ActA Is Required for Crossing of the Fetoplacental Barrier by Listeria monocytogenes

ABSTRACT The facultative intracellular bacterial pathogen Listeria monocytogenes induces severe fetal infection during pregnancy. Little is known about the molecular mechanisms allowing the maternofetal transmission of bacteria. In this work, we studied fetoplacental invasion by infecting mice with various mutants lacking virulence factors involved in the intracellular life cycle of L. monocytogenes. We found that the placenta was highly susceptible to bacteria, including avirulent bacteria, such as an L. monocytogenes mutant with an hly deletion (ΔLLO) and a nonpathogenic species, Listeria innocua, suggesting that permissive trophoblastic cells, trapping bacteria, provide a protective niche for bacterial survival. The ΔLLO mutant, which is unable to escape the phagosomal compartment of infected cells, failed to grow in the trophoblast tissue and to invade the fetus. Mutant bacteria with inlA and inlB deletion (ΔInlAB) grew in the placenta and fetus as well as did the wild-type virulent stain (EGDwt), indicating that in the murine model, internalins A and B are not involved in fetoplacental invasion by L. monocytogenes. Pregnant mice were then infected with an actA deletion (ΔActA) strain, a virulence-attenuated mutant that is unable to polymerize actin and to spread from cell to cell. With the ΔActA mutant, fetal infection occurs, but with a significant delay and restriction, and it requires a placental bacterial load 2 log units higher than that for the wild-type virulent strain. Definitive evidence for the role of ActA was provided by showing that a actA-complemented ΔActA mutant was restored in its capacity to invade fetuses. ActA-mediated cell-to-cell spreading plays a major role in the vertical transmission of L. monocytogenes to the fetus in the murine model.

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Expression of NADPH Oxidase-Dependent Resistance to Listeriosis in Mice Occurs during the First 6 to 12 Hours of Liver Infection

Infection and Immunity ◽

10.1128/iai.70.12.7179-7181.2002 ◽

2002 ◽

Vol 70 (12) ◽

pp. 7179-7181 ◽

Cited By ~ 11

Author(s):

Ronald LaCourse ◽

Lynn Ryan ◽

Robert J. North

Keyword(s):

Listeria Monocytogenes ◽

Nadph Oxidase ◽

Bacterial Load ◽

Wild Type ◽

Liver Infection

ABSTRACT Wild-type mice inoculated with Listeria monocytogenes intravenously were capable of reducing the bacterial load in their livers by 90% within 6 h. In contrast, mice with deletions of the gene for NADPH oxidase were incapable of expressing this early oxygen-dependent anti-Listeria defense and consequently showed higher levels of liver infection at later times.

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SGLT1 Deficiency Turns Listeria Infection into a Lethal Disease in Mice

Cellular Physiology and Biochemistry ◽

10.1159/000479197 ◽

2017 ◽

Vol 42 (4) ◽

pp. 1358-1365 ◽

Cited By ~ 8

Author(s):

Piyush Sharma ◽

Vishal Khairnar ◽

Ivana Vrhovac Madunić ◽

Yogesh Singh ◽

Aleksandra Pandyra ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Glucose Uptake ◽

Bacterial Load ◽

Bacterial Clearance ◽

Wild Type ◽

Hepatic Expression ◽

Cell Functions ◽

Tnf Α ◽

Listeria Infection ◽

Deficient Mice

Background: Cellular glucose uptake may involve either non-concentrative glucose carriers of the GLUT family or Na+-coupled glucose-carrier SGLT1, which accumulates glucose against glucose gradients and may thus accomplish cellular glucose uptake even at dramatically decreased extracellular glucose concentrations. SGLT1 is not only expressed in epithelia but as well in tumour cells and immune cells. Immune cell functions strongly depend on their metabolism, therefore we hypothesized that deficiency of SGLT1 modulates the defence against bacterial infection. To test this hypothesis, we infected wild type mice and gene targeted mice lacking functional SGLT1 with Listeria monocytogenes. Methods: SGLT1 deficient mice and wild type littermates were infected with 1x104 CFU Listeria monocytogenes intravenously. Bacterial titers were determined by colony forming assay, SGLT1, TNF-α, IL-6 and IL-12a transcript levels were determined by qRT-PCR, as well as SGLT1 protein abundance and localization by immunohistochemistry. Results: Genetic knockout of SGLT1 (Slc5a1–/– mice) significantly compromised bacterial clearance following Listeria monocytogenes infection with significantly enhanced bacterial load in liver, spleen, kidney and lung, and significantly augmented hepatic expression of TNF-α and IL-12a. While all wild type mice survived, all SGLT1 deficient mice died from the infection. Conclusions: SGLT1 is required for bacterial clearance and host survival following murine Listeria infection.

