Modification of the signal sequence cleavage site of listeriolysin O does not affect protein secretion but impairs the virulence of Listeria monocytogenes

Microbiology ◽  
2003 ◽  
Vol 149 (5) ◽  
pp. 1249-1255 ◽  
Author(s):  
Marie-Annick Lety ◽  
Claude Frehel ◽  
Jean-luc Beretti ◽  
Patrick Berche ◽  
Alain Charbit
Keyword(s):  
Bone Marrow ◽  
Listeria Monocytogenes ◽  
Mouse Model ◽  
Cleavage Site ◽  
Culture Supernatant ◽  
Signal Sequence ◽  
Bacterial Pathogen ◽  
Listeriolysin O ◽  
Major Virulence Factor ◽  
Signal Sequence Cleavage Site

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 phagosomal escape and bacterial infection.

scholarly journals An Inducible Cre-lox System to Analyze the Role of LLO in Listeria monocytogenes Pathogenesis

Toxins ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 38 ◽  
Author(s):  
Brittney N. Nguyen ◽  
Daniel A. Portnoy
Keyword(s):  
Bone Marrow ◽  
Listeria Monocytogenes ◽  
Mouse Model ◽  
Host Cell ◽  
Transcriptional Control ◽  
Listeriolysin O ◽  
Gene Encoding ◽  
Cell Cytosol ◽  
Host Cell Cytosol

Listeriolysin O (LLO) is a pore-forming cytolysin that allows Listeria monocytogenes to escape from phagocytic vacuoles and enter the host cell cytosol. LLO is expressed continuously during infection, but it has been a challenge to evaluate the importance of LLO secreted in the host cell cytosol because deletion of the gene encoding LLO (hly) prevents localization of L. monocytogenes to the cytosol. Here, we describe a L. monocytogenes strain (hlyfl) in which hly is flanked by loxP sites and Cre recombinase is under the transcriptional control of the L. monocytogenes actA promoter, which is highly induced in the host cell cytosol. In less than 2 h after infection of bone marrow-derived macrophages (BMMs), bacteria were 100% non-hemolytic. hlyfl grew intracellularly to levels 10-fold greater than wildtype L. monocytogenes and was less cytotoxic. In an intravenous mouse model, 90% of bacteria were non-hemolytic within three hours in the spleen and eight hours in the liver. The loss of LLO led to a 2-log virulence defect in the spleen and a 4-log virulence defect in the liver compared to WT L. monocytogenes. Thus, the production of LLO in the cytosol has significant impact on the pathogenicity of L. monocytogenes.

scholarly journals The Listeria monocytogenes hemolysin has an acidic pH optimum to compartmentalize activity and prevent damage to infected host cells

2002 ◽  
Vol 156 (6) ◽  
pp. 1029-1038 ◽  
Author(s):  
Ian J. Glomski ◽  
Margaret M. Gedde ◽  
Albert W. Tsang ◽  
Joel A. Swanson ◽  
Daniel A. Portnoy
Keyword(s):  
Listeria Monocytogenes ◽  
Bacterial Pathogen ◽  
Cytolytic Activity ◽  
Host Cells ◽  
Adaptive Mutation ◽  
Acidic Ph ◽  
Listeriolysin O ◽  
Lysosomal Hydrolases ◽  
Ph Optimum ◽  
Neutral Ph

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a phagosome and grows in the host cell cytosol. The pore-forming cholesterol-dependent cytolysin, listeriolysin O (LLO), mediates bacterial escape from vesicles and is ∼10-fold more active at an acidic than neutral pH. By swapping dissimilar residues from a pH-insensitive orthologue, perfringolysin O (PFO), we identified leucine 461 as unique to pathogenic Listeria and responsible for the acidic pH optimum of LLO. Conversion of leucine 461 to the threonine present in PFO increased the hemolytic activity of LLO almost 10-fold at a neutral pH. L. monocytogenes synthesizing LLO L461T, expressed from its endogenous site on the bacterial chromosome, resulted in a 100-fold virulence defect in the mouse listeriosis model. These bacteria escaped from acidic phagosomes and initially grew normally in cells and spread cell to cell, but prematurely permeabilized the host membrane and killed the cell. These data show that the acidic pH optimum of LLO results from an adaptive mutation that acts to limit cytolytic activity to acidic vesicles and prevent damage in the host cytosol, a strategy also used by host cells to compartmentalize lysosomal hydrolases.

scholarly journals pH-dependent Perforation of Macrophage Phagosomes by Listeriolysin O from Listeria monocytogenes

1997 ◽  
Vol 186 (7) ◽  
pp. 1159-1163 ◽  
Author(s):  
Kathryn E. Beauregard ◽  
Kyung-Dall Lee ◽  
R. John Collier ◽  
Joel A. Swanson
Keyword(s):  
Listeria Monocytogenes ◽  
Ph Dependence ◽  
Mouse Bone Marrow ◽  
Listeriolysin O ◽  
Bafilomycin A1 ◽  
Wild Type ◽  
Major Virulence Factor ◽  
Ph Dependent ◽  
Vacuolar Ph

