A Novel Role of Listeria monocytogenes Membrane Vesicles in Inhibition of Autophagy and Cell Death

Abstract

Bacterial membrane vesicle (MV) production has been mainly studied in Gram-negative species. In this study, we show that Listeria monocytogenes, a Gram-positive pathogen that causes the food-borne illness listeriosis, produces MVs both in vitro and in vivo. We found that a major virulence factor, the pore-forming hemolysin listeriolysin O (LLO), is tightly associated with the MVs, where it resides in an oxidized, inactive state. Previous studies have shown that LLO may induce cell death and autophagy. To monitor possible effects of LLO and MVs on autophagy, we performed assays for LC3 lipidation and LDH sequestration as well as analysis by confocal microscopy of HEK293 cells expressing GFP-LC3. The results revealed that MVs alone did not affect autophagy whereas they effectively abrogated autophagy induced by pure LLO or by another pore-forming toxin from Vibrio cholerae, VCC. Moreover, Listeria monocytogenes MVs significantly decreased Torin1-stimulated macroautophagy. In addition, MVs protected against necrosis of HEK293 cells caused by the lytic action of LLO. We explored the mechanisms of LLO-induced autophagy and cell death and demonstrated that the protective effect of MVs involves an inhibition of LLO-induced pore formation resulting in inhibition of autophagy and the lytic action on eukaryotic cells. Further, we determined that these MVs help bacteria to survive inside eukaryotic cells (mouse embryonic fibroblasts). Taken together, these findings suggest that intracellular release of MVs from L. monocytogenes may represent a bacterial strategy to survive inside host cells, by its control of LLO activity and by avoidance of destruction from the autophagy system during infection.

Listeria monocytogenesvirulence factors are secreted in biologically active Extracellular Vesicles

“…EVs carry significant variety of toxins for the parent bacterium, in particular LLO (17,31). We note prior studies that recovered LLO from culture supernatant through protein purification would have included the vesicular fraction and therefore those results are compatible with our findings (44).…”

“…There have been two recent reports of EVs in L. monocytogenes (17,31). In this study, we confirmed these findings, extended the observation that EVs are cytotoxic to murine macrophage-like cells, characterized the secreted EVs using simultaneous metabolite, protein, and lipid extraction (MPLEx) multi-omics approach (32,33), and evidence that EVs are secreted by intracellular bacteria into the cytosol of mammalian cells using electron microscopy and high-resolution fluorescence imaging.…”

“…We provide evidence against their formation as by-products of cell autolysis by showing major differences in lipid and protein composition from parent bacteria and imaging their release inside infected mammalian cells. These findings together with the two reports (17,31) provide compelling evidence for the notion that L. monocytogenes releases EV packed with virulence factors.…”

“…and Lactobacillus spp. (40), Bacillus subtilis (23), Pneumococcus pneumoniae (4), Streptomyces lividans (41), Clostridium perfringens (20) and Listeria monocytogenes (17,31). One advantage for toxin packaging in EVs is that it allows their delivery as a concentrated warhead that is not diluted as a function of the square of the distance from the bacterial surface.…”

“…Another intriguing aspect is whether exogenously added EVs exert their toxicity by fusing with host plasma membrane and release of contents into the host or instead EVs are first ingested by the host and trafficking in the endocytic/phagocytic compartments precedes release of toxins (31). In other bacterial pathogens (S. aureus and Legionella pneumophila), EVs were found to fuse with host cells (48) but it has been suggested that EVs from L. monocytogenes are internalized in endosomes (31). In the absence of a detailed study we consider it is possible that EVs from L. monocytogenes may simply be uptaken by endocytosis, or, given the invasive arsenal of L. monocytogenes, equally probable that EVs induce ingestion by host cells, either via internalin (InlA and InlB) interactions with lipid microdomains at the cell surface (49), or by LLO pore-triggered entry mechanisms analogous to what has been observed for L. monocytogenes cells invading human hepatocytes (9).…”