Supplementary Material for: LL-37 Triggers Formation of <b><i>Streptococcus pyogenes</i></b> Extracellular Vesicle-Like Structures with Immune Stimulatory Properties

Reports have shown that the antimicrobial peptide LL-37 is abundantly expressed but has limited bactericidal effect in <i>Streptococcus pyogenes</i> infections. At sub-inhibitory concentrations, LL-37 has been reported to alter virulence gene expression. Here, we explored the interaction of <i>S. pyogenes</i> strains with LL-37, focusing on bacterial growth, cell surface alterations and pro-inflammatory responses. Bioscreen turbidity measurements of strain 5448 cultured in the presence or absence of LL-37 confirmed the poor antimicrobial effect, and revealed a significant increase in turbidity of bacterial cultures exposed to sub-inhibitory concentrations of LL-37. However, this was not linked to increased bacterial counts. Electron microscopy of LL-37-exposed bacteria revealed the presence of vesicle-like structures on the bacterial surface. The vesicles stained positive for LL-37 and were released from the bacterial surface. Concentrated supernatants enriched in these structures had a broader protein content, including several virulence factors, compared to supernatants from untreated bacteria. The supernatants from LL-37-exposed bacteria were pro-inflammatory and elicited resistin and myeloperoxidase release from neutrophils. This is the first report on <i>S. pyogenes</i> extracellular vesicle-like structures formed at the bacterial surface in response to LL-37. The associated increased pro-inflammatory activity further implicates LL-37 as a potential factor involved in <i>S. pyogenes</i> pathogenesis.