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.

Entry of Listeria monocytogenes in Mammalian Epithelial Cells: An Updated View

“…Transient mitochondrial fragmentation, mitochondrial membrane potential loss, and drop in respiration and cellular ATP levels also correlate with the LLO-induced calcium fluxes and increased bacterial entry in HeLa cells, suggesting that the bioenergetic state of resting cells represents a barrier to L. monocytogenes invasion and the LLO-induced metabolic cell reprogramming promotes efficient bacterial internalization (Stavru et al 2011); the molecular mechanisms that link this reprogramming with phagocytosis are currently unknown. A direct role for LLO in promoting bacterial adhesion to epithelial cells has been proposed (Krawczyk-Balska and Bielecki 2005), and a recent study shows that LLO is sufficient to induce L. monocytogenes entry in HepG2 and HeLa cells in the absence of InA or InlB signaling (Vadia et al 2011). Indeed, L. innocua-expressing LLO or latex beads coated with LLO are internalized in a cholesterol-, dynamin-, tyrosine kinase-, and actin-dependent but clathrin-independent manner; interestingly, complete pore formation is critical because a LLO variant that binds to host cells but only assembles into a prepore complex fails to promote invasion.…”

“…Cite this article as Cold Spring Harb Perspect Med 2012;2:a010009 cytotoxin cooperates with InlA and InlB to potentiate efficient bacterial infection in a broad range of host cells (Vadia et al 2011).…”

The Pore-Forming Toxin Listeriolysin O Is Degraded by Neutrophil Metalloproteinase-8 and Fails To Mediate Listeria monocytogenes Intracellular Survival in Neutrophils

“…Host cell plasma membrane perforation by extracellular LLO likely plays an important role in the bacterial intracellular life cycle. As recently shown, plasma membrane perforation by extracellular LLO is sufficient to induce L. monocytogenes internalization into some epithelial cell lines (11). Extracellular LLO also controls posttranslational modifications, mitochondrial remodeling, and histone modifications during L. monocytogenes infection of epithelial cell lines (32)(33)(34).…”

“…Indeed, LLO-deficient L. monocytogenes strains remain trapped in the endocytic vesicle, are unable to divide intracellularly, and are nonvirulent in vivo (6,10). LLO mediates intracellular survival of L. monocytogenes in primary cells and immortalized cell lines from various animal species (1,(11)(12)(13). However, activated macrophages display increased resistance to infection by L. monocytogenes (14).…”

“…It is proposed that perturbation of phagosomal ionic concentrations (28), activation of host protein kinase C (29), and the cystic fibrosis transmembrane conductance regulator (30) are involved in regulating bacterial escape. A large body of evidence established that LLO is also a potent poreforming toxin when it is released in the extracellular compartment before bacterial internalization (11,31). Host cell plasma membrane perforation by extracellular LLO likely plays an important role in the bacterial intracellular life cycle.…”