{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,5,23]],"date-time":"2026-05-23T08:05:31Z","timestamp":1779523531609,"version":"3.53.1"},"posted":{"date-parts":[[2026,5,19]]},"group-title":"Microbiology","reference-count":63,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,5,19]],"date-time":"2026-05-19T00:00:00Z","timestamp":1779148800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,5,19]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  <jats:italic>Streptococcus pneumoniae<\/jats:italic>\n                  (\n                  <jats:italic>Spn<\/jats:italic>\n                  ) asymptomatically colonizes the upper respiratory tract (URT), a niche from which it can transmit to another host or cause invasive disease in the same host. The\n                  <jats:italic>in vivo<\/jats:italic>\n                  transcriptional adaptations that\n                  <jats:italic>Spn<\/jats:italic>\n                  undergoes during nasopharyngeal colonization, particularly during influenza A virus (IAV) coinfection, are poorly understood. Here, we leveraged an established infant mouse model of colonization, shedding, and transmission to perform genome-wide transcriptomic profiling of\n                  <jats:italic>Spn<\/jats:italic>\n                  during mono- and during IAV coinfection. Compared with broth-grown controls, pneumococci isolated from the URT exhibited distinct transcriptional programs, with over 200 genes differentially expressed across time points. Genes involved in carbohydrate uptake and metabolism, glycan degradation, amino sugar and nucleotide sugar metabolism, and amino acid biosynthesis were consistently enriched during colonization, highlighting metabolic adaptation to the nasopharyngeal niche. In contrast, IAV coinfection induced a markedly distinct transcriptional signature, including upregulation of branched-chain amino acid biosynthesis, bacteriocin production, and phosphate acquisition systems. Notably, the pilus islet-1 locus was upregulated during\n                  <jats:italic>Spn<\/jats:italic>\n                  -IAV coinfection. Functional studies demonstrated that while the pilus was dispensable for colonization under mono- and coinfection conditions, it promoted high-shedding events and enhanced inflammatory responses during IAV coinfection. However, reduced inflammation and reduced high shedding events from pups inoculated with a pilus-deficient mutant did not alter transmission frequency in the infant mouse model. Collectively, our findings define the\n                  <jats:italic>in vivo<\/jats:italic>\n                  transcriptional landscape of\n                  <jats:italic>Spn<\/jats:italic>\n                  during URT colonization and reveal distinct bacterial adaptations during viral coinfection, providing insight into mechanisms that influence pneumococcal persistence, inflammation, and transmission.\n                <\/jats:p>","DOI":"10.64898\/2026.05.19.726214","type":"posted-content","created":{"date-parts":[[2026,5,20]],"date-time":"2026-05-20T04:35:20Z","timestamp":1779251720000},"source":"Crossref","is-referenced-by-count":0,"title":["Influenza A Virus Coinfection Alters\n                  <i>Streptococcus pneumoniae<\/i>\n                  Gene Expression during Upper Respiratory Tract Colonization"],"prefix":"10.64898","author":[{"given":"Noah A.","family":"Nutter","sequence":"first","affiliation":[{"name":"Department of Microbiology and Immunology, Wake Forest School of Medicine, Winston-Salem, NC"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alicia","family":"Costa-Terryll","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Wake Forest School of Medicine, Winston-Salem, NC"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lance M.","family":"Miller","sequence":"additional","affiliation":[{"name":"Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, NC"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ming","family":"Leung","sequence":"additional","affiliation":[{"name":"Atrium Health Wake Forest Baptist Comprehensive Cancer Center, Winston-Salem, NC"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pengbo","family":"Zhang","sequence":"additional","affiliation":[{"name":"Atrium Health Wake Forest Baptist Comprehensive Cancer Center, Winston-Salem, NC"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel","family":"Fecko","sequence":"additional","affiliation":[{"name":"Department of Microbiology, New York University School of Medicine, New York, NY"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3737-0422","authenticated-orcid":false,"given":"M. Ammar","family":"Zafar","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Wake