Opportunistic pathogen Vibrio vulnificus causes severe systemic
infection in humans with high mortality. Although multiple
exotoxins have been characterized in V. vulnificus, their interactions and potential synergistic roles in pathogen-induced host cell death have not been investigated previously. By employing a series of multiple
exotoxin deletion mutants, we investigated whether specific
exotoxins of the pathogen functioned together to achieve severe and rapid necrotic cell death. Human epithelial cells treated with V. vulnificus with a
plpA deletion background exhibited an unusually prolonged cell blebbing, suggesting the importance of
PlpA, a
phospholipase A2, in rapid necrotic cell death by this pathogen. Additional deletion of the rtxA gene encoding the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin did not result in necrotic cell
blebs. However, if the rtxA gene was engineered to produce an effector-free MARTX toxin, the cell blebbing was observed, indicating that the pore forming activity of the MARTX toxin is sufficient, but the MARTX toxin effector domains are not necessary, for the blebbing. When a recombinant
PlpA was treated on the blebbed cells, the
blebs were completely disrupted. Consistent with this, MARTX toxin-pendent rapid release of cytosolic
lactate dehydrogenase was significantly delayed in the
plpA deletion background. Mutations in other
exotoxins such as
elastase,
cytolysin/
hemolysin, and/or extracellular
metalloprotease did not affect the
bleb formation or disruption. Together, these findings indicate that the pore forming MARTX toxin and the
phospholipase A2,
PlpA, cooperate sequentially to achieve rapid necrotic cell death by inducing cell blebbing and disrupting the
blebs, respectively.