D-alanine aminotransferase (Dat) promotes Staphylococcus aureus colonization fitness on human nasal respiratory epithelium.

Nasal colonization by Staphylococcus aureus 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 S. aureus: alanine racemase 1 (alr1) and D-alanine aminotransferase (dat). Disruption of dat reduced colonization fitness in competition with the parental strain by ≥ 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 dat::Tn mutant was 34-fold less fit than an alr1::Tn mutant. Genetic complementation with single-copy dat expressed from its native operon promoter restored parental colonization levels. Supplementation with exogenous D-alanine or L-alanine also rescued the dat::Tn colonization defect, whereas D-glutamate did not, consistent with Dat primarily producing D-alanine on nasal mucosa. Complementation with dat under control of a putative 5' 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 dat transcription. Together, these findings demonstrate that Dat is a major source of D-alanine during colonization of human nasal mucosa and is required for S. aureus fitness in this environment.