The Metalloprotease of Listeria monocytogenes Is Activated by Intramolecular Autocatalysis

ABSTRACT The metalloprotease (Mpl) of Listeria monocytogenes is a thermolysin-like protease that mediates the maturation of a broad-range phospholipase C, whose function contributes to the ability of this food-borne bacterial pathogen to survive intracellularly. Mpl is made as a proprotein that undergoes maturation by proteolytic cleavage of a large N-terminal prodomain. In this study, we identified the N terminus of mature Mpl and generated Mpl catalytic mutants to investigate the mechanism of Mpl maturation. We observed that Mpl activity was a prerequisite for maturation, suggesting a mechanism of autocatalysis. Furthermore, using a strain of L. monocytogenes expressing both the wild-type form and a catalytic mutant form of Mpl simultaneously, we determined that in vivo maturation of Mpl occurs exclusively by an intramolecular autocatalysis mechanism.

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Impact of the Listeria monocytogenes Protein InlC on Infection in Mice

Infection and Immunity ◽

10.1128/iai.01377-12 ◽

2013 ◽

Vol 81 (4) ◽

pp. 1334-1340 ◽

Cited By ~ 12

Author(s):

Nelly Leung ◽

Antonella Gianfelice ◽

Scott D. Gray-Owen ◽

Keith Ireton

Keyword(s):

Listeria Monocytogenes ◽

Mutant Strain ◽

Type Strain ◽

Wild Type Strain ◽

Bacterial Pathogen ◽

Adaptor Protein ◽

Single Amino Acid ◽

Wild Type ◽

Content Type

ABSTRACTThe bacterial pathogenListeria monocytogenescauses serious food-borne illnesses in pregnant women and the immunocompromised.L. monocytogenespromotes its internalization into host epithelial cells and then uses an F-actin-dependent motility process to spread from infected cells to surrounding healthy cells. In cultured enterocytes, efficient spread ofL. monocytogenesrequires the secreted bacterial protein InlC. InlC promotes dissemination by physically interacting with and antagonizing the function of the human adaptor protein Tuba. Here we examine the role of InlC and its interaction with host Tuba during infection in mice. The study took advantage of a single-amino-acid substitution (K173A) in InlC that impairs binding to human Tuba but does not affect InlC-mediated inhibition of the NF-κB pathway. Mice were inoculated intravenously with the wild-typeL. monocytogenesstrain EGD, an isogenic strain deleted for theinlCgene (ΔinlC), or a strain expressing K173A mutant InlC (inlC.K173A). The 50% lethal doses (LD50) for the ΔinlCorinlC.K173Amutant strain were approximately 4- or 6-fold greater than that for the wild-type strain, indicating a role forinlCin virulence. Compared to the wild-type strain, theinlC.K173Amutant strain exhibited lower bacterial loads in the liver. Histological analysis of livers indicated that the twoinlCmutant strains produced smaller foci of infection than did the wild-type strain. These smaller foci are consistent with a role for InlC in cell-to-cell spreadin vivo. Taken together, these results provide evidence that interaction of InlC with host Tuba is important for full virulence.

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Virulent Rough Filaments of Listeria monocytogenesfrom Clinical and Food Samples Secreting Wild-Type Levels of Cell-Free p60 Protein

Journal of Clinical Microbiology ◽

10.1128/jcm.38.7.2643-2648.2000 ◽

2000 ◽

Vol 38 (7) ◽

pp. 2643-2648 ◽

Cited By ~ 31

Author(s):

Neil J. Rowan ◽

Alan A. G. Candlish ◽

Andreas Bubert ◽

John G. Anderson ◽

Karl Kramer ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Hela Cells ◽

Morphological Characteristics ◽

Food Samples ◽

Food Environments ◽

Wild Type ◽

Type Form ◽

Rough Cell ◽

Multiple Cells ◽

Wild Type Form

Atypical rough cell filaments of Listeria monocytogenes(designated FR variants), isolated from clinical and food samples, form long filaments up to 96 μm in length and demonstrated wild-type levels of adherence, invasion, and cytotoxicity to human epithelial HEp-2, Caco-2, and HeLa cells. Unlike previously described avirulent rough mutants of L. monocytogenes that secrete diminished levels of the major extracellular protein p60 and that form long chains that consist of multiple cells of similar size (designated MCR variants), FR variants secreted wild-type or greater levels of p60. This study shows that virulent filamentous forms of L. monocytogenes occur in clinical and food environments and have atypical morphological characteristics compared to those of the wild-type form.

