Extracellular vesicles (EVs) are central components of bacterial secretomes, including the small, cell wall-less Mollicutes. Although EV release in Mollicutes has been reported, EV proteomic composition and function have not been explored yet. We developed a protocol for isolating EVs of the pathogens Mycoplasma mycoides subsp. capri (Mmc) and Mycoplasma (Mycoplasmopsis) bovis and examined their functionality. Proteomic analysis demonstrated that EVs mirror the proteome of the EV-producing bacteria. EVs exhibited nuclease activity, effectively digesting both circular and linear DNA. Notably, M. bovis EVs elicited immune responses in bovine primary blood cells, like those induced by live M. bovis. Our findings reveal that EVs can carry plasmids and enable their horizontal transfer, known as vesiduction. Specifically, the natural plasmid pKMK1, with an unknown transmission route, was detected in EVs of Mmc 152/93 and the tetM-containing pIVB08 plasmid was associated with EVs released by an Mmc GM12 strain carrying this plasmid. pIVB08 could be transferred via homo- and heterologous vesiduction to Mmc, M. capricolum subsp. capricolum and M. leachii. Vesiduction was impeded by membrane disruption but resisted DNase and Proteinase K treatment, suggesting that EVs protect their cargo. These findings enhance our understanding of Mollicutes EVs, particularly in host interactions and horizontal gene transfer.
