Aeromonas veronii biotype sobria serine protease activates aerolysin and cleaves EpCAM to breach the epithelial barrier.

Infections by Aeromonas can rapidly progress from mucosal colonization to invasive disease, but how these bacteria efficiently breach epithelial barriers is unclear. Here, we define a coordinated two-component virulence strategy in Aeromonas veronii biotype sobria (A. veronii sobria) involving a secreted serine protease (ASP) and aerolysin. In polarized T84 monolayers, wild-type infection caused rapid transepithelial electrical resistance (TEER) loss and degradation of junctional/adhesion proteins. An ASP-deficient strain exhibited delayed barrier collapse with reduced cleavage of ZO-1/2/3 and EpCAM, and purified ASP restored these effects, indicating ASP is required but alone insufficient for full disruption. A transposon screen identified aerolysin and secretion-related genes as key contributors to junctional damage. Mechanistically, ASP directly converted recombinant pro-aerolysin into a hemolytically active toxin in vitro, linking protease activity to toxin maturation. Functionally, ASP and aerolysin synergized to promote bacterial translocation across epithelial monolayers with marked sequence dependence; pro-aerolysin pretreatment enhanced ASP-mediated degradation of ZO proteins, claudin-7, and EpCAM. ASP further cleaved EpCAM at multiple extracellular sites, compromising adhesion and junctional homeostasis. Together, these findings establish an ASP-aerolysin pathway that couples toxin activation with junctional proteolysis to drive structural epithelial breaches.IMPORTANCEAeromonas infections can rapidly progress from intestinal colonization to invasive disease, yet the molecular steps enabling epithelial breach are unclear. We show that Aeromonas veronii sobria uses a coordinated two-factor pathway in which Aeromonas serine protease (ASP) both activates the pore-forming toxin aerolysin and directly cleaves key junctional/adhesion proteins, including ZO-1/2/3 and EpCAM. Aerolysin primes cells for enhanced ASP-mediated proteolysis in an order-dependent manner, converting TEER loss into a physical barrier rupture that permits massive bacterial translocation. Targeting ASP-aerolysin cooperation may provide a rational anti-virulence strategy.