{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,7]],"date-time":"2026-06-07T20:59:28Z","timestamp":1780865968319,"version":"3.54.1"},"posted":{"date-parts":[[2026,6,4]]},"group-title":"Microbiology","reference-count":113,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T00:00:00Z","timestamp":1780531200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,6,2]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Nasal colonization by\n                  <jats:italic>Staphylococcus aureus<\/jats:italic>\n                  is an established risk factor for invasive infection, yet bacterial determinants promoting fitness on human nasal mucosa remain incompletely defined. To identify genes required for early colonization of human nasal respiratory epithelium, we colonized human nasal epithelial organoids differentiated at air-liquid interface (HNO-ALI) with a high-density transposon (Tn) library of the methicillin-resistant USA300 strain LAC. TnSeq analysis identified 165 genes that met our threshold for candidate colonization fitness factors. Among these, genes involved in D-alanine biosynthesis and use were enriched, including two encoding the enzymes that separately synthesize D-alanine in\n                  <jats:italic>S. aureus<\/jats:italic>\n                  : alanine racemase 1 (\n                  <jats:italic>alr1)<\/jats:italic>\n                  and D-alanine aminotransferase (\n                  <jats:italic>dat)<\/jats:italic>\n                  . Disruption of\n                  <jats:italic>dat<\/jats:italic>\n                  reduced colonization fitness in competition with the parental strain by \u2265 1,000 fold across 4 different strains from clonal complexes 8, 5, and 30. In competition with the parental strain during HNO-ALI colonization, a\n                  <jats:italic>dat<\/jats:italic>\n                  ::Tn mutant was 34-fold less fit than an\n                  <jats:italic>alr1<\/jats:italic>\n                  ::Tn mutant. Genetic complementation with single-copy\n                  <jats:italic>dat<\/jats:italic>\n                  expressed from its native operon promoter restored parental colonization levels. Supplementation with exogenous D-alanine or L-alanine also rescued the\n                  <jats:italic>dat<\/jats:italic>\n                  ::Tn colonization defect, whereas D-glutamate did not, consistent with Dat primarily producing D-alanine on nasal mucosa. Complementation with\n                  <jats:italic>dat<\/jats:italic>\n                  under control of a putative 5\u2019 intra-operon promoter substantially restored colonization but failed to support growth in chemically defined medium lacking L-alanine, suggesting a new layer of environment-specific regulation of\n                  <jats:italic>dat<\/jats:italic>\n                  transcription. Together, these findings demonstrate that Dat is a major source of D-alanine during colonization of human nasal mucosa and is required for\n                  <jats:italic>S. aureus<\/jats:italic>\n                  fitness in this environment.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>AUTHOR SUMMARY<\/jats:title>\n                  <jats:p>\n                    <jats:italic>Staphylococcus aureus<\/jats:italic>\n                    is the second leading cause of death due to bacterial infection globally, and nasal colonization is a major risk factor for invasive disease. Using a physiologically relevant, host-derived model of human nasal respiratory epithelium (HNO-ALI) and TnSeq, we identified 165 candidate genes contributing to\n                    <jats:italic>S. aureus<\/jats:italic>\n                    fitness during nasal mucosal colonization. We found that D-alanine aminotransferase (Dat) is the predominant source of D-alanine during nasal colonization, whereas alanine racemase (Alr1) predominates in rich medium, revealing an environment-specific hierarchy of D-alanine biosynthesis. Disruption of\n                    <jats:italic>dat<\/jats:italic>\n                    caused a &gt; 1,000-fold defect in colonization in competition with the parental strain across multiple\n                    <jats:italic>S. aureus<\/jats:italic>\n                    clonal complexes, supporting a conserved role for\n                    <jats:italic>dat<\/jats:italic>\n                    in nasal colonization fitness. Additionally, we provide evidence that\n                    <jats:italic>dat<\/jats:italic>\n                    transcription from a previously cryptic promoter might be regulated by nasal mucosal conditions. Alr1 is proposed as an antimicrobial target in other bacterial pathogens; however, our data suggest that targeting Dat may be more effective for\n                    <jats:italic>S. aureus<\/jats:italic>\n                    nasal decolonization.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.06.01.729472","type":"posted-content","created":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T19:50:11Z","timestamp":1780602611000},"source":"Crossref","is-referenced-by-count":0,"title":["D-alanine aminotransferase (Dat) promotes\n                  <i>Staphylococcus aureus<\/i>\n                  colonization fitness on human nasal respiratory epithelium"],"prefix":"10.64898","author":[{"given":"Andrea I.","family":"Boyd","sequence":"first","affiliation":[{"name":"Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katia A.","family":"Quintanilla","sequence":"additional","affiliation":[{"name":"Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Isabel F.","family":"Escapa","sequence":"additional","affiliation":[{"name":"Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Millie A.","family":"Lewis","sequence":"additional","affiliation":[{"name":"Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leah A.","family":"Kafer","sequence":"additional","affiliation":[{"name":"Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xi-Lei","family":"Zeng","sequence":"additional","affiliation":[{"name":"Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah E.","family":"Blutt","sequence":"additional","affiliation":[{"name":"Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA"},{"name":"Alkek Center for Metagenomics and Microbiome Research, Baylor College of Medicine, Houston, Texas, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carolyn B.","family":"Ibberson","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Tennessee, Knoxville, TN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1542-1679","authenticated-orcid":false,"given":"Katherine P.","family":"Lemon","sequence":"additional","affiliation":[{"name":"Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA"},{"name":"Alkek Center for Metagenomics and Microbiome Research, Baylor College of Medicine, Houston, Texas, USA"},{"name":"Division of Infectious Diseases, Texas Children\u2019s Hospital and Department of Pediatrics Baylor College of Medicine, Houston, Texas, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026060713101831000_2026.06.01.729472v1.1","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(22)02185-7"},{"key":"2026060713101831000_2026.06.01.729472v1.2","doi-asserted-by":"publisher","DOI":"10.1056\/NEJM200101043440102"},{"key":"2026060713101831000_2026.06.01.729472v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(04)16897-9"},{"key":"2026060713101831000_2026.06.01.729472v1.4","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.30637"},{"key":"2026060713101831000_2026.06.01.729472v1.5","doi-asserted-by":"publisher","DOI":"10.1002\/14651858.CD006216.pub2"},{"key":"2026060713101831000_2026.06.01.729472v1.6","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa0808939"},{"key":"2026060713101831000_2026.06.01.729472v1.7","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa1207290"},{"key":"2026060713101831000_2026.06.01.729472v1.8","doi-asserted-by":"publisher","DOI":"10.1093\/cid\/civ901"},{"key":"2026060713101831000_2026.06.01.729472v1.9","doi-asserted-by":"publisher","DOI":"10.1084\/jem.20240002"},{"key":"2026060713101831000_2026.06.01.729472v1.10","unstructured":"WHO. 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