Molecular aspects of Listeria monocytogenes infection (original) (raw)
Microbiology, 2003
Listeriolysin O (LLO, hly-encoded), a major virulence factor secreted by the bacterial pathogen Listeria monocytogenes, is synthesized as a precursor of 529 residues. To impair LLO secretion, the four residues of the predicted signal sequence cleavage site (EA-KD) were deleted and the mutant LLO protein was expressed in a hly-negative derivative of L. monocytogenes. Unexpectedly, the mutant protein was secreted in normal amounts in the culture supernatant and was fully haemolytic. N-terminal sequencing of the secreted LLO molecule revealed that N-terminal processing of the preprotein occurred three residues downstream of the natural cleavage site. L. monocytogenes expressing this truncated LLO showed a reduced capacity to disrupt the phagosomal membranes of bone marrow macrophages and of hepatocytes; and the mutant strain showed a 100-fold decrease in virulence in the mouse model. These results suggest that the first N-terminal residues of mature LLO participate directly in phagosom...
Infection and immunity, 1999
Listeria monocytogenes secretes several proteins that have been shown to contribute to virulence. Among these is listeriolysin O (LLO), a pore-forming hemolysin that is absolutely required for virulence. Two other virulence factors are phospholipases: a phosphatidylinositol-specific phospholipase C (PI-PLC [plcA]) and a broad-range PLC (plcB). Although mutations in plcA or plcB resulted in small increases in mouse 50% lethal dose (LD50), deletions in both genes resulted in a 500-fold increase in LD50. We have examined the role of these secreted proteins in host intracellular signaling in the J774 macrophage-like cell line. Measurements of cytosolic free calcium ([Ca2+]i) have revealed a rapid spike upon exposure of these cells to wild-type L. monocytogenes. This is followed by a second peak at 5 min and a third prolonged peak with a maximal [Ca2+]i of 800 to 1,000 nM. The pattern of calcium changes was greatly altered by deletion of any of the three virulence factors. An LLO mutant ...
Journal of Biological Chemistry, 2018
Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secrete extracellular vesicles (EVs) was not pursued until recently due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following their discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteria are implicated in virulence, toxin release, and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that causes listeriosis. Here we report that L. monocytogenes produces EVs with diameters ranging from 20 to 200 nm, containing the pore-forming toxin listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Cell-free EV preparations were toxic to mammalian cells, the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC reduced LLO activity. Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multiomics we characterized protein, lipid, and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Using immunogold EM we detected LLO at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes during infection. Our findings demonstrate that L. monocytogenes uses EVs for toxin release and implicate these structures in mammalian cytotoxicity. The pathogenic Gram-positive bacterium Listeria monocytogenes is the etiological agent of listeriosis, a disease with serious consequences for pregnant women, newborns, and immunocompromised persons. Healthy individuals who have ingested large amounts of L. monocytogenes can suffer from gastroenteritis when the bacterium passes through the gastrointestinal barrier (1-4). L. monocytogenes can cause spontaneous abortions in pregnant women and meningoencephalitis by crossing the placental and blood-brain barriers, respectively (5). To invade cells, cross these barriers, and evade the immune system, L. monocytogenes has a sophisticated intracellular life cycle and pathogenic strategy (6, 7). Initially, L. monocytogenes invades various cell types, including nonphagocytic cells, by utilizing two internalins, internalin A (InlA) and internalin B (InlB), with a minor contribution by the pore-forming toxin listeriolysin O (LLO), 7 to induce uptake of the bacterium (1, 5, 8-11). Once internalized in the host vacuole, L. monocytogenes employs LLO, phosphatidylcholinespecific phospholipase (PC-PLC), and phosphatidylinositolspecific phospholipase C (PI-PLC) to disrupt the single vacuolar membrane, releasing the bacterium into the cytoplasm The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains Movie S1, Tables S1-S4, and Figs. S1-S4. Multiomics data were deposited in the MassIVE repository under accession numbers MSV000081402, MSV000081403, and MSV000081404.