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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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The Propeptide of the Metalloprotease of Listeria monocytogenes Controls Compartmentalization of the Zymogen during Intracellular Infection

Journal of Bacteriology ◽

10.1128/jb.01168-08 ◽

2009 ◽

Vol 191 (11) ◽

pp. 3594-3603 ◽

Cited By ~ 10

Author(s):

Heather S. O'Neil ◽

Brian M. Forster ◽

Kari L. Roberts ◽

Andrew J. Chambers ◽

Alan Pavinski Bitar ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Catalytic Domain ◽

Bacterial Pathogen ◽

Folding Kinetics ◽

Wild Type ◽

Semisolid Medium ◽

Mutant Proteins ◽

Intracellular Infection ◽

Infected Cells

ABSTRACT Integral to the virulence of the intracellular bacterial pathogen Listeria monocytogenes is its metalloprotease (Mpl). Mpl regulates the activity and compartmentalization of the bacterial broad-range phospholipase C (PC-PLC). Mpl is secreted as a proprotein that undergoes intramolecular autocatalysis to release its catalytic domain. In related proteases, the propeptide serves as a folding catalyst and can act either in cis or in trans. Propeptides can also influence protein compartmentalization and intracellular trafficking or decrease folding kinetics. In this study, we aimed to determine the role of the Mpl propeptide by monitoring the behavior of Mpl synthesized in the absence of its propeptide (MplΔpro) and of two Mpl single-site mutants with unstable propeptides: Mpl(H75V) and Mpl(H95L). We observed that all three Mpl mutants mediate PC-PLC activation when bacteria are grown on semisolid medium, but to a lesser extent than wild-type Mpl, indicating that, although not essential, the propeptide enhances the production of active Mpl. However, the mutant proteins were not functional in infected cells, as determined by monitoring PC-PLC maturation and compartmentalization. This defect could not be rescued by providing the propeptide in trans to the mplΔpro mutant. We tested the compartmentalization of Mpl during intracellular infection and observed that the mutant Mpl species were aberrantly secreted in the cytosol of infected cells. These data indicated that the propeptide of Mpl serves to maintain bacterium-associated Mpl and that this localization is essential to the function of Mpl during intracellular infection.

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Redundant Roles for Met Docking Site Tyrosines and the Gab1 Pleckstrin Homology Domain in InlB-Mediated Entry of Listeria monocytogenes

Infection and Immunity ◽

10.1128/iai.73.4.2061-2074.2005 ◽

2005 ◽

Vol 73 (4) ◽

pp. 2061-2074 ◽

Cited By ~ 18

Author(s):

Tumay Basar ◽

Yang Shen ◽

Keith Ireton

Keyword(s):

Listeria Monocytogenes ◽

Mammalian Cells ◽

Bacterial Pathogen ◽

Human Cell Line ◽

Pleckstrin Homology Domain ◽

Dependent Manner ◽

Docking Site ◽

Ph Domain ◽

Wild Type ◽

Pleckstrin Homology

ABSTRACT The bacterial pathogen Listeria monocytogenes causes food-borne illnesses leading to gastroenteritis, meningitis, or abortion. Listeria induces its internalization into some mammalian cells through interaction of the bacterial surface protein InlB with host Met receptor tyrosine kinase. Binding of InlB leads to phosphorylation of Met and the adapter Gab1 and to activation of host phosphoinositide (PI) 3-kinase. The mammalian ligand of Met, hepatocyte growth factor, promotes cell motility and morphogenesis in a manner dependent on phosphorylation of two docking site tyrosines at positions 1349 and 1356 in the receptor's cytoplasmic tail. Here we determined if these tyrosines were essential for Listeria entry. A derivative of the human cell line T47D stably expressing a truncated Met lacking most of its cytoplasmic domain was unable to support InlB-mediated signaling or entry. Surprisingly, cells expressing mutant Met containing phenylalanine substitutions in both tyrosines 1349 and 1356 (MetYF) allowed entry and InlB-induced Gab1 phosphorylation. However, in contrast to the situation in cells expressing wild-type Met, Gab1 phosphorylation in MetYF cells required PI 3-kinase activity. The Gab1 pleckstrin homology (PH) domain was constitutively associated with the plasma membrane of cells in a PI 3-kinase-dependent manner. Overexpression of the PH domain blocked entry of Listeria into cells expressing MetYF but not into cells expressing wild-type Met. Taken together, these results indicate that the docking site tyrosines are dispensable for internalization when membrane localization of Gab1 is constitutive. Distinct pathways of recruitment by phosphorylated tyrosines in Met and PH domain ligands in the membrane are redundant for bacterial entry.

