Many intracellular pathogens invade cells via endocytic organelles and have adapted to the drop in pH along the endocytic pathway. However, the strategy by which the host cell counteracts this pathogen adaptation remains unclear. βγ-CAT is an aerolysin-like pore-forming protein and trefoil factor complex in the frog Bombina maxima. We report here that βγ-CAT, as a host-secreted factor with an intrinsic channel-forming property, is the first example of a molecule that actively neutralizes the acidification of endocytic organelles to counteract Listeria monocytogenes infection. Immunodepletion

of endogenous βγ-CAT largely impaired the control of L. monocytogenes by frog cells. βγ-CAT elevates the pH of L. monocytogenes-containing vacuoles to limit the vacuole escape of L. monocytogenes to cytosol. Further, βγ-CAT promotes intracellular L. monocytogenes clearance via autophagy and by that the non-lytic expulsion of the bacteria from host cells. Finally, βγ-CAT attenuated the dissemination of L. monocytogenes in vivo. These findings reveal a novel host strategy and effectors that combat pathogen adaptation to acidic conditions along the endocytic pathway.