The Pore-Forming Toxin Listeriolysin O Mediates a Novel Entry Pathway of L. monocytogenes into Human Hepatocytes

Abstract

Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO) facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2). Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell.

Author Summary

Listeria monocytogenes is responsible for the severe foodborne disease listeriosis. During pathogenesis, invasion of nonphagocytic cells by L. monocytogenes is crucial for crossing the host epithelial barriers and colonization of multiple organs including the liver. In this study, we investigated the role of the pore-forming toxin listeriolysin O (LLO) in L. monocytogenes entry into human hepatocytes. LLO belongs to the largest family of bacterial pore-forming toxins called the cholesterol-dependent cytolysins and is a major virulence factor of L. monocytogenes. We observed that LLO is required for efficient entry of L. monocytogenes into hepatocytes and shed light on the molecular processes involved in this activity. Using different experimental approaches, we provide the first evidence that LLO is sufficient to induce bacterial internalization into host cells by a pore-dependent mechanism. LLO induces tyrosine kinase(s)-, dynamin-, and F-actin-dependent formation of an internalization vesicle. Similar to LLO, the pore-forming toxin pneumolysin regulates bacterial entry into host cells. Together, these findings indicate that host membrane perforation by a pore-forming toxin can be used as an invasion strategy by L. monocytogenes and raise the hypothesis that other bacteria may use a similar entry pathway.