PH-DEPENDENT PERFORATION OF MACROPHAGE PHAGOSOMES BY LISTERIOLYSIN-O FROM LISTERIA-MONOCYTOGENES

The pore-forming toxin Listeriolysin O (LLO) is a major virulence fact or implicated in escape of Listeria monocytogenes from phagocytic vacu oles. Here we describe the pH-dependence of vacuolar perforation by LL O, using the membrane-impermeant fluorophore 8-hydroxypyrene-1,3,6-tri sulfonic acid (HPTS) to monitor the pH and integrity of vacuoles in mo use bone marrow-derived macrophages. Perforation was observed when aci dic vacuoles containing wild-type L. monocytogenes displayed sudden in creases in pH and release of HPTS into the cytosol. These changes were not seen with LLO-deficient mutants. Perforation occurred at acidic v acuolar pH (4.9-6.7) and was reduced in frequency or prevented complet ely when macrophages were treated with the lysosomotropic agents ammon ium chloride or bafilomycin A(1). We conclude that acidic pH facilitat es LLO activity in vivo.