scholarly journals A critical role for phospholipase C in protective immunity conferred by listeriolysin O-deficient Listeria monocytogenes

2008 ◽  
Vol 44 (2) ◽  
pp. 159-163 ◽  
Author(s):  
Nural N. Orgun ◽  
Sing Sing Way
Keyword(s):  
Listeria Monocytogenes ◽  
Phospholipase C ◽  
Protective Immunity ◽  
Critical Role ◽  
Listeriolysin O

scholarly journals Listeria monocytogenes Phospholipase C-Dependent Calcium Signaling Modulates Bacterial Entry into J774 Macrophage-Like Cells

1999 ◽  
Vol 67 (4) ◽  
pp. 1770-1778 ◽  
Author(s):  
Sandra J. Wadsworth ◽  
Howard Goldfine
Keyword(s):  
Listeria Monocytogenes ◽  
Calcium Signaling ◽  
Phospholipase C ◽  
Virulence Factors ◽  
Intracellular Signaling ◽  
Lethal Dose ◽  
Listeriolysin O ◽  
Wild Type ◽  
J774 Macrophage ◽  
Kinetics Of

ABSTRACT 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-typeL. 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 produced none of these elevations in [Ca2+]i; however, a transient elevation was observed whenever these bacteria entered the cell. A PI-PLC mutant produced a diminished single elevation in [Ca2+]i at 15 to 30 min. A broad-range PLC mutant produced only the first calcium spike. Studies with inhibitors suggested that the first elevation arises from influx of calcium from the extracellular medium through plasma membrane channels and that the second and third elevations come from release of Ca2+ from intracellular stores. We observed that internalization of wild-type bacteria and the broad-range PLC mutant was delayed for 5 to 10 min, but the LLO and PI-PLC mutants were internalized rapidly upon infection. Inhibitors that affected calcium signaling changed the kinetics of association of wild-type bacteria with J774 cells, the kinetics of entry, and the efficiency of escape from the primary phagosome.

scholarly journals Activation of Host Phospholipases C and D in Macrophages after Infection with Listeria monocytogenes

2000 ◽  
Vol 68 (10) ◽  
pp. 5735-5741 ◽  
Author(s):  
Howard Goldfine ◽  
Sandra J. Wadsworth ◽  
Norah C. Johnston
Keyword(s):  
Listeria Monocytogenes ◽  
Intracellular Calcium ◽  
Phospholipase C ◽  
Term Treatment ◽  
Listeriolysin O ◽  
Wild Type ◽  
Murine Bone Marrow ◽  
Long Term Treatment ◽  
J774 Cells ◽  
Hydrolysis Of

ABSTRACT Infection of the J774 murine macrophage-derived cell line withListeria 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 broad-range 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 marrow-derived 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 δ in J774 cells, also inhibited the activation of PLD, but hispidin, an inhibitor of PKC βI and βII, did not. Pretreatment of J774 cells with the PLD inhibitor, 2,3-diphosphoglycerate partially inhibited escape of the bacteria from the primary phagocytic vacuole.

scholarly journals Cytolysin-Dependent Escape of the Bacterium from the Phagosome Is Required but Not Sufficient for Induction of the Th1 Immune Response against Listeria monocytogenes Infection: Distinct Role of Listeriolysin O Determined by Cytolysin Gene Replacement

2007 ◽  
Vol 75 (8) ◽  
pp. 3791-3801 ◽  
Author(s):  
Hideki Hara ◽  
Ikuo Kawamura ◽  
Takamasa Nomura ◽  
Takanari Tominaga ◽  
Kohsuke Tsuchiya ◽  
...  
Keyword(s):  
Immune Response ◽  
Listeria Monocytogenes ◽  
Virulence Factor ◽  
Protective Immunity ◽  
Gene Replacement ◽  
Listeriolysin O ◽  
Listeria Ivanovii ◽  
Ifn Γ

