Analysis of the Isoprenoid Biosynthesis Pathways in Listeria monocytogenes Reveals a Role for the Alternative 2-C-Methyl-d-Erythritol 4-Phosphate Pathway in Murine Infection

ABSTRACT Most bacteria synthesize isoprenoids through one of two essential pathways which provide the basic building block, isopentyl diphosphate (IPP): either the classical mevalonate pathway or the alternative non-mevalonate 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. However, postgenomic analyses of the Listeria monocytogenes genome revealed that this pathogen possesses the genetic capacity to produce the complete set of enzymes involved in both pathways. The nonpathogenic species Listeria innocua naturally lacks the last two genes (gcpE and lytB) of the MEP pathway, and bioinformatic analyses strongly suggest that the genes have been lost through evolution. In the present study we show that heterologous expression of gcpE and lytB in L. innocua can functionally restore the MEP pathway in this organism and confer on it the ability to induce Vγ9Vδ2 T cells. We have previously confirmed that both pathways are functional in L. monocytogenes and can provide sufficient IPP for normal growth in laboratory media (M. Begley, C. G. Gahan, A. K. Kollas, M. Hintz, C. Hill, H. Jomaa, and M. Eberl, FEBS Lett. 561:99-104, 2004). Here we describe a targeted mutagenesis strategy to create a double pathway mutant in L. monocytogenes which cannot grow in the absence of exogenously provided mevalonate, confirming the requirement for at least one intact pathway for growth. In addition, murine studies revealed that mutants lacking the MEP pathway were impaired in virulence relative to the parent strain during intraperitoneal infection, while mutants lacking the classical mevalonate pathway were not impaired in virulence potential. In vivo bioluminescence imaging also confirmed in vivo expression of the gcpE gene (MEP pathway) during murine infection.

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Toll-Like Receptor 2- and MyD88-Dependent Phosphatidylinositol 3-Kinase and Rac1 Activation Facilitates the Phagocytosis of Listeria monocytogenes by Murine Macrophages

Infection and Immunity ◽

10.1128/iai.01138-09 ◽

2010 ◽

Vol 78 (6) ◽

pp. 2857-2867 ◽

Cited By ~ 49

Author(s):

Yanna Shen ◽

Ikuo Kawamura ◽

Takamasa Nomura ◽

Kohsuke Tsuchiya ◽

Hideki Hara ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Rho Gtpases ◽

Peritoneal Macrophages ◽

Infection Model ◽

Phosphatidylinositol 3 Kinase ◽

Macrophage Phagocytosis ◽

Small Rho Gtpases ◽

Intraperitoneal Infection ◽

Phosphatidylinositol 3

ABSTRACT Toll-like receptors (TLRs) play a key role in the innate immune response by sensing bacterial ligands. The mechanisms involved in the TLR-mediated cytokine response are well established; however, the possible contribution of TLR-dependent recognition of bacteria to macrophage phagocytosis remains unclear. Listeria monocytogenes is an intracellular, parasitic, Gram-positive bacterium recognized mainly by TLR2. In this study, we investigated whether TLR2-dependent signaling is involved in the phagocytosis of L. monocytogenes by macrophages. We found no difference in the number of L. monocytogenes cells associating with wild-type (WT) and TLR2−/− macrophages 1 h after infection. However, the number of L. monocytogenes cells phagocytosed in TLR2−/− and MyD88−/− macrophages was significantly lower than that of WT macrophages. In addition, lipopolysaccharide (LPS) treatment restored impaired phagocytic activity of TLR2−/− macrophages but did not enhance the activity of MyD88−/− macrophages. The efficiency of phagocytosis was suppressed by inhibitors of phosphatidylinositol 3-kinase (PI3K) and the small Rho GTPases but not by cycloheximide. Moreover, functional activation of PI3K and Rac1 was impaired in TLR2−/− and MyD88−/− macrophages. In an in vivo infection model, we found significantly lower numbers of L. monocytogenes cells phagocytosed in peritoneal macrophages of TLR2−/− and MyD88−/− mice after intraperitoneal infection. Moreover, a lower number of bacteria were detected in the spleens of TLR2−/− mice 1 day after intravenous infection than in WT mice. These results clearly indicated that TLR2-MyD88-dependent signaling enhances the basal level of phagocytosis of L. monocytogenes by macrophages through activation of PI3K and Rac1, not by synthesis of proinflammatory cytokines or expression of phagocytic receptors.

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Dysfunctional Vγ9Vδ2 T cells are negative prognosticators and markers of dysregulated mevalonate pathway activity in chronic lymphocytic leukemia cells

Blood ◽

10.1182/blood-2012-03-417519 ◽

2012 ◽

Vol 120 (16) ◽

pp. 3271-3279 ◽

Cited By ~ 31

Author(s):

