u003cem\u003eS.pn\u003c/em\u003e after co-incubation. Additionally, RSV infection promoted \u003cem\u003eS.pn\u003c/em\u003e adhesion to airway epithelial cells in a model of RSV-secondary \u003cem\u003eS.pn\u003c/em\u003e infection. These findings are consistent with previous studies showing that RSV-induced airway damage increases host susceptibility to bacterial infections, such as \u003cem\u003eS.pn\u003c/em\u003e, through non-specific mechanisms. Notably, pharyngeal cells co-infected with RSV and \u003cem\u003eS.pn\u003c/em\u003e show an increase in the transcription of genes involved in adhesion (\u003cem\u003epsaA\u003c/em\u003e, \u003cem\u003epilus islet\u003c/em\u003e) as well as transport and binding (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). RSV infection has also been shown to increase the expression of host mediators, such as platelet-activating factor (PAF) receptor and intercellular adhesion molecule 1 (ICAM-1), which facilitate \u003cem\u003eS.pn\u003c/em\u003e adhesion (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Imaging studies using fluorescence and SEM have reported a redistribution of adherent \u003cem\u003eS.pn\u003c/em\u003e across the surface of RSV-infected epithelial cells, with dense bacterial accumulations near syncytia, suggesting viral and bacterial co-localization (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). These results indicate that RSV infection enhances S.pn adhesion to epithelial cells through multiple mechanisms, including direct viral-bacterial binding, modulation of host cell receptors, and redistribution of bacteria on epithelial surfaces. These interactions highlight the complex synergy between viral and bacterial pathogens in exacerbating respiratory infections.\u003c/p\u003e \u003cp\u003eOur results indicated that RSV enhanced \u003cem\u003eS.pn\u003c/em\u003e biofilm formation \u003cem\u003ein vitro\u003c/em\u003e, a process likely related to the observed increase in bacterial adhesion and up-regulation of peptidoglycan biosynthesis. Biofilms are highly organized bacterial communities that adhere to solid surfaces and are embedded within a self-produced extracellular matrix of polysaccharides, proteins, and DNA (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Although biofilm formation is a dynamic and continuous process, the initial adhesion phase is critical for biofilm development (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). This phase relies on specific components of the bacterial cell surface (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), including wall teichoic acid (WTA) and lipoteichoic acid (LTA), which are integral to peptidoglycan and cell membranes of Gram-positive bacteria. These structural components play pivotal roles in surface attachment and biofilm establishment (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Biofilms pose a significant clinical challenge by conferring bacterial resistance to conventional antibiotics, resulting in persistent nosocomial infections (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). This resistance is attributed to the protective matrix of the biofilm and the development of multiple antibiotic resistance mechanisms in bacteria. These challenges were evident in the antibiotic intervention experiments, where RSV\u0026thinsp;+\u0026thinsp;\u003cem\u003eS.pn\u003c/em\u003e formed the thickest biofilm and exhibited the lowest susceptibility to antibiotics. The observed reduction in antibiotic efficacy highlights the complex interplay between RSV-enhanced biofilm formation and bacterial resistance.