Forest School of Medicine, Winston-Salem, NC"},{"name":"Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026052300250707000_2026.05.19.726214v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-018-0001-8"},{"key":"2026052300250707000_2026.05.19.726214v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2007.04.088"},{"key":"2026052300250707000_2026.05.19.726214v1.3","doi-asserted-by":"publisher","DOI":"10.1111\/1469-0691.12188"},{"key":"2026052300250707000_2026.05.19.726214v1.4","doi-asserted-by":"crossref","first-page":"e0315124","DOI":"10.1128\/spectrum.03151-24","article-title":"A global epidemic serotype 14 Streptococcus pneumoniae switching to non-vaccine types","volume":"13","year":"2025","journal-title":"Microbiol Spectr"},{"key":"2026052300250707000_2026.05.19.726214v1.5","doi-asserted-by":"publisher","DOI":"10.1093\/infdis\/jis703"},{"key":"2026052300250707000_2026.05.19.726214v1.6","doi-asserted-by":"publisher","DOI":"10.1016\/S1473-3099(24)00158-0"},{"key":"2026052300250707000_2026.05.19.726214v1.7","first-page":"9990","article-title":"High-resolution analysis of the pneumococcal transcriptome under a wide range of infection-relevant conditions","volume":"46","year":"2018","journal-title":"Nucleic Acids Res"},{"key":"2026052300250707000_2026.05.19.726214v1.8","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.00228-09"},{"key":"2026052300250707000_2026.05.19.726214v1.9","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.01125-09"},{"key":"2026052300250707000_2026.05.19.726214v1.10","doi-asserted-by":"crossref","unstructured":"Maestro B , Sanz JM . 2016. Choline Binding Proteins from Streptococcus pneumoniae: A Dual Role as Enzybiotics and Targets for the Design of New Antimicrobials. Antibiotics (Basel) 5.","DOI":"10.3390\/antibiotics5020021"},{"key":"2026052300250707000_2026.05.19.726214v1.11","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2020.01543"},{"key":"2026052300250707000_2026.05.19.726214v1.12","doi-asserted-by":"publisher","DOI":"10.1016\/j.chom.2016.12.005"},{"key":"2026052300250707000_2026.05.19.726214v1.13","doi-asserted-by":"crossref","first-page":"2006","DOI":"10.1128\/iai.57.7.2006-2013.1989","article-title":"Effect of Streptococcus pneumoniae on human respiratory epithelium in vitro","volume":"57","year":"1989","journal-title":"Infect Immun"},{"key":"2026052300250707000_2026.05.19.726214v1.14","doi-asserted-by":"crossref","first-page":"lqae039","DOI":"10.1093\/nargab\/lqae039","article-title":"A comprehensive analysis of pneumococcal two-component system regulatory networks","volume":"6","year":"2024","journal-title":"NAR Genom Bioinform"},{"key":"2026052300250707000_2026.05.19.726214v1.15","doi-asserted-by":"crossref","first-page":"e0037522","DOI":"10.1128\/iai.00375-22","article-title":"The Two-Component System YesMN Promotes Pneumococcal Host-to-Host Transmission and Regulates Genes Involved in Zinc Homeostasis","volume":"91","year":"2023","journal-title":"Infect Immun"},{"key":"2026052300250707000_2026.05.19.726214v1.16","doi-asserted-by":"publisher","DOI":"10.1186\/s40168-023-01640-9"},{"key":"2026052300250707000_2026.05.19.726214v1.17","doi-asserted-by":"crossref","first-page":"e0344722","DOI":"10.1128\/spectrum.03447-22","article-title":"Impact of Pneumococcal and Viral Pneumonia on the Respiratory and Intestinal Tract Microbiomes of Mice","volume":"11","year":"2023","journal-title":"Microbiol Spectr"},{"key":"2026052300250707000_2026.05.19.726214v1.18","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0195098"},{"key":"2026052300250707000_2026.05.19.726214v1.19","doi-asserted-by":"publisher","DOI":"10.1086\/591708"},{"key":"2026052300250707000_2026.05.19.726214v1.20","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1003238"},{"key":"2026052300250707000_2026.05.19.726214v1.21","doi-asserted-by":"publisher","DOI":"10.1016\/j.chom.2014.06.005"},{"key":"2026052300250707000_2026.05.19.726214v1.22","doi-asserted-by":"crossref","first-page":"xtac007","DOI":"10.1093\/femsmc\/xtac007","article-title":"Secondary infection with Streptococcus pneumoniae decreases influenza virus replication and is linked to severe disease","volume":"3","year":"2022","journal-title":"FEMS Microbes"},{"key":"2026052300250707000_2026.05.19.726214v1.23","doi-asserted-by":"crossref","unstructured":"Ortigoza MB , Blaser SB , Zafar MA , Hammond AJ , Weiser JN . 