Dual Hyaluronidase Genes hysA and hysA^νSaβ Enhance MRSA ST398 Skin Infection

Methicillin-resistant Staphylococcus aureus (MRSA) ST398 carries two hyaluronidase genes, hysA and its homologue hysA^νSaβ, the latter located on the genomic island νSaβ. However, the prevalence of hysA^νSaβ and its contribution to virulence remain unclear. Here, we report that the hysA^νSaβ gene is present in 18.3% (4707/25,752) of S. aureus in the NCBI database, with ST398 being the most prevalent sequence type (30.9%, 1457/4707). In ST398, the hysA^νSaβ gene is flanked by IS21 and IS3, with >99.0% nucleotide identity across strains, suggesting horizontal acquisition. In a mouse skin infection model, a wild-type ST398 MRSA strain carrying both hysA and hysA^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, hysA and hysA^νSaβ enhanced the survival of MRSA ST398 inside RAW 264.7 macrophages and neutrophils. In vitro, a double knockout strain (ΔhysA-ΔhysA^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 ΔhysA-ΔhysA^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 hysA and hysA^νSaβ in MRSA ST398 pathogenesis, suggesting that these hyaluronidases are potential targets for antistaphylococcal therapy.