Reduced Oxygen Condition Is Associated with Genome-Wide Expression Changes in Mastitis-Lineage Staphylococcus aureus During In Vitro Invasion into a Mammary Cell Line

Staphylococcus aureus (S. aureus) bovine mastitis is a significant public health issue. Despite enormous efforts, important gaps remain regarding host–microenvironmental factors. How intramammary reduced oxygen modulates S. aureus transcription in bovine mammary epithelial cells (MECs) remains unclear. We examined oxygen-associated transcriptional changes in a bovine-mammary adapted S. aureus clone following internalization into MECs and identified functional category enrichments under Normal-O₂ and Reduced-O₂ exposures. Bovine MAC-T monolayers were infected with a dominant bovine mastitis isolate under Normal-O₂ or Reduced-O₂ conditions. Triplicate infection experiments were performed for each oxygen condition. Each condition included matched non-reacted bacterial controls maintained under the same gas condition but without MAC-T exposure serving as the reference condition for expression calling. RNA was extracted and profiled using a high-throughput qRT-PCR platform covering genome-wide loci. Expression calls were mapped to curated BioQT roles and interpreted descriptively. Results indicated 211 loci were upregulated and 99 were downregulated under Normal-O₂ conditions, versus 53 upregulated and 35 downregulated under Reduced-O₂ conditions, relative to their non-reacted controls. Under Normal-O₂ conditions, regulated loci covered multiple functional roles, including cellular processes, transport/binding proteins, regulatory functions, and energy metabolism with downregulated loci enriched in transport/binding and cell-envelope categories. Under Reduced-O₂ conditions, upregulated loci were abundant in cellular process annotations dominated by pathogenesis/toxin-related functions, whereas downregulated loci were enriched in nucleotide biosynthetic and DNA/cell division categories. Thus, this reveals oxygen-associated shifts in the transcriptional response of intramammary S. aureus in MAC-T cells. Normal-O₂ conditions were associated with broader category representation, whereas Reduced-O₂ conditions yielded a narrower distribution enriched for selected toxin/pathogenesis- and iron/cation-associated annotations. These oxygen-linked transcriptional-shifts highlight candidate pathways for the intramammary adaptation of S. aureus, potential diagnostic markers, anti-virulence strategies, and targeted therapeutics.