2018. An Infant Mouse Model of Influenza Virus Transmission Demonstrates the Role of Virus-Specific Shedding, Humoral Immunity, and Sialidase Expression by Colonizing Streptococcus pneumoniae. mBio 9.","DOI":"10.1128\/mBio.02359-18"},{"key":"2026052300250707000_2026.05.19.726214v1.24","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.00416-16"},{"key":"2026052300250707000_2026.05.19.726214v1.25","doi-asserted-by":"publisher","DOI":"10.1172\/JCI57762"},{"key":"2026052300250707000_2026.05.19.726214v1.26","doi-asserted-by":"publisher","DOI":"10.1126\/science.1061217"},{"key":"2026052300250707000_2026.05.19.726214v1.27","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2164-13-734"},{"key":"2026052300250707000_2026.05.19.726214v1.28","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2010428117"},{"key":"2026052300250707000_2026.05.19.726214v1.29","doi-asserted-by":"crossref","first-page":"e0082323","DOI":"10.1128\/spectrum.00823-23","article-title":"In Vivo Genome-Wide Gene Expression Profiling Reveals That Haemophilus influenzae Purine Synthesis Pathway Benefits Its Infectivity within the Airways","volume":"11","year":"2023","journal-title":"Microbiol Spectr"},{"key":"2026052300250707000_2026.05.19.726214v1.30","doi-asserted-by":"crossref","unstructured":"Zafar MA , Hammond AJ , Hamaguchi S , Wu WS , Kono M , Zhao LL , Weiser JN . 2019. Identification of Pneumococcal Factors Affecting Pneumococcal Shedding Shows that the dlt Locus Promotes Inflammation and Transmission. Mbio 10.","DOI":"10.1128\/mBio.01032-19"},{"key":"2026052300250707000_2026.05.19.726214v1.31","doi-asserted-by":"crossref","unstructured":"Zafar MA , Hamaguchi S , Zangari T , Cammer M , Weiser JN . 2017. Capsule Type and Amount Affect Shedding and Transmission of Streptococcus pneumoniae. mBio 8.","DOI":"10.1128\/mBio.00989-17"},{"key":"2026052300250707000_2026.05.19.726214v1.32","doi-asserted-by":"crossref","first-page":"e0209688","DOI":"10.1371\/journal.pone.0209688","article-title":"Deletion of genes involved in the ketogluconate metabolism, Entner-Doudoroff pathway, and glucose dehydrogenase increase local and invasive virulence phenotypes in Streptococcus pneumoniae","volume":"14","year":"2019","journal-title":"PLoS One"},{"key":"2026052300250707000_2026.05.19.726214v1.33","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1008931"},{"key":"2026052300250707000_2026.05.19.726214v1.34","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1038\/s41541-021-00413-5","article-title":"Pneumococcal capsule blocks protection by immunization with conserved surface proteins","volume":"6","year":"2021","journal-title":"NPJ Vaccines"},{"key":"2026052300250707000_2026.05.19.726214v1.35","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.01775-05"},{"key":"2026052300250707000_2026.05.19.726214v1.36","doi-asserted-by":"crossref","first-page":"e1013562","DOI":"10.1371\/journal.ppat.1013562","article-title":"The serine protease HtrA regulates Group B Streptococcus virulence and affects the host response to infection","volume":"21","year":"2025","journal-title":"PLoS Pathog"},{"key":"2026052300250707000_2026.05.19.726214v1.37","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.72.3.1618-1625.2004"},{"key":"2026052300250707000_2026.05.19.726214v1.38","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.72.6.3584-3591.2004"},{"key":"2026052300250707000_2026.05.19.726214v1.39","doi-asserted-by":"crossref","first-page":"1270848","DOI":"10.3389\/fcimb.2023.1270848","article-title":"Pilus of Streptococcus pneumoniae: structure, function and vaccine potential","volume":"13","year":"2023","journal-title":"Front Cell Infect Microbiol"},{"key":"2026052300250707000_2026.05.19.726214v1.40","doi-asserted-by":"crossref","first-page":"e0305023","DOI":"10.1128\/spectrum.03050-23","article-title":"Time-resolved RNA-seq analysis to unravel the in vivo competence induction by Streptococcus pneumoniae during pneumonia-derived sepsis","volume":"12","year":"2024","journal-title":"Microbiol Spectr"},{"key":"2026052300250707000_2026.05.19.726214v1.41","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-016-1054-5"},{"key":"2026052300250707000_2026.05.19.726214v1.42","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.05756-11"},{"key":"2026052300250707000_2026.05.19.726214v1.43","doi-asserted-by":"crossref","first-page":"578533","DOI":"10.3389\/fmicb.2020.578533","article-title":"SP_0916 Is an Arginine Decarboxylase That Catalyzes the Synthesis of Agmatine, Which Is Critical for Capsule Biosynthesis in Streptococcus pneumoniae","volume":"11","year":"2020","journal-title":"Front Microbiol"},{"key":"2026052300250707000_2026.05.19.726214v1.44","doi-asserted-by":"crossref","first-page":"1060583","DOI":"10.3389\/fmicb.2022.1060583","article-title":"Global transcriptional responses of pneumococcus to human blood components and cerebrospinal fluid","volume":"13","year":"2022","journal-title":"Front Microbiol"},{"key":"2026052300250707000_2026.05.19.726214v1.45","doi-asserted-by":"crossref","unstructured":"Ritchie ND , Evans TJ . 