Comparison of listeriolysin O and phospholipases PlcA and PlcB activities, and initial intracellular growth capability among food and clinical strains ofListeria monocytogenes

2018 ◽  
Vol 124 (3) ◽  
pp. 899-909 ◽  
Author(s):  
M. Kanki ◽  
H. Naruse ◽  
K. Kawatsu
Keyword(s):  
Listeriolysin O ◽  
Intracellular Growth ◽  
Clinical Strains ◽  
Oflisteria Monocytogenes

scholarly journals Role of Listeriolysin O in Cell-to-Cell Spread ofListeria monocytogenes

2000 ◽  
Vol 68 (2) ◽  
pp. 999-1003 ◽  
Author(s):  
Margaret M. Gedde ◽  
Darren E. Higgins ◽  
Lewis G. Tilney ◽  
Daniel A. Portnoy
Keyword(s):  
Bacterial Pathogen ◽  
Listeriolysin O ◽  
Genetic Approach ◽  
Wild Type ◽  
Intracellular Growth ◽  
Double Membrane ◽  
Vacuolar Compartment ◽  
Oflisteria Monocytogenes ◽  
Cell Spread

ABSTRACT Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a host vacuolar compartment and grows rapidly in the cytosol. Listeriolysin O (LLO) is a secreted pore-forming protein essential for the escape of L. monocytogenes from the vacuole formed upon initial internalization. However, its role in intracellular growth and cell-to-cell spread events has not been testable by a genetic approach. In this study, purified six-His-tagged LLO (HisLLO) was noncovalently coupled to the surface of nickel-treated LLO-negative mutants. Bound LLO mediated vacuolar escape in approximately 2% of the mutants. After 5.5 h of growth, cytosolic bacteria were indistinguishable from wild-type bacteria with regard to formation of pseudopod-like extensions, here termed listeriopods, and spread to adjacent cells. However, bacteria in adjacent cells failed to multiply and were found in double-membrane vacuoles. Addition of bound LLO to mutants lacking LLO and two distinct phospholipases C (PLCs) also resulted in spread to adjacent cells, but these triple mutants became trapped in multiple-membrane vacuoles that are reminiscent of autophagocytic vacuoles. These studies show that neither LLO nor the PLCs are necessary for listeriopod formation and uptake of bacteria into neighboring cells but that LLO is required for the escape ofL. monocytogenes from the double-membrane vacuole that forms upon cell-to-cell spread.

scholarly journals Stress-Induced ClpP Serine Protease ofListeria monocytogenes Is Essential for Induction of Listeriolysin O-Dependent Protective Immunity

2001 ◽  
Vol 69 (8) ◽  
pp. 4938-4943 ◽  
Author(s):  
Olivier Gaillot ◽  
Søren Bregenholt ◽  
Francis Jaubert ◽  
James P. Di Santo ◽  
Patrick Berche
Keyword(s):  
Immune Response ◽  
Serine Protease ◽  
Protective Immunity ◽  
Listeriolysin O ◽  
Intracellular Growth ◽  
Lethal Challenge ◽  
Protective Antigens ◽  
Oflisteria Monocytogenes

ABSTRACT The stress-induced protease ClpP is required for virulence of the facultative intracellular pathogen Listeria monocytogenes. We previously found that in the absence of ClpP, the virulence of this pathogen was strongly reduced, mainly due to the decreased production of functional listeriolysin O (LLO), a major immunodominant virulence factor promoting intracellular growth. In this work, a clpP deletion mutant of L. monocytogenes was used to study the generation of anti-Listeria protective immunity. We found that ClpP is required for the intracellular growth of L. monocytogenes in resident macrophages in vivo. Mice infected with doses as high as 106 clpP mutant bacteria were not protected against a lethal challenge of wild-type bacteria and did not develop any detectable LLO-specific cytolytic T cells or antibodies, suggesting that the amount of LLO produced in infected mice under these conditions was too low to induce a specific immune response. However, in contrast to the results obtained with a mutant with a disrupted hly gene, this lack of protection was overcome by inoculation of very high infecting doses ofclpP mutant bacteria (5 × 108), thus producing sufficient amounts of LLO to stimulate anti-Listeria immunity. The role of ClpP was confirmed by showing that anti-Listeria immunity was restored in mice infected with a clpP-complemented mutant. These results indicate that the stress-induced serine protease ClpP is a potential target for modulating the presentation of protective antigens such as LLO and thereby the immune response against L. monocytogenes.

