ABSTRACTListeria monocytogenesis a facultative intracellular bacterial pathogen that causes serious disease in immunocompromised individuals, pregnant women, and neonates. Bacterial virulence is mediated by the expression of specific gene products that facilitate entry into host cells and enable bacterial replication; the majority of these gene products are regulated by a transcriptional activator known as PrfA.L. monocytogenesstrains containingprfAE77K orprfAG155S mutations exhibit increased expression of virulence genes in broth culture and are hypervirulent in mice. To define the scope of the influences of theprfAE77K andprfAG155S mutations onL. monocytogenespathogenesis, multiple aspects of bacterial invasion and intracellular growth were examined. Enhanced bacterial invasion of host epithelial cells was dependent on the expression of a number of surface proteins previously associated with invasion, including InlA, InlB, and ActA. In addition to these surface proteins, increased production of thehly-encoded secreted hemolysin listeriolysin O (LLO) was also found to significantly enhance bacterial invasion into epithelial cell lines for bothprfAmutant strains. AlthoughprfAE77K andprfAG155S strains were similar in their invasive phenotypes, the infection of epithelial cells withprfAE77K strains resulted in host cell plasma membrane damage, whereasprfAG155S strains did not alter plasma membrane integrity. Bacterial infection of human epithelial cells, in which the production of LLO is not required for bacterial entry into the cytosol, indicated thatprfAE77K cytotoxic effects were mediated via LLO. BothprfAE77K andprfAG155S strains were more efficient than wild-type bacteria in gaining access to the host cell cytosol and in initiating the polymerization of host cell actin, and both were capable of mediating LLO-independent lysis of host cell vacuoles in cell lines for whichL. monocytogenesvacuole disruption normally requires LLO activity. These experiments illuminate the diverse facets ofL. monocytogenespathogenesis that are significantly enhanced by the constitutive activation of PrfA viaprfAmutations and underscore the critical role of this protein in promotingL. monocytogenesvirulence.