The causative agent of listeriosis, Listeria monocytogenes, is a frequent contaminant of fruits, vegetables, cheeses, and processed foods. In certain high risk groups, L. monocytogenes can cause potentially fatal infections. The bacterium can traverse the intestinal epithelial barrier and spread throughout the body via the bloodstream and lymphatic system to infect the liver, cross the blood-brain barrier, and in pregnant women, the placental barrier. L. monocytogenes is able to cross these biological barriers due to its ability to proliferate within certain phagocytic and nonphagocytic cells. Bacterial invasins, most notably internalin (InlA) and InlB, bind to receptors on the surface of nonphagocytic cells to induce endocytosis of the bacterium through a zipper-like mechanism. L. monocytogenes can then escape from the endocytic vacuole and enter the cytosol through the activity of the pore-forming toxin listeriolysin O (LLO). In the cytosol, the bacterium can replicate and disseminate to adjacent cells by cell-to-cell spreading.
LLO belongs to the cholesterol-dependent cytolysin (CDC) family of bacterial pore-forming toxins. These toxins are secreted as monomers, oligomerize on cholesterol-rich membranes, and form large transmembrane pores. In addition to mediating vacuolar escape, LLO stimulates signaling pathways in host cells. We hypothesized that the signaling activity of extracellular LLO affects the intracellular lifecycle of L. monocytogenes. Using quantitative immunofluorescence microscopy to assess the importance of LLO for bacterial association to and internalization into epithelial cells, we found that LLO is required for efficient L. monocytogenes internalization into hepatocytes, and is sufficient to induce internalization of noninvasive bacteria and polystyrene beads. Using novel LLO variants that bind to host cells but are unable to form the pore complex, we demonstrated that pore formation was required for LLO-mediated internalization.
The CDC streptolysin O is known to stimulate a Ca2+-dependent membrane repair response that involves endocytosis of the toxin from the perforated plasma membrane. LLO stimulates a Ca2+-dependent membrane repair response, but this response is insufficient to account for L. monocytogenes internalization. LLO-mediated internalization and membrane repair both require the influx of extracellular Ca2+, but differ in their requirement for F-actin and K+ efflux. Surprisingly, using ionophores to stimulate Ca2+ and K+ fluxes, as happens when the plasma membrane is perforated by LLO, was sufficient to induce internalization of large particles. LLO mediates bacterial internalization in all human hepatocytes we tested and in additional epithelial cell lines. However, LLO does not induce bacterial internalization in all epithelial cells that it perforates.
In light of this data, LLO emerges as a significant invasion factor expressed by L. monocytogenes, which is coexpressed with InlA and InlB during infection. With the same approaches used to assess the role of LLO, we determined the contribution of InlA and InlB to bacterial association, internalization, and intracellular viability in nonphagocytic cells that express the InlA and InlB receptors. We found significant variability in the involvement of each of these invasins in bacterial association, internalization, and intracellular viability, demonstrating that the activity of invasins is not governed solely by the presence or absence of their receptors.