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Synergism of imipenem with fosfomycin associated with the active cell wall recycling and heteroresistance in Acinetobacter calcoaceticus-baumannii complex

Scientific Reports ◽

10.1038/s41598-021-04303-7 ◽

2022 ◽

Vol 12 (1) ◽

Author(s):

Uthaibhorn Singkham-in ◽

Tanittha Chatsuwan

Keyword(s):

Cell Wall ◽

De Novo ◽

Acinetobacter Calcoaceticus ◽

Active Cell ◽

Wild Type ◽

Cell Wall Metabolism ◽

Carbapenem Resistant ◽

Type Form ◽

Wild Type Form ◽

Cell Wall Recycling

AbstractThe carbapenem-resistant Acinetobacter calcoaceticus-baumannii (ACB) complex has become an urgent threat worldwide. Here, we determined antibiotic combinations and the feasible synergistic mechanisms against three couples of ACB (A. baumannii (AB250 and A10), A. pittii (AP1 and AP23), and A. nosocomialis (AN4 and AN12)). Imipenem with fosfomycin, the most effective in the time-killing assay, exhibited synergism to all strains except AB250. MurA, a fosfomycin target encoding the first enzyme in the de novo cell wall synthesis, was observed with the wild-type form in all isolates. Fosfomycin did not upregulate murA, indicating the MurA-independent pathway (cell wall recycling) presenting in all strains. Fosfomycin more upregulated the recycling route in synergistic strain (A10) than non-synergistic strain (AB250). Imipenem in the combination dramatically downregulated the recycling route in A10 but not in AB250, demonstrating the additional effect of imipenem on the recycling route, possibly resulting in synergism by the agitation of cell wall metabolism. Moreover, heteroresistance to imipenem was observed in only AB250. Our results indicate that unexpected activity of imipenem on the active cell wall recycling concurrently with the presence of heteroresistance subpopulation to imipenem may lead to the synergism of imipenem and fosfomycin against the ACB isolates.

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

Journal of Experimental Medicine ◽

10.1084/jem.186.7.1159 ◽

1997 ◽

Vol 186 (7) ◽

pp. 1159-1163 ◽

Cited By ~ 173

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.

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DNA Vaccination Protects Mice against Challenge with Listeria monocytogenes Expressing the Hepatitis C Virus NS3 Protein

Infection and Immunity ◽

10.1128/iai.71.11.6372-6380.2003 ◽

2003 ◽

Vol 71 (11) ◽

pp. 6372-6380 ◽

Cited By ~ 12

Author(s):

Benjamin E. Simon ◽

Kenneth A. Cornell ◽

Tina R. Clark ◽

Sunwen Chou ◽

Hugo R. Rosen ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Listeria Monocytogenes ◽

Immune Responses ◽

Bacterial Pathogen ◽

Cell Responses ◽

Ns3 Protein ◽

Plasmid Dna Vaccine ◽

In Vivo Induction

ABSTRACT The goal of this study was to develop a new surrogate challenge model for use in evaluating protective cell-mediated immune responses against hepatitis C virus (HCV) antigens. The use of recombinant Listeria monocytogenes organisms which express HCV antigens provides novel tools with which to assay such in vivo protection, as expression of immunity against this hepatotropic bacterial pathogen is dependent on antigen-specific CD8+ T lymphocytes. A plasmid DNA vaccine encoding a ubiquitin-NS3 fusion protein was generated, and its efficacy was confirmed by in vivo induction of NS3-specific, gamma interferon-secreting T cells following vaccination of BALB/c mice. These immunized mice also exhibited specific in vivo protection against subsequent challenge with a recombinant L. monocytogenes strain (TC-LNS3) expressing the NS3 protein. Notably, sublethal infection of naive mice with strain TC-LNS3 induced similar NS3-specific T-cell responses. These findings suggest that recombinant strains of L. monocytogenes expressing HCV antigens should prove useful for evaluating, or even inducing, protective immune responses against HCV antigens.

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Metal oxide nanoparticle-functionalized sebacic acid-grafted PHEAM nanocarriers for enriched activity of metronidazole against food borne bacteria: in vitro and in vivo study

New Journal of Chemistry ◽

10.1039/c8nj03718c ◽

2018 ◽

Vol 42 (22) ◽

pp. 18437-18447 ◽

Cited By ~ 1

Author(s):

Murugesan Gowri ◽

Kannan Suganya ◽

Nachimuthu Latha ◽

Marudhamuthu Murugan ◽

Mukhtar Ahmed ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Metal Oxide ◽

Sebacic Acid ◽

In Vivo Study ◽

Metal Oxide Nanoparticle ◽

Food Borne ◽

Oxide Nanoparticle

Food borne infection is a serious complication caused by Listeria monocytogenes (L. monocytogenes), a dangerous bacteria.

