Release of extracellular vesicles (EV) by Gram-negative and positive bacteria is being frequently reported. EV are nano-sized, membrane-derived, non-self-replicating, spherical structures shed into the extracellular environment that could play a role in bacteria-host interactions. Evidence of EV production in bacteria belonging to the class Mollicutes, which are wall-less, is mainly restricted to the genus Acholeplasma and is scanty for the Mycoplasma genus that comprises major human and animal pathogens. Here EV release by six Mycoplasma (sub)species of clinical importance was investigated. EV were obtained under nutritional stress conditions, purified by ultracentrifugation and observed by electron microscopy. The membrane proteins of EV from three different species were further identified by mass spectrometry as a preliminary approach to determining their potential role in host-pathogen interactions. EV were shown to be released by all six (sub)species although their quantities and sizes (30–220 nm) were very variable. EV purification was complicated by the minute size of viable mycoplasmal cells. The proteins of EV-membranes from three (sub)species included major components of host-pathogen interactions, suggesting that EV could contribute to make the host-pathogen interplay more complex. The process behind EV release has yet to be deciphered, although several observations demonstrated their active release from the plasma membrane of living cells. This work shed new light on old concepts of “elementary bodies” and “not-cell bound antigens”.

Rationale: Metabolic syndrome (MetS) is a cluster of interrelated risk factors for cardiovascular diseases and atherosclerosis. Circulating levels of large extracellular vesicles (lEVs), submicrometer-sized vesicles released from plasma membrane, from MetS patients were shown to induce endothelial dysfunction but their role in early stage of atherosclerosis and on vascular smooth muscle cells (SMC) remain to be fully elucidated. Objective: To determine the mechanisms by which lEVs lead to the progression of atherosclerosis in the setting of MetS. Methods and Results: Proteomic analysis revealed that the small GTPase, Rap1 was overexpressed in lEVs from MetS patients compared to those from non-MetS (nMetS) subjects. Rap1 was in GTP-associated active state in both types of lEVs and Rap1-lEVs levels correlated with increased cardiovascular risks including stenosis. MetS-lEVs, but not nMetS-lEVs, increased Rap1-dependent endothelial cell permeability. MetS-lEVs significantly promoted migration and proliferation of human aortic SMC and increased expression of pro-inflammatory molecules and activation of ERK5/p38 pathways. Neutralization of Rap1 by specific antibody or pharmacological inhibition of Rap1 with GGTi-298, completely prevented the effects of lEVs from MetS patients. HFD-fed ApoE -/- mice displayed an increased expression of Rap1 both in aortas and circulating lEVs. lEVs accumulated in plaque atherosclerotic lesions depending on the progression of atherosclerosis. lEVs from HFD-fed ApoE -/- mice, but not those from mice fed with a standard diet, enhanced SMC proliferation. Human atherosclerotic lesions were enriched in lEVs expressing Rap1. Conclusions: These data demonstrate that Rap1 carried by MetS-lEVs participates in the enhanced SMC proliferation, migration, pro-inflammatory profile and activation of ERK5/p38 pathways leading to vascular inflammation and remodeling, and atherosclerosis. These results highlight that Rap1 carried by MetS-lEVs may be a novel determinant of diagnostic value for cardiometabolic risk factors and suggest Rap1 as a promising therapeutic target against the development of atherosclerosis.