Dual Hyaluronidase Genes <i>hysA</i> and <i>hysA</i> ^νSaβ Enhance MRSA ST398 Skin Infection

Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) ST398 carries two hyaluronidase genes, <i>hysA</i> and its homologue <i>hysA</i>^νSaβ, the latter located on the genomic island νSaβ. However, the prevalence of <i>hysA</i>^νSaβ and its contribution to virulence remain unclear. Here, we report that the <i>hysA</i>^νSaβ gene is present in 18.3% (4707/25,752) of <i>S. aureus</i> in the NCBI database, with ST398 being the most prevalent sequence type (30.9%, 1457/4707). In ST398, the <i>hysA</i>^νSaβ gene is flanked by IS<i>21</i> and IS<i>3</i>, with >99.0% nucleotide identity across strains, suggesting horizontal acquisition. In a mouse skin infection model, a wild-type ST398 MRSA strain carrying both <i>hysA</i> and <i>hysA</i>^νSaβ formed significantly larger abscesses than isogenic mutants lacking one or both hyaluronidase genes. Wild-type infection led to a higher bacterial load and sustained induction of chemokines (CCL5, CXCL1, CCL4) and pro-inflammatory cytokines (IL-1β, IL-6, IL-33), resulting in prolonged neutrophil recruitment and severe inflammation. Consistently, <i>hysA</i> and <i>hysA</i>^νSaβ enhanced the survival of MRSA ST398 inside RAW 264.7 macrophages and neutrophils. <i>In vitro</i>, a double knockout strain (Δ<i>hysA</i>-Δ<i>hysA</i>^νSaβ) grew more slowly with hyaluronic acid (HA) as the sole carbon source, accompanied by intracellular accumulation of specific amino acids (proline, valine, threonine, and phenylalanine) and downregulation of amino acid biosynthesis pathways. Moreover, RAW 264.7 macrophages infected with Δ<i>hysA</i>-Δ<i>hysA</i>^νSaβ showed a marked upregulation of the oxidative phosphorylation (OXPHOS) pathway compared to uninfected controls, suggesting an enhanced cellular metabolic and inflammatory response that could improve bacterial clearance. Our findings highlight the functionally redundant roles of <i>hysA</i> and <i>hysA</i>^νSaβ in MRSA ST398 pathogenesis, suggesting that these hyaluronidases are potential targets for antistaphylococcal therapy.