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

Mycoplasma pneumoniae (Mp) causes both pulmonary and extrapulmonary infections in humans, and its virulence is largely attributed to the community-acquired respiratory distress syndrome (CARDS) toxin. CARDS toxin is transiently expressed during Mp growth in the serum-rich SP4 medium, detected predominantly in the cytoplasm, with none secreted into the medium. Here, we show that CARDS toxin synthesis has increased at least fivefold during Mp growth in serum-free minimal media, with approximately 40% of the toxin being released into the extracellular environment. In infected human alveolar epithelial cells, the toxin is visualized both in association with Mp and in free form around the perinuclear region, confirming its secretion. Using density gradient centrifugation, transmission electron microscopy, and immunoblotting, we demonstrate that Mp secretes extracellular vesicles enriched with CARDS toxin. Approximately 35% of the secreted toxin is associated with vesicles, while the remainder exists in a free-soluble form. The vesicle-bound toxin shows increased resistance to proteolytic degradation compared to the free-soluble toxin. Both forms of the purified toxin are biologically active and induce varying degrees of cytotoxicity in human epithelial cells based on their distinct trafficking patterns, which highlights their distinct roles in host-cell interactions. Together, these findings reveal a dual mode of CARDS toxin secretion as a key feature of Mp pathogenicity, providing a significant benefit in toxin delivery and thereby enhancing virulence, expanding the scope of infection.