ABSTRACT Listeria monocytogenes evades the antimicrobial mechanisms of macrophages by escaping from the phagosome into the cytosolic space via a unique cytolysin that targets the phagosomal membrane, listeriolysin O (LLO), encoded by hly. Gamma interferon (IFN-γ), which is known to play a pivotal role in the induction of Th1-dependent protective immunity in mice, appears to be produced, depending on the bacterial virulence factor. To determine whether the LLO molecule (the major virulence factor of L. monocytogenes) is indispensable or the escape of bacteria from the phagosome is sufficient to induce IFN-γ production, we first constructed an hly-deleted mutant of L. monocytogenes and then established isogenic L. monocytogenes mutants expressing LLO or ivanolysin O (ILO), encoded by ilo from Listeria ivanovii. LLO-expressing L. monocytogenes was highly capable of inducing IFN-γ production and Listeria-specific protective immunity, while the hly-deleted mutant was not. In contrast, the level of IFN-γ induced by ILO-expressing L. monocytogenes was significantly lower both in vitro and in vivo, despite the ability of this strain to escape the phagosome and the intracellular multiplication at a level equivalent to that of LLO-expressing L. monocytogenes. Only a negligible level of protective immunity was induced in mice against challenge with LLO- and ILO-expressing L. monocytogenes. These results clearly show that escape of the bacterium from the phagosome is a prerequisite but is not sufficient for the IFN-γ-dependent Th1 response against L. monocytogenes, and some distinct molecular nature of LLO is indispensable for the final induction of IFN-γ that is essentially required to generate a Th1-dependent immune response.

scholarly journals Requirement of the Listeria monocytogenes Broad-Range Phospholipase PC-PLC during Infection of Human Epithelial Cells

2003 ◽  
Vol 185 (21) ◽  
pp. 6295-6307 ◽  
Author(s):  
Angelika Gründling ◽  
Mark D. Gonzalez ◽  
Darren E. Higgins
Keyword(s):  
Listeria Monocytogenes ◽  
Epithelial Cells ◽  
Host Cell ◽  
Phospholipase C ◽  
Expression System ◽  
Broth Culture ◽  
Listeriolysin O ◽  
Proteolytic Activation ◽  
Human Epithelial Cells ◽  
Cell Spread

ABSTRACT In this study, we investigated the requirement of the Listeria monocytogenes broad-range phospholipase C (PC-PLC) during infection of human epithelial cells. L. monocytogenes is a facultative intracellular bacterial pathogen of humans and a variety of animal species. After entering a host cell, L. monocytogenes is initially surrounded by a membrane-bound vacuole. Bacteria promote their escape from this vacuole, grow within the host cell cytosol, and spread from cell to cell via actin-based motility. Most infection studies with L. monocytogenes have been performed with mouse cells or an in vivo mouse model of infection. In all mouse-derived cells tested, the pore-forming cytolysin listeriolysin O (LLO) is absolutely required for lysis of primary vacuoles formed during host cell entry. However, L. monocytogenes can escape from primary vacuoles in the absence of LLO during infection of human epithelial cell lines Henle 407, HEp-2, and HeLa. Previous studies have shown that the broad-range phospholipase C, PC-PLC, promotes lysis of Henle 407 cell primary vacuoles in the absence of LLO. Here, we have shown that PC-PLC is also required for lysis of HEp-2 and HeLa cell primary vacuoles in the absence of LLO expression. Furthermore, our results indicated that the amount of PC-PLC activity is critical for the efficiency of vacuolar lysis. In an LLO-negative derivative of L. monocytogenes strain 10403S, expression of PC-PLC has to increase before or upon entry into human epithelial cells, compared to expression in broth culture, to allow bacterial escape from primary vacuoles. Using a system for inducible PC-PLC expression in L. monocytogenes, we provide evidence that phospholipase activity can be increased by elevated expression of PC-PLC or Mpl, the enzyme required for proteolytic activation of PC-PLC. Lastly, by using the inducible PC-PLC expression system, we demonstrate that, in the absence of LLO, PC-PLC activity is not only required for lysis of primary vacuoles in human epithelial cells but is also necessary for efficient cell-to-cell spread. We speculate that the additional requirement for PC-PLC activity is for lysis of secondary double-membrane vacuoles formed during cell-to-cell spread.

scholarly journals Listeriolysin O Suppresses Phospholipase C-Mediated Activation of the Microbicidal NADPH Oxidase to Promote Listeria monocytogenes Infection