\u003c/p\u003e \u003cp\u003eFurthermore, our findings also indicated a potential association between RSV infection and \u003cem\u003eS.pn\u003c/em\u003e adhesion, mediated by RSV G glycoproteins expressed on infected epithelial cells. These glycoproteins serve as adhesion receptors for \u003cem\u003eS.pn\u003c/em\u003e, facilitating bacterial adhesion (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Notably, this enhanced adhesion appears to be independent of the pneumococcal strain or of variability in RSV G glycoprotein sequences (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Pull-down assays and mass spectrometry confirmed direct binding between RSV G glycoproteins and \u003cem\u003eS.pn\u003c/em\u003e, consistent with earlier studies reporting increased bacterial adhesion and invasiveness in a mouse model due to this interaction (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). The interaction between RSV G glycoprotein and pneumococcus has also been linked to significant changes in the pneumococcal transcriptome, including the up-regulation of key virulence-related genes (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). This study identified specific \u003cem\u003eS.pn\u003c/em\u003e membrane proteins that interacted with RSV G glycoproteins, including the late competence ComGG protein and the ABC transporter substrate-binding protein. Among these, the ABC transporter substrate-binding protein demonstrated a higher binding affinity, underscoring its potential role in mediating bacterial adhesion and virulence.\u003c/p\u003e \u003cp\u003eThe conventional view is that viral infections are self-limiting and require minimal intervention has been challenged, particularly in the context of RSV infections. The use of broad-spectrum antiviral drugs, such as interferons, remains highly debated. Although the virus itself is not typically harmful to the host, clinical evidence strongly supports a positive correlation between \u003cem\u003eS.pn\u003c/em\u003e and RSV disease severity (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e), \u003csup\u003e,\u003c/sup\u003e(\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Our study also confirmed that RSV enhanced susceptibility to \u003cem\u003eS.pn\u003c/em\u003e infection and exacerbated disease severity. Notably, early and aggressive antiviral therapy appeared to be beneficial in mitigating secondary bacterial infections. However, the lack of specific antiviral treatments for RSV-infected individuals presents a significant clinical challenge. Furthermore, while some studies have reported that antibiotics improve outcomes in children with RSV-associated respiratory failure, highlighting the role of bacterial co-infections in aggravating viral disease severity (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), other studies have failed to identify a clear benefit of antibiotics in RSV disease management (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Until more definitive evidence emerges, clinicians must exercise careful judgment in the use of antibiotics in these cases.\u003c/p\u003e \u003cp\u003eBeta-lactam antibiotics remain the first choice for managing \u003cem\u003eS.pn\u003c/em\u003e infections, particularly in co-infection scenarios. Of note, our study demonstrated that RSV co-incubation up-regulated \u003cem\u003eS.pn\u003c/em\u003e peptidoglycan biosynthesis\u0026mdash;a critical target for penicillin-based antimicrobials. This suggests that penicillin-based treatments, such as penicillin and amoxicillin, may be especially effective in patients co-infected with RSV and \u003cem\u003eS.pn\u003c/em\u003e. This highlights the potential for prioritizing these drugs in clinical practice.While current RSV vaccines primarily focus on the RSV fusion (F) glycoprotein (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e), our results underscore the importance of RSV G glycoproteins in the pathogenic interaction between RSV and \u003cem\u003eS.pn\u003c/em\u003e. Given the critical role of RSV G glycoproteins in facilitating bacterial adherence and virulence, it is worth considering whether vaccine development targeting this glycoprotein could simultaneously protect against RSV and \u003cem\u003eS.pn\u003c/em\u003e co-infections, offering a broader scope of prevention.