The increase in mucin exocytosis and the upregulation of MUC genes encoding for membrane-bound mucins induced by the thiol-activated exotoxin listeriolysin O is a host cell defence response that inhibits the cell-entry of Listeria monocytogenes

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.

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Listeria monocytogenes Exploits Normal Host Cell Processes to Spread from Cell to Cell✪

The Journal of Cell Biology ◽

10.1083/jcb.146.6.1333 ◽

1999 ◽

Vol 146 (6) ◽

pp. 1333-1350 ◽

Cited By ~ 114

Author(s):

Jennifer R. Robbins ◽

Angela I. Barth ◽

Hélène Marquis ◽

Eugenio L. de Hostos ◽

W. James Nelson ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Host Cell ◽

Mdck Cells ◽

Recipient Cell ◽

Cell Monolayer ◽

Bacterial Pathogen ◽

Host Cells ◽

Turnover Rates ◽

Membrane Bound ◽

Intercellular Spread

The bacterial pathogen, Listeria monocytogenes, grows in the cytoplasm of host cells and spreads intercellularly using a form of actin-based motility mediated by the bacterial protein ActA. Tightly adherent monolayers of MDCK cells that constitutively express GFP-actin were infected with L. monocytogenes, and intercellular spread of bacteria was observed by video microscopy. The probability of formation of membrane-bound protrusions containing bacteria decreased with host cell monolayer age and the establishment of extensive cell-cell contacts. After their extension into a recipient cell, intercellular membrane-bound protrusions underwent a period of bacterium-dependent fitful movement, followed by their collapse into a vacuole and rapid vacuolar lysis. Actin filaments in protrusions exhibited decreased turnover rates compared with bacterially associated cytoplasmic actin comet tails. Recovery of motility in the recipient cell required 1–2 bacterial generations. This delay may be explained by acid-dependent cleavage of ActA by the bacterial metalloprotease, Mpl. Importantly, we have observed that low levels of endocytosis of neighboring MDCK cell surface fragments occurs in the absence of bacteria, implying that intercellular spread of bacteria may exploit an endogenous process of paracytophagy.

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Relative Roles of Listeriolysin O, InlA, and InlB inListeria monocytogenesUptake by Host Cells

Infection and Immunity ◽

10.1128/iai.00555-18 ◽

2018 ◽

Vol 86 (10) ◽

Cited By ~ 10

Author(s):

Christopher C. Phelps ◽

Stephen Vadia ◽

Eusondia Arnett ◽

Yubo Tan ◽

Xiaoli Zhang ◽

...

Keyword(s):

Endothelial Cells ◽

Host Cell ◽

Cell Types ◽

Bacterial Attachment ◽

Host Cells ◽

Listeriolysin O ◽

Gene Encoding ◽

Content Type ◽

Membrane Perforation ◽

Genes Encoding

ABSTRACTListeria monocytogenesis a facultative intracellular pathogen that infects a wide variety of cells, causing the life-threatening disease listeriosis.L. monocytogenesvirulence factors include two surface invasins, InlA and InlB, known to promote bacterial uptake by host cells, and the secreted pore-forming toxin listeriolysin O (LLO), which disrupts the phagosome to allow bacterial proliferation in the cytosol. In addition, plasma membrane perforation by LLO has been shown to facilitateL. monocytogenesinternalization into epithelial cells. In this work, we tested the host cell range and importance of LLO-mediatedL. monocytogenesinternalization relative to the canonical invasins, InlA and InlB. We measured the efficiencies ofL. monocytogenesassociation with and internalization into several human cell types (hepatocytes, cytotrophoblasts, and endothelial cells) using wild-type bacteria and isogenic single, double, and triple deletion mutants for the genes encoding InlA, InlB and LLO. No role for InlB was detected in any tested cells unless the InlB expression level was substantially enhanced, which was achieved by introducing a mutation (prfA*) in the gene encoding the transcription factor PrfA. In contrast, InlA and LLO were the most critical invasion factors, although they act in a different manner and in a cell-type-dependent fashion. As expected, InlA facilitates both bacterial attachment and internalization in cells that express its receptor, E-cadherin. LLO promotesL. monocytogenesinternalization into hepatocytes, but not into cytotrophoblasts and endothelial cells. Finally, LLO and InlA cooperate to increase the efficiency of host cell invasion byL. monocytogenes.