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Differentiation of propeptide residues regulating the compartmentalization, maturation and activity of the broad-range phospholipase C of Listeria monocytogenes

Biochemical Journal ◽

10.1042/bj20100557 ◽

2010 ◽

Vol 432 (3) ◽

pp. 557-566 ◽

Cited By ~ 10

Author(s):

Emily R. Slepkov ◽

Alan Pavinski Bitar ◽

Hélène Marquis

Keyword(s):

Cell Wall ◽

Listeria Monocytogenes ◽

Amino Acid ◽

Enzymatic Activity ◽

Phospholipase C ◽

Bacterial Pathogen ◽

Amino Acid Residues ◽

C Terminus ◽

N Terminus ◽

Vacuolar Acidification

The intracellular bacterial pathogen Listeria monocytogenes secretes a broad-range phospholipase C enzyme called PC-PLC (phosphatidylcholine phospholipase C) whose compartmentalization and enzymatic activity is regulated by a 24-amino-acid propeptide (Cys28–Ser51). During intracytosolic multiplication, bacteria accumulate the proform of PC-PLC at their membrane–cell-wall interface, whereas during cell-to-cell spread vacuolar acidification leads to maturation and rapid translocation of PC-PLC across the cell wall in a manner that is dependent on Mpl, the metalloprotease of Listeria. In the present study, we generated a series of propeptide mutants to determine the minimal requirement to prevent PC-PLC enzymatic activity and to identify residues regulating compartmentalization and maturation. We found that a single residue at position P1 (Ser51) of the cleavage site is sufficient to prevent enzymatic activity, which is consistent with P1′ (Trp52) being located within the active-site pocket. We observed that mutants with deletions at the N-terminus, but not the C-terminus, of the propeptide are translocated across the cell wall more effectively than wild-type PC-PLC at a physiological pH, and that individual amino acid residues within the N-terminus influence Mpl-mediated maturation of PC-PLC at acidic pH. However, deletion of more than 75% of the propeptide was required to completely prevent Mpl-mediated maturation of PC-PLC. These results indicate that the N-terminus of the propeptide regulates PC-PLC compartmentalization and that specific residues within the N-terminus influence the ability of Mpl to mediate PC-PLC maturation, although a six-residue propeptide is sufficient for Mpl to mediate PC-PLC maturation.

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Compartmentalization of the Broad-Range Phospholipase C Activity to the Spreading Vacuole Is Critical for Listeria monocytogenes Virulence

Infection and Immunity ◽

10.1128/iai.01001-06 ◽

2006 ◽

Vol 75 (1) ◽

pp. 44-51 ◽

Cited By ~ 27

Author(s):

P. S. Marie Yeung ◽

Yoojin Na ◽

Amanda J. Kreuder ◽

Hélène Marquis

Keyword(s):

Listeria Monocytogenes ◽

Phospholipase C ◽

Membrane Damage ◽

Bacterial Pathogen ◽

Host Cells ◽

Virulence Attenuation ◽

Infected Cells ◽

Host Cell Membranes ◽

Cell Spread

ABSTRACT Listeria monocytogenes is a bacterial pathogen that multiplies in the cytosol of host cells and spreads directly from cell to cell by using an actin-based mechanism of motility. The broad-range phospholipase C (PC-PLC) of L. monocytogenes contributes to bacterial escape from vacuoles formed upon cell-to-cell spread. PC-PLC is made as an inactive proenzyme whose activation requires cleavage of an N-terminal propeptide. During infection, PC-PLC is activated specifically in acidified vacuoles. To assess the importance of compartmentalizing PC-PLC activity during infection, we created a mutant that makes constitutively active PC-PLC (the plcBΔpro mutant). Results from intracellular growth and cell-to-cell spread assays showed that the plcBΔpro mutant was sensitive to gentamicin, suggesting that unregulated PC-PLC activity causes damage to host cell membranes. This was confirmed by the observation of a twofold increase in staining of live infected cells by a non-membrane-permeant DNA fluorescent dye. However, membrane damage was not sufficient to cause cell lysis and was dependent on bacterial cell-to-cell spread, suggesting that damage was localized to bacterium-containing filopodia. Using an in vivo competitive infection assay, we observed that the plcBΔpro mutant was outcompeted up to 200-fold by the wild-type strain in BALB/c mice. Virulence attenuation was greater when mice were infected orally than when they were infected intravenously, presumably because the plcBΔpro mutant was initially outcompeted in the intestines, reducing the number of mutant bacteria reaching the liver and spleen. Together, these results emphasize the importance for L. monocytogenes virulence of compartmentalizing the activity of PC-PLC during infection.

