Dual mode of delivery of <i>Mycoplasma pneumoniae</i> CARDS toxin determines the toxin trafficking pathway and cytotoxicity in host cells

<i>Mycoplasma pneumoniae</i> (<i>Mp</i>) is a significant human respiratory pathogen whose systemic effects are largely caused by the CARDS toxin. Our study provides new insights into the secretion and delivery of CARDS toxin. We demonstrate that <i>Mp</i> highly expresses and selectively releases CARDS toxin when grown in serum-free minimal media and during interaction with host cells. Importantly, <i>Mp</i> utilizes two distinct pathways to secrete CARDS toxin: extracellular vesicles (EVs) and a non-EV-mediated route. Compared to the free-soluble toxin, the vesicle-associated CARDS toxin resists proteolytic degradation, which likely enhances its ability to act on distant sites. Both forms of toxin exhibit varying degrees of cytotoxicity on human epithelial cells, highlighting their distinct roles in host-cell interactions. These findings reveal that <i>Mp</i> uses multiple strategies to propagate its virulence factors effectively. Understanding these delivery mechanisms provides potential new targets for developing therapeutic interventions to counteract <i>Mp</i> pathogenesis, particularly those mediated by CARDS toxin.