Human neutrophil α-defensin HNP1 interacts with bacterial cell wall components to promote biofilm formation by Staphylococcus aureus

<i>Staphylococcus aureus</i> is a major opportunistic pathogen whose ability to form biofilms significantly enhances its persistence and antibiotic resistance. We previously found that human neutrophil peptide 1 (HNP1), a member of the α-defensin family, promotes biofilm formation in <i>Acinetobacter baumannii</i>. Here, we demonstrate that HNP1 enhances biofilm formation across a range of Gram-positive and Gram-negative bacteria, with particularly strong effects in <i>S. aureus</i>, which increases its tolerance to multiple antibiotics. Mechanistically, HNP1 promotes bacterial adhesion and self-aggregation, facilitating the early stages of biofilm development. Transcriptomic analysis revealed minimal activation of canonical biofilm-associated genes, suggesting that HNP1 acts through a non-transcriptional mechanism. Notably, HNP1 was found to interact with <i>S. aureus</i> cell wall components, peptidoglycan and wall teichoic acid, which mainly triggers biofilm formation. These findings expand the role of HNP1 in promoting biofilm formation, which may have implications for understanding infection persistence and antibiotic tolerance.