\u003c/p\u003e \u003cp\u003eIn conclusion, our study showed that RSV infection influenced susceptibility to \u003cem\u003eS.pn\u003c/em\u003e infections and disease severity through both host-dependent and host-independent mechanisms. Notably, RSV directly interacted with \u003cem\u003eS.pn\u003c/em\u003e, increasing bacterial adherence, enhancing bacterial virulence, reducing neutrophil-mediated bacterial clearance, and intensifying pulmonary inflammation. Furthermore, RSV infection facilitated \u003cem\u003eS.pn\u003c/em\u003e adhesion to airway epithelial cells, with RSV G glycoproteins playing a key role in this pathogenic process. These findings provide valuable insights for clinicians to rationally select anti-infective therapies, emphasizing the utility of penicillin-based antimicrobials in managing co-infections. Additionally, the study offers a theoretical foundation for the development of pathogen-targeted treatments, including vaccines against RSV G glycoproteins, which could reduce the burden of co-infections involving RSV and S.pn.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLu Li contributed to the study design, experiments operation, data analysis, data interpretation, preparation of tables/figures and writing of the report. Xin Long contributed to the study design, data acquisition, data analysis, data interpretation and preparation of figures. Yuncheng Wang and Peiru Shi contributed to the data acquisition, data analysis, and data interpretation. Yushun Wan contributed to the study design, instruction in experimental techniques, data interpretation and writing of the report. Enmei Liu conceived the idea for the study and critically reviewed the manuscript for intellectual content. Yu Deng conceived the idea as well as secured funding for the study, and critically reviewed the manuscript for intellectual content. All authors make an approval of the version to be submitted.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was funded by a National key Research and Development Program of China [2022YFC2704900]; Natural Science Foundation of Chongqing, China[cstc2019jycj-msxmX0858]; CQMU Program for Youth Innovation in Future Medicine. The funding bodies had no part in the design, conduct, analysis or interpretation of this study or the decision to submit this paper for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that supports the findings of this study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments involving animals were carried out in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Animal Experiments of Chongqing Medical University (Permit number: SYXK-(YU) 2012-0001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no competing interests exist.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe thank Prof. Yushun Wan for excellent technical support, and Christine Watts for her support in writing this article. \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e strains D39 was kindly provided by Xuemei Zhang (Chongqing, China).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMcAllister DA, Liu L, Shi T, Chu Y, Reed C, Burrows J, et al. Global, regional, and national estimates of pneumonia morbidity and mortality in children younger than 5 years between 2000 and 2015: a systematic analysis. \u003cem\u003eLancet Glob Health\u003c/em\u003e 2019;7(1):e47-e57. \u003c/li\u003e\n\u003cli\u003eLi Y, Wang X, Blau DM, Caballero MT, Feikin DR, Gill CJ, et al. Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in children younger than 5 years in 2019: a systematic analysis. \u003cem\u003eLancet\u003c/em\u003e 2022;399(10340):2047-64. \u003c/li\u003e\n\u003cli\u003eShibata T, Makino A, Ogata R, Nakamura S, Ito T, Nagata K, et al. Respiratory syncytial virus infection exacerbates pneumococcal pneumonia via Gas6/Axl-mediated macrophage polarization. \u003cem\u003eJ Clin Invest\u003c/em\u003e \u003c/li\u003e\n\u003cli\u003eMorpeth SC, Munywoki P, Hammitt LL, Bett A, Bottomley C, Onyango CO, et al. Impact of viral upper respiratory tract infection on the concentration of nasopharyngeal pneumococcal carriage among Kenyan children. \u003cem\u003eSci Rep\u003c/em\u003e 2018;8(1):11030. \u003c/li\u003e\n\u003cli\u003eAmpofo K, Bender J, Sheng X, Korgenski K, Daly J, Pavia AT, et al. Seasonal invasive pneumococcal disease in children: role of preceding respiratory viral infection. \u003cem\u003ePediatrics\u003c/em\u003e 2008;122(2):229-37. \u003c/li\u003e\n\u003cli\u003eWeinberger DM, Grant LR, Steiner CA, Weatherholtz R, Santosham M, Viboud C, et al. Seasonal drivers of pneumococcal disease incidence: impact of bacterial carriage and viral activity. \u003cem\u003eClin Infect Dis\u003c/em\u003e 2014;58(2):188-94. \u003c/li\u003e\n\u003cli\u003eBen-Shimol S, Greenberg D, Hazan G, Shemer-Avni Y, Givon-Lavi N, Dagan R. Seasonality of both bacteremic and nonbacteremic pneumonia coincides with viral lower respiratory tract infections in early childhood, in contrast to nonpneumonia invasive pneumococcal disease, in the pre-pneumococcal conjugate vaccine era. \u003cem\u003eClin Infect Dis\u003c/em\u003e 2015;60(9):1384-7.\u003c/li\u003e\n\u003cli\u003eStark JM, Stark MA, Colasurdo GN, LeVine AM. Decreased bacterial clearance from the lungs of mice following primary respiratory syncytial virus infection. \u003cem\u003eJ Med Virol\u003c/em\u003e \u003c/li\u003e\n\u003cli\u003eBourassa MH, Lands LC. Preventative therapies for respiratory Syncytial virus (RSV) in children: Where are we now? \u003cem\u003ePaediatr Respir Rev\u003c/em\u003e 2024;49:24-7.\u003c/li\u003e\n\u003cli\u003eLong X, Shi PR, Luo ZX, Luo J, Ren L, Liu EM, et al. Impact of Streptococcus pneumoniae colonization in upper airway on the clinical manifestations of children with respiratory syncytial virus infection. \u003cem\u003eZhonghua Er Ke Za Zhi\u003c/em\u003e 2022;60(7):694-9. \u003c/li\u003e\n\u003cli\u003eKrishnamurthy A, Kyd J. The roles of epithelial cell contact, respiratory bacterial interactions and phosphorylcholine in promoting biofilm formation by Streptococcus pneumoniae and nontypeable Haemophilus influenzae. \u003cem\u003eMicrobes Infect\u003c/em\u003e 2014;16(8):640-7. \u003c/li\u003e\n\u003cli\u003eAnderson EM, Sychantha D, Brewer D, Clarke AJ, Geddes-McAlister J, Khursigara CM. Peptidoglycomics reveals compositional changes in peptidoglycan between biofilm- and planktonic-derived Pseudomonas aeruginosa. \u003cem\u003eJ Biol Chem\u003c/em\u003e 2020;295(2):504-16. \u003c/li\u003e\n\u003cli\u003eHament JM, Aerts PC, Fleer A, Van Dijk H, Harmsen T, Kimpen JL, et al. Enhanced adherence of Streptococcus pneumoniae to human epithelial cells infected with respiratory syncytial virus. \u003cem\u003ePediatr Res\u003c/em\u003e 2004;55(6):972-8. \u003c/li\u003e\n\u003cli\u003eLevine S, Klaiber-Franco R, Paradiso PR. Demonstration that glycoprotein G is the attachment protein of respiratory syncytial virus. \u003cem\u003eJ Gen Virol\u003c/em\u003e 1987;68 ( Pt 9):2521-4. \u003c/li\u003e\n\u003cli\u003eJumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. Highly accurate protein structure prediction with AlphaFold. \u003cem\u003eNature\u003c/em\u003e 2021;596(7873):583-9. \u003c/li\u003e\n\u003cli\u003eKadioglu A, Gingles NA, Grattan K, Kerr A, Mitchell TJ, Andrew PW. Host cellular immune response to pneumococcal lung infection in mice. \u003cem\u003eInfect Immun\u003c/em\u003e 2000;68(2):492-501. \u003c/li\u003e\n\u003cli\u003eBou Ghanem EN, Clark S, Roggensack SE, McIver