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FOG-1 Directly Binds to a Chromatin Remodeling and Deacetylase-Containing Complex To Mediate Transcriptional Repression by GATA-1.

Blood ◽

10.1182/blood.v104.11.355.355 ◽

2004 ◽

Vol 104 (11) ◽

pp. 355-355

Author(s):

Wei Hong ◽

Minako Nakazawa ◽

Ying-Yu Chen ◽

Rajashree Kori ◽

Carrie Rakowski ◽

...

Keyword(s):

Transcriptional Repression ◽

Histone Deacetylation ◽

Gene Repression ◽

Single Point Mutation ◽

Mutant Form ◽

Erythroid Cells ◽

Binding Studies ◽

Nucleosome Remodeling ◽

N Terminus

Abstract Terminal erythroid maturation requires coordinated activation of erythroid marker genes and repression of genes associated with the undifferentiated state. These gene expression patterns are mediated by the concerted action of the erythroid transcription factor GATA-1 and its cofactor FOG-1 that can activate or repress transcription depending on promoter context. We and others showed previously that one mechanism by which FOG-1 functions is to facilitate GATA-1 association with certain DNA target sites in vivo. Using gene complementation studies of GATA-1-ablated erythroid cells, we show that at several GATA-1-repressed target genes (c-kit, c-myc and GATA-2) FOG-1 is dispensable for GATA-1 occupancy in vivo but essential for gene repression and histone deacetylation. To examine how FOG-1 functions as co-repressor we performed affinity chromatography, conventional protein purification and in vitro binding studies to identify proteins that bind FOG-1. We discovered that FOG-1 directly associates with the nucleosome remodeling and histone deacetylase complex NURD. This interaction is mediated by a small conserved domain at the N-terminus of FOG-1 and the MTA-1 subunit of NURD. Association of FOG-1 with NURD occurs in vivo and depends on an intact N-terminus of FOG-1. A series of point mutations across the N-terminus of FOG-1 revealed a tight correlation between NURD binding and transcriptional repression. In particular, a single point mutation at the N-terminus of FOG-1 that abrogated NURD binding also blocked gene repression by FOG-1. Finally, the ability of GATA-1 to repress transcription was impaired in erythroid cells expressing a mutant form of FOG-1 that is defective for NURD binding. Together, these studies show that FOG-1 and very likely other FOG proteins are bona fide co-repressors that link GATA proteins to histone deacetylation and nucleosome remodeling via a novel protein interaction module.

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A non-catalytic histidine residue influences the function of the metalloprotease of Listeria monocytogenes

Microbiology ◽

10.1099/mic.0.071779-0 ◽

2014 ◽

Vol 160 (1) ◽

pp. 142-148 ◽

Cited By ~ 1

Author(s):

Brian M. Forster ◽

Alan Pavinski Bitar ◽

Hélène Marquis

Keyword(s):

Cell Wall ◽

Listeria Monocytogenes ◽

Amino Acid ◽

Positive Charge ◽

Bacterial Pathogen ◽

Histidine Residue ◽

Amino Acid Residues ◽

Intracellular Growth ◽

N Terminus ◽

Membrane Bound

Mpl, a thermolysin-like metalloprotease, and PC-PLC, a phospholipase C, are synthesized as proenzymes by the intracellular bacterial pathogen Listeria monocytogenes. During intracellular growth, L. monocytogenes is temporarily confined in a membrane-bound vacuole whose acidification leads to Mpl autolysis and Mpl-mediated cleavage of the PC-PLC N-terminal propeptide. Mpl maturation also leads to the secretion of both Mpl and PC-PLC across the bacterial cell wall. Previously, we identified negatively charged and uncharged amino acid residues within the N terminus of the PC-PLC propeptide that influence the ability of Mpl to mediate the maturation of PC-PLC, suggesting that these residues promote the interaction of the PC-PLC propeptide with Mpl. In the present study, we identified a non-catalytic histidine residue (H226) that influences Mpl secretion across the cell wall and its ability to process PC-PLC. Our results suggest that a positive charge at position 226 is required for Mpl functions other than autolysis. Based on the charge requirement at this position, we hypothesize that this residue contributes to the interaction of Mpl with the PC-PLC propeptide.

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