A Method for Purification of Listeriolysin O from a Hypersecretor Strain ofListeria monocytogenes

1999 ◽  
Vol 15 (2) ◽  
pp. 243-245 ◽  
Author(s):  
Cherie M. Walton ◽  
Catherine H. Wu ◽  
George Y. Wu
Keyword(s):  
Listeriolysin O ◽  
Oflisteria Monocytogenes

scholarly journals Mutagenesis of Active-Site Histidines ofListeria monocytogenes Phosphatidylinositol-Specific Phospholipase C: Effects on Enzyme Activity and Biological Function

1999 ◽  
Vol 67 (1) ◽  
pp. 182-186 ◽  
Author(s):  
Trudi Bannam ◽  
Howard Goldfine
Keyword(s):  
Active Site ◽  
Site Directed Mutagenesis ◽  
Intracellular Pathogen ◽  
Wild Type ◽  
Intracellular Growth ◽  
Oflisteria Monocytogenes ◽  
Culture Supernatants ◽  
Derivatives Of ◽  
Cell Spread ◽  
Active Site Histidine

ABSTRACT Listeria monocytogenes, a gram-positive facultative intracellular pathogen, produces two distinct phospholipases C. PC-PLC, encoded by plcB, is a broad-range phospholipase, whereas PI-PLC, encoded by plcA, is specific for phosphatidylinositol. It was previously shown that PI-PLC plays a role in efficient escape of L. monocytogenes from the primary phagosome. To further understand the function of PI-PLC in intracellular growth, site-directed mutagenesis of plcA was performed. Two potential active-site histidine residues were mutated independently to alanine, serine, and phenylalanine. With the exception of the activity of the enzyme containing H38F, which was unstable, the PI-PLC enzyme activities of culture supernatants containing each mutant enzyme were <1% of wild-type activity. In addition, the levels of expression of the mutant PI-PLC proteins were equivalent to wild-type expression. Derivatives of L. monocytogenes containing these specific plcA mutations were found to have phenotypes similar to that of the plcA deletion strain in an assay for escape from the primary vacuole, in intracellular growth in a murine macrophage cell line, and in a plaquing assay for cell-to-cell spread. Thus, catalytic activity of PI-PLC is required for all its intracellular functions.

scholarly journals Inducible Control of Virulence Gene Expression in Listeria monocytogenes: Temporal Requirement of Listeriolysin O during Intracellular Infection

2002 ◽  
Vol 184 (21) ◽  
pp. 5935-5945 ◽  
Author(s):  
Christina E. Dancz ◽  
Andrea Haraga ◽  
Daniel A. Portnoy ◽  
Darren E. Higgins
Keyword(s):  
Listeria Monocytogenes ◽  
Cell Line ◽  
Broth Culture ◽  
Listeriolysin O ◽  
Virulence Determinants ◽  
Intracellular Growth ◽  
Iptg Induction ◽  
Intracellular Infection ◽  
Dose Dependent ◽  
Cell Spread

ABSTRACT We have constructed a lac repressor/operator-based system to tightly regulate expression of bacterial genes during intracellular infection by Listeria monocytogenes. An L. monocytogenes strain was constructed in which expression of listeriolysin O was placed under the inducible control of an isopropyl-β-d-thiogalactopyranoside (IPTG)-dependent promoter. Listeriolysin O (LLO) is a pore-forming cytolysin that mediates lysis of L. monocytogenes-containing phagosomes. Using hemolytic-activity assays and Western blot analysis, we demonstrated dose-dependent IPTG induction of LLO during growth in broth culture. Moreover, intracellular growth of the inducible-LLO (iLLO) strain in the macrophage-like cell line J774 was strictly dependent upon IPTG. We have further shown that iLLO bacteria trapped within primary phagocytic vacuoles can be induced to escape into the cytosol following addition of IPTG to the cell culture medium, thus yielding the ability to control bacterial escape from the phagosome and the initiation of intracellular growth. Using the iLLO strain in plaque-forming assays, we demonstrated an additional requirement for LLO in facilitating cell-to-cell spread in L2 fibroblasts, a nonprofessional phagocytic cell line. Furthermore, the efficiency of cell-to-cell spread of iLLO bacteria in L2 cells was IPTG dose dependent. The potential use of this system for determining the temporal requirements of additional virulence determinants of intracellular pathogenesis is discussed.