SR, Alcaide P, Haydon PG, et al. Extracellular Adenosine Protects against Streptococcus pneumoniae Lung Infection by Regulating Pulmonary Neutrophil Recruitment. \u003cem\u003ePLoS Pathog\u003c/em\u003e. 2015;11(8):e1005126. \u003c/li\u003e\n\u003cli\u003eDomon H, Oda M, Maekawa T, Nagai K, Takeda W, Terao Y. Streptococcus pneumoniae disrupts pulmonary immune defence via elastase release following pneumolysin-dependent neutrophil lysis. \u003cem\u003eSci Rep\u003c/em\u003e 2016;6:38013. \u003c/li\u003e\n\u003cli\u003eRusskamp NF, Ruemmler R, Roewe J, Moore BB, Ward PA, Bosmann M. Experimental design of complement component 5a-induced acute lung injury (C5a-ALI): a role of CC-chemokine receptor type 5 during immune activation by anaphylatoxin. \u003cem\u003eFaseb j\u003c/em\u003e 2015;29(9):3762-72. \u003c/li\u003e\n\u003cli\u003eBosmann M, Ward PA. Role of C3, C5 and anaphylatoxin receptors in acute lung injury and in sepsis. \u003cem\u003eAdv Exp Med Biol\u003c/em\u003e 2012;946:147-59. \u003c/li\u003e\n\u003cli\u003ePark I, Kim M, Choe K, Song E, Seo H, Hwang Y, et al. Neutrophils disturb pulmonary microcirculation in sepsis-induced acute lung injury. \u003cem\u003eEur Respir J\u003c/em\u003e 2019;53(3). \u003c/li\u003e\n\u003cli\u003eDomon H, Nagai K, Maekawa T, Oda M, Yonezawa D, Takeda W, et al. Neutrophil Elastase Subverts the Immune Response by Cleaving Toll-Like Receptors and Cytokines in Pneumococcal Pneumonia. \u003cem\u003eFront Immunol\u003c/em\u003e 2018;9:732. \u003c/li\u003e\n\u003cli\u003eLu YJ, Gross J, Bogaert D, Finn A, Bagrade L, Zhang Q, et al. Interleukin-17A mediates acquired immunity to pneumococcal colonization. \u003cem\u003ePLoS Pathog\u003c/em\u003e 2008;4(9):e1000159. \u003c/li\u003e\n\u003cli\u003eRitchie ND, Ritchie R, Bayes HK, Mitchell TJ, Evans TJ. IL-17 can be protective or deleterious in murine pneumococcal pneumonia. \u003cem\u003ePLoS Pathog\u003c/em\u003e 2018;14(5):e1007099. \u003c/li\u003e\n\u003cli\u003eBerry AM, Paton JC. Additive attenuation of virulence of Streptococcus pneumoniae by mutation of the genes encoding pneumolysin and other putative pneumococcal virulence proteins. \u003cem\u003eInfect Immun\u003c/em\u003e 2000;68(1):133-40.\u003c/li\u003e\n\u003cli\u003eMahdi LK, Van der Hoek MB, Ebrahimie E, Paton JC, Ogunniyi AD. Characterization of Pneumococcal Genes Involved in Bloodstream Invasion in a Mouse Model. \u003cem\u003ePLoS One\u003c/em\u003e 2015;10(11):e0141816. \u003c/li\u003e\n\u003cli\u003eSkovsted IC, Kerrn MB, Sonne-Hansen J, Sauer LE, Nielsen AK, Konradsen HB, et al. Purification and structure characterization of the active component in the pneumococcal 22F polysaccharide capsule used for adsorption in pneumococcal enzyme-linked immunosorbent assays. \u003cem\u003eVaccine\u003c/em\u003e 2007;25(35):6490-500. \u003c/li\u003e\n\u003cli\u003eJohnson JW, Fisher JF, Mobashery S. Bacterial cell-wall recycling. \u003cem\u003eAnn N Y Acad Sci\u003c/em\u003e 2013;1277(1):54-75.\u003c/li\u003e\n\u003cli\u003eKimaro Mlacha SZ, Peret TC, Kumar N, Romero-Steiner S, Dunning Hotopp JC, Ishmael N, et al. Transcriptional adaptation of pneumococci and human pharyngeal cells in the presence of a virus infection. \u003cem\u003eBMC Genomics\u003c/em\u003e 2013;14:378. \u003c/li\u003e\n\u003cli\u003eAvadhanula V, Rodriguez CA, Devincenzo JP, Wang Y, Webby RJ, Ulett GC, et al. Respiratory viruses augment the adhesion of bacterial pathogens to respiratory epithelium in a viral species- and cell type-dependent manner. \u003cem\u003eJ Virol\u003c/em\u003e 2006;80(4):1629-36. \u003c/li\u003e\n\u003cli\u003eYokota S, Okabayashi T, Hirakawa S, Tsutsumi H, Himi T, Fujii N. Clarithromycin suppresses human respiratory syncytial virus infection-induced Streptococcus pneumoniae adhesion and cytokine production in a pulmonary epithelial cell line. \u003cem\u003eMediators