Membrane vesicles derived from Streptococcus suis serotype 2 induce cell pyroptosis in endothelial cells via the NLRP3/Caspase-1/ GSDMD pathway

Streptococcus suis serotype 2 ( S . suis 2) is a zoonotic pathogen that clinically causes severe swine and human infections (such as meningitis, endocarditis, and septicemia). In order to cause widespread diseases in different organs, S . suis 2 must colonize the host, break the blood barrier, and cause exaggerated inflammation. In the last few years, most studies have focused on a single virulence factor and its influences on the host. Membrane vesicles (MVs) can be actively secreted into the extracellular environment contributing to bacteria -host interactions. Gramnegative bacteria -derived outer membrane vesicles (OMVs) were recently shown to activate host Caspase-11mediated non -canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide (LPS), causing host cell pyroptosis. However, little is known about the effect of the MVs from S . suis 2 (Gram-positive bacteria without LPS) on cell pyroptosis. Thus, we investigated the molecular mechanism by which S . suis 2 MVs participate in endothelial cell pyroptosis. In this study, we used proteomics, electron scanning microscopy, fluorescence microscope, Western blotting, and bioassays, to investigate the MVs secreted by S . suis 2. First, we demonstrated that S . suis 2 secreted MVs with an average diameter of 72.04 nm, and 200 proteins in MVs were identified. Then, we showed that MVs were transported to cells via mainly dynamin-dependent endocytosis. The S . suis 2 MVs activated NLRP3/Caspase-1/GSDMD canonical inflammasome signaling pathway, resulting in cell pyroptosis, but it did not activate the Caspase-4/-5 pathway. More importantly, endothelial cells produce large amounts of reactive oxygen species (ROS) and lost their mitochondrial membrane potential under induction by S . suis 2 MVs. The results in this study suggest for the first time that MVs from S . suis 2 were internalized by endothelial cells via mainly dynamin-dependent endocytosis and might promote NLRP3/Caspase-1/GSDMD pathway by mitochondrial damage, which produced mtDNA and ROS under induction, leading to the pyroptosis of endothelial cells.