2011 ◽  
Vol 10 (6) ◽  
pp. 627-634 ◽  
Author(s):  
Grace Y. Lam ◽  
Ramzi Fattouh ◽  
Aleixo M. Muise ◽  
Sergio Grinstein ◽  
Darren E. Higgins ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Nadph Oxidase ◽  
Phospholipase C ◽  
Listeriolysin O ◽  
Listeria Monocytogenes Infection

Listeria monocytogeneslisteriolysin O and phosphatidylinositol-specific phospholipase C affect adherence to epithelial cells

2005 ◽  
Vol 51 (9) ◽  
pp. 745-751 ◽  
Author(s):  
Agata Krawczyk-Balska ◽  
Jacek Bielecki
Keyword(s):  
Listeria Monocytogenes ◽  
Epithelial Cells ◽  
Phospholipase C ◽  
Listeriolysin O ◽  
Cell Responses ◽  
Bacterial Binding ◽  
Intracellular Ca ◽  
Infected Cells ◽  
Host Parasite ◽  
Phagosomal Membrane

Listeria monocytogenes, a foodborn intracellular animal and human pathogen, produces several exotoxins contributing to virulence. Among these are listeriolysin O (LLO), a pore-forming cholesterol-dependent hemolysin, and a phosphatidylinositol-specific phospholipase C (PI-PLC). LLO is known to play an important role in the escape of bacteria from the primary phagocytic vacuole of macrophages, and PI-PLC supports this process. Evidence is accumulating that LLO and PI-PLC are multifunctional virulence factors with many important roles in the host-parasite interaction other than phagosomal membrane disruption. LLO and PI-PLC may induce a number of host cell responses by modulating signal transduction of infected cells via intracellular Ca2+levels and the metabolism of phospholipids. This would result in the activation of host phospholipase C and protein kinase C. In the present study, using Bacillus sub tilis strains expressing LLO, PI-PLC, and simultaneously LLO and PI-PLC, we show that LLO and PI-PLC enhance bacterial binding to epithelial cells Int407, with LLO being necessary and PI-PLC playing an accessory role. The results of this work suggest that these two listerial proteins act on epithelial cells prior to internalization.Key words: Listeria monocytogenes, listeriolysin O, phosphatidylinositol-specific phospholipase C, Bacillus subtilis, adherence.

scholarly journals Characterization of Listeria monocytogenes Expressing Anthrolysin O and Phosphatidylinositol-Specific Phospholipase C from Bacillus anthracis

2005 ◽  
Vol 73 (10) ◽  
pp. 6639-6646 ◽  
Author(s):  
Zhengyu Wei ◽  
Pamela Schnupf ◽  
Mathilde A. Poussin ◽  
Lauren A. Zenewicz ◽  
Hao Shen ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Bacillus Anthracis ◽  
Host Cell ◽  
Phospholipase C ◽  
Host Cells ◽  
Listeriolysin O ◽  
Intracellular Growth ◽  
Cell Cytosol ◽  
Anthrolysin O ◽  
Cell Spread

ABSTRACT Two virulence factors of Listeria monocytogenes, listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC), mediate escape of this pathogen from the phagocytic vacuole of macrophages, thereby allowing the bacterium access to the host cell cytosol for growth and spread to neighboring cells. We characterized their orthologs from Bacillus anthracis by expressing them in L. monocytogenes and characterizing their contribution to bacterial intracellular growth and cell-to-cell spread. We generated a series of L. monocytogenes strains expressing B. anthracis anthrolysin O (ALO) and PI-PLC in place of LLO and L. monocytogenes PI-PLC, respectively. We found that ALO was active at both acidic and neutral pH and could functionally replace LLO in mediating escape from a primary vacuole; however, ALO exerted a toxic effect on the host cell by damaging the plasma membrane. B. anthracis PI-PLC, unlike the L. monocytogenes ortholog, had high activity on glycosylphosphatidylinositol-anchored proteins. L. monocytogenes expressing B. anthracis PI-PLC showed significantly decreased efficiencies of escape from a phagosome and in cell-to-cell spread. We further compared the level of cytotoxicity to host cells by using mutant strains expressing ALO in combination either with L. monocytogenes PI-PLC or with B. anthracis PI-PLC. The results demonstrated that the mutant strain expressing the combination of ALO and B. anthracis PI-PLC caused less damage to host cells than the strain expressing ALO and L. monocytogenes PI-PLC. The present study indicates that LLO and L. monocytogenes PI-PLC has adapted for L. monocytogenes intracellular growth and virulence and suggests that ALO and B. anthracis PI-PLC may have a role in B. anthracis pathogenesis.