Inflamm\u003c/em\u003e 2012;2012:528568. \u003c/li\u003e\n\u003cli\u003eCosterton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. \u003cem\u003eScience\u003c/em\u003e 1999;284(5418):1318-22. \u003c/li\u003e\n\u003cli\u003eKhan F, Pham DTN, Kim YM. Alternative strategies for the application of aminoglycoside antibiotics against the biofilm-forming human pathogenic bacteria. \u003cem\u003eAppl Microbiol Biotechnol\u003c/em\u003e 2020;104(5):1955-76. \u003c/li\u003e\n\u003cli\u003eKhan F, Jeong GJ, Javaid A, Thuy Nguyen Pham D, Tabassum N, Kim YM. Surface adherence and vacuolar internalization of bacterial pathogens to the Candida spp. cells: Mechanism of persistence and propagation. \u003cem\u003eJ Adv Res\u003c/em\u003e 2023;53:115-36. \u003c/li\u003e\n\u003cli\u003eJeong GJ, Khan F, Tabassum N, Cho KJ, Kim YM. Controlling biofilm and virulence properties of Gram-positive bacteria by targeting wall teichoic acid and lipoteichoic acid. \u003cem\u003eInt J Antimicrob Agents\u003c/em\u003e 2023;62(4):106941. \u003c/li\u003e\n\u003cli\u003eYan Z, Huang M, Melander C, Kjellerup BV. Dispersal and inhibition of biofilms associated with infections. \u003cem\u003eJ Appl Microbiol\u003c/em\u003e. 2020;128(5):1279-88. \u003c/li\u003e\n\u003cli\u003eAvadhanula V, Wang Y, Portner A, Adderson E. Nontypeable Haemophilus influenzae and Streptococcus pneumoniae bind respiratory syncytial virus glycoprotein. \u003cem\u003eJ Med Microbiol\u003c/em\u003e 2007;56(Pt 9):1133-7. \u003c/li\u003e\n\u003cli\u003eBrealey JC, Sly PD, Young PR, Chappell KJ. Analysis of phylogenetic diversity and in vitro adherence characteristics of respiratory syncytial virus and Streptococcus pneumoniae clinical isolates obtained during pediatric respiratory co-infections. \u003cem\u003eMicrobiology (Reading)\u003c/em\u003e 2020;166(1):63-72. \u003c/li\u003e\n\u003cli\u003eHament JM, Aerts PC, Fleer A, van Dijk H, Harmsen T, Kimpen JL, et al. Direct binding of respiratory syncytial virus to pneumococci: a phenomenon that enhances both pneumococcal adherence to human epithelial cells and pneumococcal invasiveness in a murine model. \u003cem\u003ePediatr Res\u003c/em\u003e 2005;58(6):1198-203. \u003c/li\u003e\n\u003cli\u003eSmith CM, Sandrini S, Datta S, Freestone P, Shafeeq S, Radhakrishnan P, et al. Respiratory syncytial virus increases the virulence of Streptococcus pneumoniae by binding to penicillin binding protein 1a. A new paradigm in respiratory infection. \u003cem\u003eAm J Respir Crit Care Med\u003c/em\u003e 2014;190(2):196-207. \u003c/li\u003e\n\u003cli\u003eBrealey JC, Chappell KJ, Galbraith S, Fantino E, Gaydon J, Tozer S, et al. Streptococcus pneumoniae colonization of the nasopharynx is associated with increased severity during respiratory syncytial virus infection in young children. \u003cem\u003eRespirology\u003c/em\u003e 2018;23(2):220-7. \u003c/li\u003e\n\u003cli\u003eDiaz-Diaz A, Bunsow E, Garcia-Maurino C, Moore-Clingenpeel M, Naples J, Juergensen A, et al. Nasopharyngeal Codetection of Haemophilus influenzae and Streptococcus pneumoniae Shapes Respiratory Syncytial Virus Disease Outcomes in Children. \u003cem\u003eJ Infect Dis\u003c/em\u003e 2022;225(5):912-23. \u003c/li\u003e\n\u003cli\u003eEsposito S, Zampiero A, Terranova L, Ierardi V, Ascolese B, Daleno C, et al. Pneumococcal bacterial load colonization as a marker of mixed infection in children with alveolar community-acquired pneumonia and respiratory syncytial virus or rhinovirus infection. \u003cem\u003ePediatr Infect Dis J\u003c/em\u003e 2013;32(11):1199-204. \u003c/li\u003e\n\u003cli\u003eShein SL, Kong M, McKee B, O\u0026apos;Riordan M, Toltzis P, Randolph AG. Antibiotic Prescription in Young Children With Respiratory Syncytial Virus-Associated Respiratory Failure and Associated Outcomes. \u003cem\u003ePediatr Crit Care Med\u003c/em\u003e 