Release of extracellular vesicles (EVs) is a common feature among eukaryotes, 
archaea, and bacteria. However, the biogenesis and downstream biological effects 
of EVs released from gram-positive bacteria remain poorly characterized. Here, 
we report that EVs purified from a community-associated methicillin-resistant 
Staphylococcus aureus strain were internalized into human macrophages in vitro 
and that this process was blocked by inhibition of the dynamin-dependent 
endocytic pathway. Human macrophages responded to S. aureus EVs by TLR2 
signaling and activation of NLRP3 inflammasomes through K+ efflux, leading to 
the recruitment of ASC and activation of caspase-1. Cleavage of pro-interleukin 
(IL)-1β, pro-IL-18, and gasdermin-D by activated caspase-1 resulted in the 
cellular release of the mature cytokines IL-1β and IL-18 and induction of 
pyroptosis. Consistent with this result, a dose-dependent cytokine response was 
detected in the extracellular fluids of mice challenged intraperitoneally with 
S. aureus EVs. Pore-forming toxins associated with S. aureus EVs were critical 
for NLRP3-dependent caspase-1 activation of human macrophages, but not for TLR2 
signaling. In contrast, EV-associated lipoproteins not only mediated TLR2 
signaling to initiate the priming step of NLRP3 activation but also modulated EV 
biogenesis and the toxin content of EVs, resulting in alterations in IL-1β, 
IL-18, and caspase-1 activity. Collectively, our study describes mechanisms by 
which S. aureus EVs induce inflammasome activation and reveals an unexpected 
role of staphylococcal lipoproteins in EV biogenesis. EVs may serve as a novel 
secretory pathway for S. aureus to transport protected cargo in a concentrated 
form to host cells during infections to modulate cellular functions.