2019. Dual RNA-seq in Streptococcus pneumoniae Infection Reveals Compartmentalized Neutrophil Responses in Lung and Pleural Space. mSystems 4.","DOI":"10.1128\/mSystems.00216-19"},{"key":"2026052300250707000_2026.05.19.726214v1.46","doi-asserted-by":"crossref","unstructured":"Minhas V , Aprianto R , McAllister LJ , Wang H , David SC , McLean KT , Comerford I , McColl SR , Paton JC , Veening JW , Trappetti C. 2020. In vivo dual RNA-seq reveals that neutrophil recruitment underlies differential tissue tropism of Streptococcus pneumoniae. Communications Biology 3.","DOI":"10.1038\/s42003-020-1018-x"},{"key":"2026052300250707000_2026.05.19.726214v1.47","doi-asserted-by":"crossref","unstructured":"Bath J , Bjanes E , Goekeri C , Hsiao J , Uzun D , Nouailles G , Nizet V , Ribbeck K. 2024. Mucins protect against Streptococcus pneumoniae virulence by suppressing pneumolysin expression. J Clin Invest 134.","DOI":"10.1172\/JCI182769"},{"key":"2026052300250707000_2026.05.19.726214v1.48","doi-asserted-by":"crossref","first-page":"e0104823","DOI":"10.1128\/msystems.01048-23","article-title":"The host transcriptional response to superinfection by influenza A virus and Streptococcus pneumoniae","volume":"9","year":"2024","journal-title":"mSystems"},{"key":"2026052300250707000_2026.05.19.726214v1.49","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.00721-09"},{"key":"2026052300250707000_2026.05.19.726214v1.50","doi-asserted-by":"crossref","unstructured":"Kilian M , Tettelin H. 2019. Identification of Virulence-Associated Properties by Comparative Genome Analysis of Streptococcus pneumoniae, S. pseudopneumoniae, S. mitis, Three S. oralis Subspecies, and S. infantis. mBio 10.","DOI":"10.1128\/mBio.02520-19"},{"key":"2026052300250707000_2026.05.19.726214v1.51","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0033320"},{"key":"2026052300250707000_2026.05.19.726214v1.52","doi-asserted-by":"publisher","DOI":"10.1021\/bi300762p"},{"key":"2026052300250707000_2026.05.19.726214v1.53","doi-asserted-by":"crossref","unstructured":"Amblar M , Zaballos A , de la Campa AG . 2022. Role of PatAB Transporter in Efflux of Levofloxacin in Streptococcus pneumoniae. Antibiotics (Basel) 11.","DOI":"10.3390\/antibiotics11121837"},{"key":"2026052300250707000_2026.05.19.726214v1.54","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0511017103"},{"key":"2026052300250707000_2026.05.19.726214v1.55","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M112.398875"},{"key":"2026052300250707000_2026.05.19.726214v1.56","doi-asserted-by":"crossref","first-page":"151449","DOI":"10.1016\/j.ijmm.2020.151449","article-title":"Pilus islets and the clonal spread of piliated Streptococcus pneumoniae: A review","volume":"310","year":"2020","journal-title":"Int J Med Microbiol"},{"key":"2026052300250707000_2026.05.19.726214v1.57","doi-asserted-by":"publisher","DOI":"10.1086\/656333"},{"key":"2026052300250707000_2026.05.19.726214v1.58","doi-asserted-by":"crossref","first-page":"599","DOI":"10.1016\/j.mex.2018.05.011","article-title":"Optimization of phenol-chloroform RNA extraction","volume":"5","year":"2018","journal-title":"MethodsX"},{"key":"2026052300250707000_2026.05.19.726214v1.59","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/bts635"},{"key":"2026052300250707000_2026.05.19.726214v1.60","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btt656"},{"key":"2026052300250707000_2026.05.19.726214v1.61","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-014-0550-8"},{"key":"2026052300250707000_2026.05.19.726214v1.62","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.00224-15"},{"key":"2026052300250707000_2026.05.19.726214v1.63","doi-asserted-by":"crossref","unstructured":"Siegel SJ , Tamashiro E , Weiser JN . 2015. Clearance of Pneumococcal Colonization in Infants Is Delayed through Altered Macrophage Trafficking. Plos Pathogens 11.","DOI":"10.1371\/journal.ppat.1005004"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.05.19.726214","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,23]],"date-time":"2026-05-23T07:25:16Z","timestamp":1779521116000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.05.19.726214"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,19]]},"references-count":63,"URL":"https:\/\/doi.org\/10.64898\/2026.05.19.726214","relation":{},"subject":[],"published":{"date-parts":[[2026,5,19]]},"subtype":"preprint"}}