The pore-forming toxin listeriolysin O (LLO) is a major virulence factor implicated in escape of Listeria monocytogenes from phagocytic vacuoles. Here we describe the pH-dependence of vacuolar perforation by LLO, using the membrane-impermeant fluorophore 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) to monitor the pH and integrity of vacuoles in mouse bone marrow–derived macrophages. Perforation was observed when acidic vacuoles containing wild-type L. monocytogenes displayed sudden increases in pH and release of HPTS into the cytosol. These changes were not seen with LLO-deficient mutants. Perforation occurred at acidic vacuolar pH (4.9–6.7) and was reduced in frequency or prevented completely when macrophages were treated with the lysosomotropic agents ammonium chloride or bafilomycin A1. We conclude that acidic pH facilitates LLO activity in vivo.

Virulence of Listeria monocytogenes Propagated in NaCl Containing Media at 4, 25 and 37°C

1994 ◽  
Vol 57 (6) ◽  
pp. 475-478 ◽  
Author(s):  
ERIC R. MYERS ◽  
SCOTT E. MARTIN
Keyword(s):  
Superoxide Dismutase ◽  
Listeria Monocytogenes ◽  
Sodium Chloride ◽  
Mouse Model ◽  
Virulence Factor ◽  
Growth Temperature ◽  
Lethal Dose ◽  
Listeriolysin O ◽  
Sod Activity ◽  
Approximate Lethal Dose

Virulence, as determined in a mouse model, and virulence factor activities of catalase (CA), superoxide dismutase (SOD) and listeriolysin O (LLO), was examined in Listeria monocytogenes 10403S. Cells were propagated in media containing various concentrations of sodium chloride (NaCl) at 4, 25 and 37°C. Strain 10403S exhibited significant increases in CA activity and LLO when grown in media containing 428 mM of NaCl at 37°C. The CA activities at 4 and 25°C were significantly less, and the cells exhibited similar increases and decreases as cells grown at 37°C. When comparing the growth temperatures, the CA activity decreased as the growth temperature decreased. The SOD activity was significantly increased only when cells were propagated in media containing either 428 or 1,112 mM of NaCl. The SOD activity increased as the growth temperature decreased. No LLO activity was detected when cells were grown at 4 and 25°C. The production of these enzymes appeared to be thermoregulated. In addition, approximate lethal dose (ALD50) values were determined after intragastric (i.g.) and intraperitoneal (i.p.) infection. Each method of infection indicated that LLO was required for virulence, while growth in salt containing media, growth at 4°C, or the production of higher levels of CA, SOD and LLO did not appear to influence the virulence of L. monocytogenes.

scholarly journals The Posttranslocation Chaperone PrsA2 Contributes to Multiple Facets of Listeria monocytogenes Pathogenesis

2009 ◽  
Vol 77 (7) ◽  
pp. 2612-2623 ◽  
Author(s):  
Francis Alonzo ◽  
Gary C. Port ◽  
Min Cao ◽  
Nancy E. Freitag
Keyword(s):  
Listeria Monocytogenes ◽  
Gene Product ◽  
Bacterial Pathogen ◽  
Virulence Gene ◽  
Host Cells ◽  
Listeriolysin O ◽  
Virulence Gene Expression ◽  
High Concentrations ◽  
Host Infection ◽  
Cell Spread

ABSTRACT Listeria monocytogenes is an intracellular bacterial pathogen whose virulence depends on the regulated expression of numerous secreted bacterial factors. As for other gram-positive bacteria, many proteins secreted by L. monocytogenes are translocated across the bacterial membrane in an unfolded state to the compartment existing between the membrane and the cell wall. This compartment presents a challenging environment for protein folding due to its high density of negative charge, high concentrations of cations, and low pH. We recently identified PrsA2 as a gene product required for L. monocytogenes virulence. PrsA2 was identified based on its increased secretion by strains containing a mutationally activated form of prfA, the key regulator of L. monocytogenes virulence gene expression. The prsA2 gene product is one of at least two predicted peptidyl-prolyl cis/trans-isomerases encoded by L. monocytogenes; these proteins function as posttranslocation protein chaperones and/or foldases. In this study, we demonstrate that PrsA2 plays a unique and important role in L. monocytogenes pathogenesis by promoting the activity and stability of at least two critical secreted virulence factors: listeriolysin O (LLO) and a broad-specificity phospholipase. Loss of PrsA2 activity severely attenuated virulence in mice and impaired bacterial cell-to-cell spread in host cells. In contrast, mutants lacking prsA1 resembled wild-type bacteria with respect to intracellular growth and cell-to-cell spread as well as virulence in mice. PrsA2 is thus distinct from PrsA1 in its unique requirement for the stability and full activity of L. monocytogenes-secreted factors that contribute to host infection.