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Interleukin-21 reduces Listeria monocytogenes secondary infection via CD8+ effector memory T cells

10.21203/rs.3.rs-41839/v1 ◽

2020 ◽

Author(s):

Md. Yeashin Gazi ◽

Yuji Takeda ◽

Hidetoshi Nara ◽

Akemi Araki ◽

Nobuhito Nemoto ◽

...

Keyword(s):

T Cells ◽

Listeria Monocytogenes ◽

T Cell ◽

Primary Infection ◽

Bacterial Load ◽

Challenge Infection ◽

Effector Memory ◽

Intracellular Bacteria ◽

Wild Type ◽

Secondary Challenge

Abstract Background/Purpose: Interleukin-21 (IL-21), which is a member of the common γ-chain cytokine family, is mainly produced by CD4+ T cells and has broad impact on immune responses. IL-21 isoform is a splicing variant of IL-21 and is functionally similar to conventional IL-21. We established IL-21 isoform transgenic (IL-21isoTg) mouse, which constitutively expresses IL-21 isoform specifically in T cells. IL-21isoTg mouse possesses high amount of CD8+ T cells in normal physiological condition. The purpose of this study is to determine whether CD8+ T cells in the IL-21isoTg mouse work against intracellular bacteria infection.Methods: Wild type (WT) and IL-21isoTg mouse are orally inoculated Listeria monocytogenes (L. monocytogenes) on day 0, and 15 days after primary infection. Bacterial load in each organs, and T cell responses are analyzed.Results: IL-21isoTg and wild type (WT) mouse had similar bacterial load after L. monocytogenes primary infection. On the other hand, after secondary challenge infection, IL-21isoTg mouse exhibited reduced bacterial load in some organs compared to WT. Analysis of T cell response after primary infection showed that IL-21isoTg mouse induced higher levels of CD8+ effector memory T (TEM) cells than WT.Conclusion: IL-21-induced CD8+ TEM cells might eventually reduce the bacterial load in organs after secondary challenge infection in IL-21isoTg mouse. To the best of our knowledge, this is the first study to show that IL-21 is a pivotal factor involved in eliminating intracellular bacteria, probably through CD8+ TEM cells.

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Cell competition between wild-type and JAK2V617F mutant cells prevents disease relapse after stem cell transplantation in a murine model of myeloproliferative neoplasm

Experimental Hematology and Oncology ◽

10.1186/s40164-021-00241-2 ◽

2021 ◽

Vol 10 (1) ◽

Author(s):

Haotian Zhang ◽

Melissa Castiglione ◽

Lei Zheng ◽

Huichun Zhan

Keyword(s):

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Murine Model ◽

Molecular Mechanisms ◽

Myeloproliferative Neoplasms ◽

Disease Relapse ◽

Wild Type ◽

Cell Competition ◽

Mutant Cells

AbstractDisease relapse after allogeneic stem cell transplantation is a major cause of treatment-related morbidity and mortality in patients with myeloproliferative neoplasms (MPNs). The cellular and molecular mechanisms for MPN relapse are not well understood. Here, we established a murine model of MPN relapse, in which ~ 60% of the MPN recipient mice develop disease relapse after receiving stem cell transplantation with wild-type marrow donor. Using this model, we find that impaired wild-type cell function is associated with MPN disease relapse. We also show that competition between wild-type and JAK2V617F mutant cells can modulate the immune cell composition and PD-L1 expression induced by the JAK2V617F oncogene. These results suggest that cell competition between wild-type donor cells and JAK2V617F mutant recipient cells can prevent MPN disease relapse after stem cell transplantation.