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

Infection and Immunity ◽

10.1128/iai.73.10.6639-6646.2005 ◽

2005 ◽

Vol 73 (10) ◽

pp. 6639-6646 ◽

Cited By ~ 25

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.

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Gliding Motility Leads to Active Cellular Invasion by Cryptosporidium parvum Sporozoites

Infection and Immunity ◽

10.1128/iai.73.9.5379-5387.2005 ◽

2005 ◽

Vol 73 (9) ◽

pp. 5379-5387 ◽

Cited By ~ 67

Author(s):

Dawn M. Wetzel ◽

Joann Schmidt ◽

Mark S. Kuhlenschmidt ◽

J. P. Dubey ◽

L. David Sibley

Keyword(s):

Host Cell ◽

Cryptosporidium Parvum ◽

Diarrheal Disease ◽

Cytochalasin D ◽

Gliding Motility ◽

Cell Entry ◽

Host Cells ◽

Cellular Invasion ◽

Butanedione Monoxime ◽

Membrane Bound

ABSTRACT We examined gliding motility and cell invasion by an early-branching apicomplexan, Cryptosporidium parvum, which causes diarrheal disease in humans and animals. Real-time video microscopy demonstrated that C. parvum sporozoites undergo circular and helical gliding, two of the three stereotypical movements exhibited by Toxoplasma gondii tachyzoites. C. parvum sporozoites moved more rapidly than T. gondii sporozoites, which showed the same rates of motility as tachyzoites. Motility by C. parvum sporozoites was prevented by latrunculin B and cytochalasin D, drugs that depolymerize the parasite actin cytoskeleton, and by the myosin inhibitor 2,3-butanedione monoxime. Imaging of the initial events in cell entry by Cryptosporidium revealed that invasion occurs rapidly; however, the parasite does not enter deep into the cytosol but rather remains at the cell surface in a membrane-bound compartment. Invasion did not stimulate rearrangement of the host cell cytoskeleton and was inhibited by cytochalasin D, even in host cells that were resistant to the drug. Our studies demonstrate that C. parvum relies on a conserved actin-myosin motor for motility and active penetration of its host cell, thus establishing that this is a widely conserved feature of the Apicomplexa.

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Listeriolysin O as a Reporter To Identify Constitutive and In Vivo-Inducible Promoters in the Pathogen Listeria monocytogenes

Infection and Immunity ◽

10.1128/iai.68.6.3242-3250.2000 ◽

2000 ◽

Vol 68 (6) ◽

pp. 3242-3250 ◽

Cited By ~ 32

Author(s):

Iharilalao Dubail ◽

Patrick Berche ◽

Alain Charbit

Keyword(s):

Listeria Monocytogenes ◽

Host Cells ◽

Transcriptional Analysis ◽

Listeriolysin O ◽

Gram Positive ◽

Genome Database ◽

Inducible Promoters ◽

Genes Encoding

ABSTRACT Listeria monocytogenes is a facultative intracellular gram-positive bacterium capable of growing in the cytoplasm of infected host cells. Bacterial escape from the phagosomal vacuole of infected cells is mainly mediated by the pore-forming hemolysin listeriolysin O (LLO) encoded by hly. LLO-negative mutants of L. monocytogenes are avirulent in the mouse model. We have developed a genetic system with hly as a reporter gene allowing the identification of both constitutive and in vivo-inducible promoters of this pathogen. Genomic libraries were created by randomly inserting L. monocytogenes chromosomal fragments upstream of the promoterless hly gene cloned into gram-positive and gram-negative shuttle vectors and expressed in an LLO-negative mutant strain. With this hly-based promoter trap system, combined with access to the L. monocytogenes genome database, we identified 20 in vitro-transcribed genes, including genes encoding (i) p60, a previously known virulence gene, (ii) a putative new hemolysin, and (iii) two proteins of the general protein secretion pathway. By using the hly-based system as an in vivo expression technology tool, nine in vivo-induced loci of L. monocytogenes were identified, including genes encoding (i) the previously known in vivo-inducible phosphatidylinositol phospholipase C and (ii) a putative N-acetylglucosamine epimerase, possibly involved in teichoic acid biosynthesis. The use of hly as a reporter is a simple and powerful alternative to classical methods for transcriptional analysis to monitor promoter activity in L. monocytogenes.