Virulence potential of Listeria monocytogenes strains recovered from pigs in Spain

2020 ◽  
Vol 187 (11) ◽  
pp. e101-e101
Author(s):  
Jaime Gómez-Laguna ◽  
Fernando Cardoso-Toset ◽  
Jazmín Meza-Torres ◽  
Javier Pizarro-Cerdá ◽  
Juan J Quereda
Keyword(s):  
Listeria Monocytogenes ◽  
Virulence Factors ◽  
Bacterial Pathogen ◽  
Listeriolysin O ◽  
Actin Assembly ◽  
Virulence Potential ◽  
Serotype 1 ◽  
Internalin B ◽  
Meat Samples ◽  
Internalin A

BackgroundListeria monocytogenes is a foodborne bacterial pathogen that causes listeriosis, an infectious disease in animals and people, with pigs acting as asymptomatic reservoirs. In August 2019 an outbreak associated with the consumption of pork meat caused 222 human cases of listeriosis in Spain. Determining the diversity as well as the virulence potential of strains from pigs is important to public health.MethodsThe behaviour of 23 L monocytogenes strains recovered from pig tonsils, meat and skin was compared by studying (1) internalin A, internalin B, listeriolysin O, actin assembly-inducing protein and PrfA expression levels, and (2) their invasion and intracellular growth in eukaryotic cells.ResultsMarked differences were found in the expression of the selected virulence factors and the invasion and intracellular replication phenotypes of L monocytogenes strains. Strains obtained from meat samples and belonging to serotype 1/2a did not have internalin A anchored to the peptidoglycan. Some strains expressed higher levels of the studied virulence factors and invaded and replicated intracellularly more efficiently than an epidemic L monocytogenes reference strain (F2365).ConclusionThis study demonstrates the presence of virulent L monocytogenes strains with virulent potential in pigs, with valuable implications in veterinary medicine and food safety.

scholarly journals A Role for Taok2 in Listeria monocytogenes Vacuolar Escape

2020 ◽  
Author(s):  
Juan J Quereda ◽  
Camille Morel ◽  
Noelia Lopez-Montero ◽  
Jason Ziveri ◽  
Steven Rolland ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Kinase Activity ◽  
Bacterial Pathogen ◽  
Host Factors ◽  
Host Cells ◽  
Listeriolysin O ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Activity Inhibition ◽  
Interfering Rna

Abstract The bacterial pathogen Listeria monocytogenes invades host cells, ruptures the internalization vacuole, and reaches the cytosol for replication. A high-content small interfering RNA (siRNA) microscopy screen allowed us to identify epithelial cell factors involved in L. monocytogenes vacuolar rupture, including the serine/threonine kinase Taok2. Kinase activity inhibition using a specific drug validated a role for Taok2 in favoring L. monocytogenes cytoplasmic access. Furthermore, we showed that Taok2 recruitment to L. monocytogenes vacuoles requires the presence of pore-forming toxin listeriolysin O. Overall, our study identified the first set of host factors modulating L. monocytogenes vacuolar rupture and cytoplasmic access in epithelial cells.

scholarly journals Listeria monocytogenes Mutants That Fail To Compartmentalize Listerolysin O Activity Are Cytotoxic, Avirulent, and Unable To Evade Host Extracellular Defenses

2003 ◽  
Vol 71 (12) ◽  
pp. 6754-6765 ◽  
Author(s):  
Ian J. Glomski ◽  
Amy L. Decatur ◽  
Daniel A. Portnoy
Keyword(s):  
Listeria Monocytogenes ◽  
Extracellular Fluid ◽  
Bacterial Pathogen ◽  
Cytolytic Activity ◽  
Mouse Serum ◽  
Listeriolysin O ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Cell Cytosol ◽  
Host Cell Cytosol

ABSTRACT Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a phagosome and grows in the host cell cytosol. Escape of the bacterium from the phagosome to the cytosol is mediated by the bacterial pore-forming protein listeriolysin O (LLO). LLO has multiple mechanisms that optimize activity in the phagosome and minimize activity in the host cytosol. Mutants that fail to compartmentalize LLO activity are cytotoxic and have reduced virulence. We sought to determine why cytotoxic bacteria have attenuated virulence in the mouse model of listeriosis. In this study, we constructed a series of strains with mutations in LLO and with various degrees of cytotoxicity. We found that the more cytotoxic the strain in cell culture, the less virulent it was in mice. Induction of neutropenia increased the relative virulence of the cytotoxic strains 100-fold in the spleen and 10-fold in the liver. The virulence defect was partially restored in neutropenic mice by adding gentamicin, an antibiotic that kills extracellular bacteria. Additionally, L. monocytogenes grew more slowly in extracellular fluid (mouse serum) than within tissue culture cells. We concluded that L. monocytogenes controls the cytolytic activity of LLO to maintain its nutritionally rich intracellular niche and avoid extracellular defenses of the host.

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