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 Stimulating Respiratory Activity Primes Anaerobically Grown Listeria monocytogenes for Subsequent Intracellular Infections

Pathogens ◽  
2018 ◽  
Vol 7 (4) ◽  
pp. 96 ◽  
Author(s):  
Nathan Wallace ◽  
Erica Rinehart ◽  
Yvonne Sun
Keyword(s):  
Listeria Monocytogenes ◽  
Respiratory Activity ◽  
Growth Conditions ◽  
Growth Defect ◽  
Listeriolysin O ◽  
Low Oxygen ◽  
Intracellular Growth ◽  
Aerobic Conditions ◽  
Intracellular Infections ◽  
Cell Spread

Listeria monocytogenes (L. monocytogenes) is a Gram-positive, enteric pathogen and the causative agent of listeriosis. During transition through the gastrointestinal tract, L. monocytogenes routinely encounters suboxic conditions. However, how the exposure to the low oxygen environment affects subsequent pathogenesis is not completely understood. Our lab previously reported that anaerobically grown L. monocytogenes exhibited an intracellular growth defect in macrophages even though the infection took place under aerobic conditions. This phenotype suggests that prior growth conditions have a prolonged effect on the outcome of subsequent intracellular infection. In this study, to further investigate the mechanisms that contribute to the compromised intracellular growth after anaerobic exposure, we hypothesized that the lack of respiratory activity under anaerobic conditions prevented anaerobically grown L. monocytogenes to establish subsequent intracellular growth under aerobic conditions. To test this hypothesis, respiratory activity in anaerobically grown L. monocytogenes was stimulated by exogenous fumarate and subsequent intracellular pathogenesis was assessed. The results showed that fumarate supplementation significantly increased the respiratory activity of anaerobically grown L. monocytogenes and rescued the subsequent intracellular growth defect, likely through promoting the production of listeriolysin O, phagosomal escape, and cell-cell spread. This study highlights the importance of respiratory activity in L. monocytogenes in modulating the outcome of subsequent intracellular infections.

scholarly journals Expression of Listeriolysin O and ActA by Intracellular and Extracellular Listeria monocytogenes

1999 ◽  
Vol 67 (1) ◽  
pp. 131-139 ◽  
Author(s):  
Marlena A. Moors ◽  
Brian Levitt ◽  
Philip Youngman ◽  
Daniel A. Portnoy
Keyword(s):  
Listeria Monocytogenes ◽  
Bacillus Pumilus ◽  
Luria Bertani ◽  
Listeriolysin O ◽  
Intracellular Growth ◽  
Chloramphenicol Resistance ◽  
Growth Environment ◽  
Transcriptional Reporter ◽  
J774 Cells ◽  
Reporter Gene System

ABSTRACT Listeria monocytogenes requires listeriolysin O (LLO) and ActA, the products of hly and actA, respectively, to establish a productive intracellular infection. LLO is essential for vacuolar lysis and entry into the cytosol, while ActA is required for bacterial spread to adjacent cells. We have used a transcriptional reporter gene system to compare the expression ofactA and hly during intracellular growth to that during growth in broth cultures. The hly andactA genes were transcriptionally fused toEscherichia coli lacZ and Bacillus pumilus cat-86 (cat), and the fusions were integrated in single copies into the L. monocytogenes chromosome. A chloramphenicol resistance assay indicated that the hlyfusion but not the actA fusion was significantly activated in Luria-Bertani (LB) broth, and this finding correlated with LLO and ActA levels detectable in broth cultures. Quantitation of promoter activity on the basis of β-galactosidase activity revealed up to 10-fold-higher level of expression of the hly fusion relative to the actA fusion in LB broth. In contrast, both fusions were active in the cytosol of J774 cells, and the activity of the actA fusion was approximately 3-fold higher than that of the hly fusion under these conditions. However, quantitative immunoprecipitation of ActA and LLO from infected J774 cells demonstrated approximately 70-fold more cytosolic ActA than cytosolic LLO. Finally, in comparison to induction in broth cultures,actA was highly induced (226-fold) and hly was moderately induced (20-fold) in J774 cells. Collectively, these results indicate that actA and hly are differentially regulated in response to the growth environment and that both genes are preferentially expressed during intracellular growth. Further, while the lower level of production of ActA than of LLO in broth can be accounted for by transcriptional regulation, the relative abundance of intracellular ActA compared to that of intracellular LLO is a function of additional, possibly host-mediated, factors.

scholarly journals Critical role of the N-terminal residues of listeriolysin O in phagosomal escape and virulence ofListeria monocytogenes

2002 ◽  
Vol 46 (2) ◽  
pp. 367-379 ◽  
Author(s):  
Marie-Annick Lety ◽  
Claude Frehel ◽  
Patrick Berche ◽  
Alain Charbit
Keyword(s):  
Critical Role ◽  
Listeriolysin O ◽  
Oflisteria Monocytogenes
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