Vancomycin-stressed Staphylococcus aureus extracellular vesicles hijack macrophage autophagy to enhance bacterial...

Methicillin-resistant Staphylococcus aureus (MRSA) is a major multidrug-resistant human pathogen. Vancomycin has long been the cornerstone therapy for invasive MRSA infections. Although nearly all MRSA isolates are susceptible to vancomycin in standard antimicrobial susceptibility testing, vancomycin treatment failure in MRSA bloodstream infections approaches 30%, and the underlying mechanisms remain poorly defined. Here, we show that subinhibitory concentrations of vancomycin promote MRSA survival within macrophages by hijacking host macroautophagy/autophagy. Using in vitro and in vivo models, we demonstrate that vancomycin exacerbates S. aureus-induced autophagic flux blockade, facilitating bacterial persistence. Mechanistically, this effect is not caused directly by vancomycin but by extracellular vesicles (EVs) secreted by S. aureus under conditions of vancomycin stress. These EVs exhibit a potent capacity to disrupt autophagosome-lysosome fusion, leading to the accumulation of autophagosomes. Proteomic profiling revealed a significant enrichment of the toxin α-hemolysin (Hla) in EVs derived from vancomycin-stressed bacteria. Deletion of the hla gene in S. aureus substantially attenuates the autophagy-disrupting capacity of vancomycin-induced EVs. Furthermore, recombinant α-hemolysin directly impaired autophagic flux in macrophages. RNA-sequencing and mechanistic analyses identified the PI3K-AKT pathway as a key signaling axis downstream of Hla, and this mechanism was further confirmed to mediate the autophagic degradation blockade induced by EVs from vancomycin-stressed S. aureus. Collectively, vancomycin triggers S. aureus to secrete EVs enriched with α-hemolysin, which potently inhibit autophagosome-lysosome fusion and promote intracellular MRSA survival. These findings provide critical insights into vancomycin treatment failure and identify bacterial EVs and α-hemolysin as potential therapeutic targets.Abbreviations: AKT/protein kinase B: AKT serine/threonine kinase; CFU: colony-forming units; EVs: extracellular vesicles; GFP: green fluorescent protein; MAP1LC3B-II/LC3B-II: microtubule associated protein 1 light chain 3 beta-II; MOI: multiplicity of infection; MRSA: methicillin-resistantStaphylococcus aureus; p-AKT: phosphorylated AKT; p-PI3K: phosphorylated phosphoinositide 3-kinase; PI3K: phosphoinositide 3-kinase;S. aureus:Staphylococcus aureus; SQSTM1/p62: sequestosome 1; VAN: vancomycin.