2019;20(2):101-9. \u003c/li\u003e\n\u003cli\u003eJuv\u0026eacute;n T, Mertsola J, Waris M, Leinonen M, Ruuskanen O. Clinical response to antibiotic therapy for community-acquired pneumonia. \u003cem\u003eEur J Pediatr\u003c/em\u003e 2004;163(3):140-4. \u003c/li\u003e\n\u003cli\u003eFarley R, Spurling GK, Eriksson L, Del Mar CB. Antibiotics for bronchiolitis in children under two years of age. \u003cem\u003eCochrane Database Syst Rev\u003c/em\u003e 2014;2014(10):Cd005189. \u003c/li\u003e\n\u003cli\u003eKneyber MC, Kimpen JL. Antibiotics in RSV bronchiolitis: still no evidence of effect. \u003cem\u003eEur Respir J\u003c/em\u003e 2007;29(6):1285. \u003c/li\u003e\n\u003cli\u003eCrank MC, Ruckwardt TJ, Chen M, Morabito KM, Phung E, Costner PJ, et al. A proof of concept for structure-based vaccine design targeting RSV in humans. \u003cem\u003eScience\u003c/em\u003e 2019;365(6452):505-9.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Respiratory syncytial virus, Streptococcus pneumoniae, pneumonia, G glycoprotein, co-infection","lastPublishedDoi":"10.21203/rs.3.rs-5978497/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5978497/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eCo-infection with RSV and \u003cem\u003eS.pn\u003c/em\u003e is linked to severe, often fatal pneumonia, with unclear molecular mechanisms. This study investigates whether RSV and \u003cem\u003eS.pn\u003c/em\u003e interaction enhances pneumococcal pathogenicity and explores the underlying molecular mechanisms.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe co-incubated \u003cem\u003eS.pn\u003c/em\u003e with RSV and transnasally infected mice, using RNA-seq and proteomics to analyze bacterial pathogenesis. Airway epithelial cells were infected with RSV and subsequently challenged with \u003cem\u003eS.pn\u003c/em\u003e. We constructed an in vitro biofilm model for further study. Confocal microscopy and Western blotting investigated the association between the RSV G glycoprotein and \u003cem\u003eS.pn\u003c/em\u003e. Mass spectrometry and pull-down assays identified surface proteins involved in direct binding.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAfter incubation with RSV, \u003cem\u003eS.pn\u003c/em\u003e exhibited a significant increase in inflammatory response and adherence to epithelial cells, as well as enhanced virulence in a murine pneumonia model. These effects were associated with extensive changes in the proteomics of \u003cem\u003eS.pn\u003c/em\u003e and significantly upregulated expression of peptidoglycan biosynthesis genes. Additionally, we found that the RSV G glycoprotein binds to the maltodextrin ABC transporter substrate-binding protein of \u003cem\u003eS.pn\u003c/em\u003e, which may explain how RSV infection enhances \u003cem\u003eS.pn\u003c/em\u003e adhesion to cells.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe direct interaction between the RSV G glycoprotein and \u003cem\u003eS.pn\u003c/em\u003e, leading to increased bacterial pathogenicity and more severe disease outcomes, represents a novel paradigm in respiratory infections.\u003c/p\u003e\u003ch2\u003eClinical trial number\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e","manuscriptTitle":"Respiratory syncytial virus G glycoprotein promotes Streptococcus pneumoniae-induced severe pneumonia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-17 16:48:51","doi":"10.21203/rs.3.rs-5978497/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9346f86c-bd07-49eb-aebc-b4a081e5ee91","owner":[],"postedDate":"February 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-10T12:09:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-17 16:48:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5978497","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5978497","identity":"rs-5978497","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}