scholarly journals Differences in Gamma Interferon Production Induced by Listeriolysin O and Ivanolysin O Result in Different Levels of Protective Immunity in Mice Infected with Listeria monocytogenes and Listeria ivanovii

2003 ◽  
Vol 71 (5) ◽  
pp. 2447-2454 ◽  
Author(s):  
Terumi Kimoto ◽  
Ikuo Kawamura ◽  
Chikara Kohda ◽  
Takamasa Nomura ◽  
Kohsuke Tsuchiya ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Protective Immunity ◽  
Lethal Dose ◽  
Gamma Interferon ◽  
Listeriolysin O ◽  
Serum Samples ◽  
Listeria Ivanovii ◽  
Ifn Γ ◽  
Different Levels

ABSTRACT Two pathogenic species in the genus Listeria, Listeria monocytogenes and Listeria ivanovii, are characterized by the production of hemolysins belonging to cholesterol-dependent cytolysins, listeriolysin O (LLO) and ivanolysin O (ILO), respectively. LLO, produced by L. monocytogenes, is able to induce gamma interferon (IFN-γ) production and contributes to the generation of Th1-dependent protective immunity. On the other hand, nothing is known about the role of ILO, produced by L. ivanovii, in this regard. In this study, we immunized mice with 0.1 50% lethal dose (LD50) of L. monocytogenes and L. ivanovii. Protective immunity against a challenge with 10 LD50 was generated in mice infected with L. monocytogenes, whereas L. ivanovii infection did not induce protection. After immunization, the level of IFN-γ in serum samples was increased in mice given L. monocytogenes but not in those given L. ivanovii. To determine the IFN-γ-inducing activity of cytolysins, recombinant protein was constructed. Recombinant ILO exhibited significantly lower IFN-γ-inducing activity than LLO. By comparing the IFN-γ-inducing activity of a chimera incorporating LLO and ILO, it was found that domains 1 to 3 of LLO were critical for IFN-γ-inducing activity while the counterpart in ILO was unable to induce cytokine production. These results suggested that the weak ability of ILO to induce IFN-γ production is responsible for the failure of L. ivanovii to generate effective protective immunity.

A comparison of selected methods for measuring the virulence properties ofListeriaspp.

2006 ◽  
Vol 52 (4) ◽  
pp. 301-307 ◽  
Author(s):  
Sally Chiu ◽  
Paul B Vanderlinde ◽  
Gary A Dykes
Keyword(s):  
Listeria Monocytogenes ◽  
Phospholipase C ◽  
Virulence Genes ◽  
Listeriolysin O ◽  
Wide Range ◽  
Virulence Protein ◽  
Associated Proteins ◽  
Polymerase Chain ◽  
Virulence Properties

The comparative ability of different methods to assess virulence of Listeria species was investigated in ten Listeria strains. All strains were initially subjected to pulsed-field gel electrophoresis analysis to determine their relatedness. Virulence characteristics were subsequently tested for by (i) determining the presence of six virulence genes by polymerase chain reaction; (ii) testing for the production of listeriolysin O, phosphatidylcholine phospholipase C, and phosphatidylinositol-specific phospholipase C; (iii) investigating the hydrophobicity of the strains; (iv) determining the strains ability to attach to, enter, and replicate within the Caco-2 cells. Variations in most of the virulence characteristics were obvious across the strains for the range of tests performed. A wide range of anomalous results among methods were apparent. In particular, the presence of virulence genes was found to be unrelated to the production of virulence-associated proteins in vitro, while virulence protein production and hydrophobicity in Listeria monocytogenes were found to be unrelated or marginally related, respectively, to the ability to invade the Caco-2 cell line. It was concluded that the methods investigated were unable to consistently and unequivocally measure the differences in the virulence properties of the strains.Key words: Listeria monocytogenes, virulence, pathogenicity.

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