Listeriolysin O: a key protein ofListeria monocytogeneswith multiple functions
FEMS Microbiology Reviews, 2006
Cholesterol-dependent cytolysins (CDCs) are produced by a large number of pathogenic Gram-positive bacteria. Most of these single-chain proteins are secreted in the extracellular medium. Among the species producing CDCs, only two species belonging to the genus Listeria (Listeria monocytogenes and Listeria ivanovii) are able to multiply intracellularly and release their toxins in the phagosomal compartment of the infected host cell. This review provides an updated overview on the importance of listeriolysin O (LLO) in the pathogenicity of L. monocytogenes, focusing mainly on two aspects: (1) the structure-function relationship of LLO and (2) its role in intra-and extracellular signalling. We first examine the specific sequence determinants, or protein domains, that make this cytolysin so well adapted to the intracellular lifestyle of L. monocytogenes. The roles that LLO has in cellular signalling events in the context of relations to pathogenesis are also discussed.
Surface proteins and the pathogenic potential of Listeria monocytogenes
Trends in Microbiology, 2002
Listeria monocytogenes is a ubiquitous Grampositive, rod-shaped, non-capsulated, nonsporulating, facultative intracellular bacterium that causes severe food-borne infections in humans and animals. Human listeriosis is characterized by a high mortality rate (>20%), and immunosuppressed individuals, pregnant women, foetuses and neonates are most susceptible to Listeria infections. Listeria disseminates from the intestine to the blood, the inner organs and eventually to the brain and foetoplacental unit after crossing the intestinal, the blood-brain and the foeto-placental barriers. In the infected host, Listeria is mostly intracellular owing to its capacity to survive within phagocytes and induce its phagocytosis by non-phagocytic cells.
Activation of Host Phospholipases C and D in Macrophages after Infection with Listeria monocytogenes
Infection and Immunity, 2000
Infection of the J774 murine macrophage-derived cell line with Listeria monocytogenes results in several elevations of intracellular calcium during the first 15 min of infection. These appear to result from the actions of secreted bacterial proteins, including phosphatidylinositol-specific phospholipase C (PI-PLC), a broadrange phospholipase C, and listeriolysin O (LLO) (S. J. Wadsworth and H. Goldfine, Infect. Immun. 67:1770-1778, 1999). We have measured hydrolysis of host PI and the activation of host polyphosphoinositide-specific PLC and host phospholipase D (PLD) during infection with wild-type and mutant L. monocytogenes. Elevated hydrolysis of host PI occurred within the first 10 min of infection and was dependent on both bacterial PI-PLC and LLO, both of which were required for the earliest elevations of intracellular calcium in the host cell. A more rapid hydrolysis of host PI was observed at 30 min after infection, at the time when wild-type bacteria have been internalized. Activation of host PLC, also occurred in the first 10 min of infection but was not dependent on the presence of bacterial PI-PLC. Similar observations were made in murine bone marrowderived macrophages. In J774 cells, activation of host PLD was observed after 20 min of infection and was dependent on bacterial LLO. Mutants in the bacterial phospholipases produced levels of PLD activation similar to those produced by the wild type. Phorbol myristate acetate (PMA) also activated host PLD, while long-term treatment with PMA resulted in loss of the ability of L. monocytogenes to activate host PLD, suggesting an involvement of protein kinase C (PKC) in the activation of PLD. Rottlerin, an inhibitor of PKC ␦ in J774 cells, also inhibited the activation of PLD, but hispidin, an inhibitor of PKC I and II, did not. Pretreatment of J774 cells with the PLD inhibitor, 2,3-diphosphoglycerate partially inhibited escape of the bacteria from the primary phagocytic vacuole.