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The Metalloprotease of Listeria monocytogenes Is Activated by Intramolecular Autocatalysis

Journal of Bacteriology ◽

10.1128/jb.00852-07 ◽

2007 ◽

Vol 190 (1) ◽

pp. 107-111 ◽

Cited By ~ 25

Author(s):

Alan Pavinski Bitar ◽

Min Cao ◽

Hélène Marquis

Keyword(s):

Listeria Monocytogenes ◽

Bacterial Pathogen ◽

Mutant Form ◽

Wild Type ◽

Food Borne ◽

Large N ◽

N Terminus ◽

Type Form ◽

Wild Type Form

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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Genes Associated with Desiccation and Osmotic Stress in Listeria monocytogenes as Revealed by Insertional Mutagenesis

Applied and Environmental Microbiology ◽

10.1128/aem.01134-15 ◽

2015 ◽

Vol 81 (16) ◽

pp. 5350-5362 ◽

Cited By ~ 22

Author(s):

Patricia A. Hingston ◽

Marta J. Piercey ◽

Lisbeth Truelstrup Hansen

Keyword(s):

Listeria Monocytogenes ◽

Osmotic Stress ◽

Energy Production ◽

Insertional Mutagenesis ◽

Molecular Mechanisms ◽

Damage Control ◽

Membrane Lipid ◽

Lipid Biosynthesis ◽

Wild Type ◽

Content Type

ABSTRACTListeria monocytogenesis a foodborne pathogen whose survival in food processing environments may be associated with its tolerance to desiccation. To probe the molecular mechanisms used by this bacterium to adapt to desiccation stress, a transposon library of 11,700L. monocytogenesmutants was screened, using a microplate assay, for strains displaying increased or decreased desiccation survival (43% relative humidity, 15°C) in tryptic soy broth (TSB). The desiccation phenotypes of selected mutants were subsequently assessed on food-grade stainless steel (SS) coupons in TSB plus 1% glucose (TSB-glu). Single transposon insertions in mutants exhibiting a change in desiccation survival of >0.5 log CFU/cm2relative to that of the wild type were determined by sequencing arbitrary PCR products. Strain morphology, motility, and osmotic stress survival (in TSB-glu plus 20% NaCl) were also analyzed. The initial screen selected 129 desiccation-sensitive (DS) and 61 desiccation-tolerant (DT) mutants, out of which secondary screening on SS confirmed 15 DT and 15 DS mutants. Among the DT mutants, seven immotile and flagellum-less strains contained transposons in genes involved in flagellum biosynthesis (fliP,flhB,flgD,flgL) and motor control (motB,fliM,fliY), while others harbored transposons in genes involved in membrane lipid biosynthesis, energy production, potassium uptake, and virulence. The genes that were interrupted in the 15 DS mutants included those involved in energy production, membrane transport, protein metabolism, lipid biosynthesis, oxidative damage control, and putative virulence. Five DT and 14 DS mutants also demonstrated similar significantly (P< 0.05) different survival relative to that of the wild type when exposed to osmotic stress, demonstrating that some genes likely have similar roles in allowing the organism to survive the two water stresses.

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Cell competition between wild-type and JAK2V617F mutant cells prevents disease relapse after stem cell transplantation in a murine model of myeloproliferative neoplasm

10.1101/2021.08.24.457589 ◽

2021 ◽

Author(s):

Haotian Zhang ◽

Melissa Castiglione ◽

Lei Zheng ◽

Huichun Zhan

Keyword(s):

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Murine Model ◽

Molecular Mechanisms ◽

Myeloproliferative Neoplasms ◽

Disease Relapse ◽

Wild Type ◽

Cell Competition ◽

Mutant Cells

Disease relapse after allogeneic stem cell transplantation is a major cause of treatment-related morbidity and mortality in patients with myeloproliferative neoplasms (MPNs). The cellular and molecular mechanisms for MPN relapse are not well understood. Here, we established a murine model of MPN relapse, in which ~60% of the MPN recipient mice develop disease relapse after receiving stem cell transplantation with wild-type marrow donor. Using this model, we find that impaired wild-type cell function is associated with MPN disease relapse. We also show that competition between wild-type and JAK2V617F mutant cells can modulate the immune cell composition and PD-L1 expression induced by the JAK2V617F oncogene. These results suggest that cell competition between wild-type donor cells and JAK2V617F mutant recipient cells can prevent MPN disease relapse after stem cell transplantation.

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