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Listeria monocytogenes Stimulates Mucus Exocytosis in Cultured Human Polarized Mucosecreting Intestinal Cells through Action of Listeriolysin O

Infection and Immunity ◽

10.1128/iai.66.8.3673-3681.1998 ◽

1998 ◽

Vol 66 (8) ◽

pp. 3673-3681 ◽

Cited By ~ 34

Author(s):

Marie-Hélène Coconnier ◽

Elyess Dlissi ◽

Myriam Robard ◽

Christian L. Laboisse ◽

Jean-Louis Gaillard ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Direct Action ◽

Membrane Vesicle ◽

Regulatory System ◽

Cell Entry ◽

Intracellular Pathogen ◽

Listeriolysin O ◽

Stimulatory Action ◽

Isogenic Mutants ◽

Stimulation Of

ABSTRACT When the intracellular pathogen Listeria monocytogenesinfects cultured human mucosecreting polarized HT29-MTX cells apically, it induces the stimulation of mucus exocytosis without cell entry. Using a set of isogenic mutants and purified listeriolysin O (LLO), we identified the L. monocytogenes thiol-activated exotoxin LLO as the agonist of mucus secretion. We demonstrated that the LLO-induced mucus exocytosis did not result from the LLO membrane-damaging activity. We found that LLO-induced mucus exocytosis is an event requiring the binding of LLO to a brush border-associated receptor and membrane oligomerization of the exotoxin. By a pharmacological approach, we demonstrated that no regulatory system or intracellular transducing signal known to be involved in control of mucin exocytosis was activated by LLO. Based on the present data, the stimulatory action of LLO on mucin exocytosis could be accounted for either by an unknown signaling system which remains to be determined or by direct action of LLO with the membrane vesicle components involved in the intracellular vesicular transport of mucins.

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The cell biology of Listeria monocytogenes infection

The Journal of Cell Biology ◽

10.1083/jcb.200205009 ◽

2002 ◽

Vol 158 (3) ◽

pp. 409-414 ◽

Cited By ~ 283

Author(s):

Daniel A. Portnoy ◽

Victoria Auerbuch ◽

Ian J. Glomski

Keyword(s):

Listeria Monocytogenes ◽

Host Cell ◽

Cell Biology ◽

Productive Infection ◽

Acquired Immunity ◽

Listeriolysin O ◽

Listeria Monocytogenes Infection ◽

Infected Cells ◽

Fail Safe

Listeria monocytogenes has emerged as a remarkably tractable pathogen to dissect basic aspects of cell biology, intracellular pathogenesis, and innate and acquired immunity. In order to maintain its intracellular lifestyle, L. monocytogenes has evolved a number of mechanisms to exploit host processes to grow and spread cell to cell without damaging the host cell. The pore-forming protein listeriolysin O mediates escape from host vacuoles and utilizes multiple fail-safe mechanisms to avoid causing toxicity to infected cells. Once in the cytosol, the L. monocytogenes ActA protein recruits host cell Arp2/3 complexes and enabled/vasodilator-stimulated phosphoprotein family members to mediate efficient actin-based motility, thereby propelling the bacteria into neighboring cells. Alteration in any of these processes dramatically reduces the ability of the bacteria to establish a productive infection in vivo.

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Early Events During Invasion of J774 Cells by Listeria Monocytogenes

Microscopy and Microanalysis ◽

10.1017/s1431927600029214 ◽

2001 ◽

Vol 7 (S2) ◽

pp. 628-629

Author(s):

Paul Webster

Keyword(s):

Listeria Monocytogenes ◽

Protein Composition ◽

The Elderly ◽

Farm Animals ◽

Listeriolysin O ◽

Cell Cytoplasm ◽

Bacterial Surface ◽

J774 Cells ◽

Membrane Bound ◽

Gain Access

The gram positive bacillus, Listeria monocytogenesis a contaminant of the food we eat and although it is a common pathogen in farm animals L. monocytogenesdoes not normally infect humans. However, immunocompromised individuals, infants and the elderly are susceptible to infection. The bacteria, taken into membrane bound phagosomes, use a thiol-activated cytolysin listeriolysin O to disrupt the membrane (LLO) and gain access to the cell cytoplasm. Subsequent actin polymerizations on the bacterial surface induce motility in the cell and long cytoplasmic protrusions at the cell surface that spread bacteria to neighboring cells.L. monocytogenesenters cells by phagocytosis. By using short infection times of 5 min or less, it was possible to examine early interactions between J774 cells and L. monocytogenes.This study shows that during the early stages of internalization the bacteria are able to modify the protein composition of the forming phagosome membrane.

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