Biochemistry, 2009
Listeria monocytogenes phosphatidylinositol-specific phospholipase C (PI-PLC) plays a critical role in escape of this human pathogen from host cell vacuoles. Unlike classical bacterial PI-PLCs, the L. monocytogenes enzyme has very weak activity on glycosylphosphatidylinositol (GPI)-anchored proteins. Previous crystal structure analysis has revealed that a small -strand (Vb) is present in Bacillus cereus PI-PLC and is absent in the enzyme from L. monocytogenes. This Vb -strand in B. cereus PI-PLC forms contacts with the glycan linker of GPI anchors, which presumably increases its activity on GPI-anchored proteins. In this study, we show that, of all known bacterial PI-PLCs, those from listeriae are the only ones that lack the -strand. Expression by L. monocytogenes of B. cereus PI-PLC, which has strong activity on GPI-anchored proteins, inhibited bacterial escape from a vacuole and cell-to-cell spread, resulting in greatly reduced virulence in mice. Deletion of the Vb -strand from B. cereus PI-PLC abolished its ability to cleave GPI-anchored proteins, decreased its inhibitory effects, and increased its virulence in mice. These results strongly suggest that L. monocytogenes PI-PLC has evolved as an important determinant of L. monocytogenes pathogenesis by absence of the Vb -strand, thus leading to greatly reduced activity on GPI-anchored proteins.
Infection and Immunity, 1990
We identified nonhemolytic mutants of Listeria monocytogenes that were severely deficient in their ability to invade mammalian nonprofessional phagocytes. These mutants were generated spontaneously or by means of transposon Tn916 mutagenesis. In terms of their extracellular proteins, the noninvasive mutants were deficient not only in the sulfhydryl-activated hemolysin (listeriolysin) but also in an antigenically unrelated extracellular protein with an apparent molecular weight of 32,000 which could induce opacity in egg yolk and is considered to be a phospholipase. Our results suggest the existence of a common genetic control between the expression of listeriolysin and that of other determinants, including a phospholipase and determinants involved in the ability of L. monocytogenes to enter mammalian cells.
Optimization of Listeriolysin O Production by Listeria Monocytogenes
Journal of Food Safety, 1994
Listeria monocytogenes is a pathogenic bacterium that invades epithelial cells by activating host signaling cascades, which promote bacterial engulfment within a phagosome. The pore-forming toxin listeriolysin O (LLO), which is required for bacteria phagosomal escape, has also been associated with the activation of several signaling pathways when secreted by extracellular bacteria, including Ca 2+ influx and promotion of L. monocytogenes entry. Quantitative host surfaceome analysis revealed significant quantitative remodeling of a defined set of cell surface glycoproteins upon LLO treatment, including a subset previously identified to play a role in the L. monocytogenes infection process. Our data further shows that the lysosomal-associated membrane proteins LAMP-1 and LAMP-2 are translocated to the cellular surface and those LLO-induced Ca 2+ fluxes are required to trigger the surface relocalization of LAMP-1. Finally, we identify late endosomes/lysosomes as the major donor compartments of LAMP-1 upon LLO treatment and by perturbing their function, we suggest that these organelles participate in L. monocytogenes invasion.
Infection and immunity, 1998
The secreted broad-range phosphatidylcholine (PC)-preferring phospholipase C (PC-PLC) of Listeria monocytogenes plays a role in the bacterium's ability to escape from phagosomes and spread from cell to cell. Based on comparisons with two orthologs, Clostridium perfringens alpha-toxin and Bacillus cereus PLC (PLCBc), we generated PC-PLC mutants with altered enzymatic activities and substrate specificities and analyzed them for biological function in tissue culture and mouse models of infection. Two of the conserved active-site zinc-coordinating histidines were confirmed by single amino acid substitutions H69G and H118G, which resulted in proteins inactive in broth culture and unstable intracellularly. Substitutions D4E and H56Y remodeled the PC-PLC active site to more closely resemble the PLCBc active site, while a gene replacement resulted in L. monocytogenes secreting PLCBc. All of these mutants yielded similar amounts of active enzyme as wild-type PC-PLC both in broth culture ...