Marta Coscia ◽

Candida Vitale ◽

Silvia Peola ◽

Myriam Foglietta ◽

Micol Rigoni ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

Mevalonate Pathway ◽

Variable Region ◽

Lymphocytic Leukemia ◽

Effector Memory ◽

Pathway Activity ◽

Vγ9vδ2 T Cells ◽

Time To First Treatment

Abstract The role of Vγ9Vδ2 T cells in chronic lymphocytic leukemia (CLL) is unexplored, although these cells have a natural inclination to react against B-cell malignancies. Proliferation induced by zoledronic acid was used as a surrogate of γδ TCR-dependent stimulation to functionally interrogate Vγ9Vδ2 T cells in 106 untreated CLL patients. This assay permitted the identification of responder and low-responder (LR) patients. The LR status was associated with greater baseline counts of Vγ9Vδ2 T cells and to the expansion of the effector memory and terminally differentiated effector memory subsets. The tumor immunoglobulin heavy chain variable region was more frequently unmutated in CLL cells of LR patients, and the mevalonate pathway, which generates Vγ9Vδ2 TCR ligands, was more active in unmutated CLL cells. In addition, greater numbers of circulating regulatory T cells were detected in LR patients. In multivariate analysis, the LR condition was an independent predictor of shorter time-to-first treatment. Accordingly, the time-to-first treatment was significantly shorter in patients with greater baseline numbers of total Vγ9Vδ2 T cells and effector memory and terminally differentiated effector memory subpopulations. These results unveil a clinically relevant in vivo relationship between the mevalonate pathway activity of CLL cells and dys-functional Vγ9Vδ2 T cells.

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Exploring Listeria monocytogenes Transcriptomes in Correlation with Divergence of Lineages and Virulence as Measured in Galleria mellonella

Applied and Environmental Microbiology ◽

10.1128/aem.01370-19 ◽

2019 ◽

Vol 85 (21) ◽

Cited By ~ 2

Author(s):

Bo-Hyung Lee ◽

Dominique Garmyn ◽

Laurent Gal ◽

Cyprien Guérin ◽

Laurent Guillier ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Galleria Mellonella ◽

Fine Tuning ◽

Host Cells ◽

Gene Repertoire ◽

Transcript Levels ◽

Content Type ◽

Correlation Analyses ◽

Virulence Potential

ABSTRACT As for many opportunistic pathogens, the virulence potential of Listeria monocytogenes is highly heterogeneous between isolates and correlated, to some extent, with phylogeny and gene repertoires. In sharp contrast with copious data on intraspecies genome diversity, little is known about transcriptome diversity despite the role of complex genetic regulation in pathogenicity. The current study implemented RNA sequencing to characterize the transcriptome profiles of 33 isolates under optimal in vitro growth conditions. Transcript levels of conserved single-copy genes were comprehensively explored from several perspectives, including phylogeny, in silico-predicted virulence category based on epidemiological multilocus sequence typing (MLST) data, and in vivo virulence phenotype assessed in Galleria mellonella. Comparing baseline transcriptomes between isolates was intrinsically more complex than standard genome comparison because of the inherent plasticity of gene expression in response to environmental conditions. We show that the relevance of correlation analyses and their statistical power can be enhanced by using principal-component analysis to remove the first level of irrelevant, highly coordinated changes linked to growth phase. Our results highlight the major contribution of transcription factors with key roles in virulence to the diversity of transcriptomes. Divergence in the basal transcript levels of a substantial fraction of the transcriptome was observed between lineages I and II, echoing previously reported epidemiological differences. Correlation analysis with in vivo virulence identified numerous sugar metabolism-related genes, suggesting that specific pathways might play roles in the onset of infection in G. mellonella. IMPORTANCE Listeria monocytogenes is a multifaceted bacterium able to proliferate in a wide range of environments from soil to mammalian host cells. The accumulated genomic data underscore the contribution of intraspecies variations in gene repertoire to differential adaptation strategies between strains, including infection and stress resistance. It seems very likely that the fine-tuning of the transcriptional regulatory network is also a key component of the phenotypic diversity, albeit more difficult to investigate than genome content. Some studies reported incongruity in the basal transcriptome between isolates, suggesting a putative relationship with phenotypes, but small isolate numbers hampered proper correlation analyses with respect to their characteristics. The present study is the embodiment of the promising approach that consists of analyzing correlations between transcriptomes and various isolate characteristics. Statistically significant correlations were found with phylogenetic groups, epidemiological evidence of virulence potential, and virulence in Galleria mellonella larvae used as an in vivo model.

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Listeria monocytogenes-infected human monocytic derived dendritic cells activate Vγ9Vδ2 T cells independently of HMBPP production

Scientific Reports ◽

10.1038/s41598-021-95908-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Alejandro F. Alice ◽

Gwen Kramer ◽

Shelly Bambina ◽

Keith S. Bahjat ◽

Michael J. Gough ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Listeria Monocytogenes ◽

T Cell ◽

Γδ T Cells ◽

Mep Pathway ◽

Cell Recognition ◽

T Cell Recognition ◽

Infected Cells ◽

Vγ9vδ2 T Cells

AbstractGamma-delta (γδ) T cells express T cell receptors (TCR) that are preconfigured to recognize signs of pathogen infection. In primates, γδ T cells expressing the Vγ9Vδ2 TCR innately recognize (E)-4-hydroxy-3-methyl-but- 2-enyl pyrophosphate (HMBPP), a product of the 2-C-methyl-D-erythritol 4- phosphate (MEP) pathway in bacteria that is presented in infected cells via interaction with members of the B7 family of costimulatory molecules butyrophilin (BTN) 3A1 and BTN2A1. In humans, Listeria monocytogenes (Lm) vaccine platforms have the potential to generate potent Vγ9Vδ2 T cell recognition. To evaluate the activation of Vγ9Vδ2 T cells by Lm-infected human monocyte-derived dendritic cells (Mo-DC) we engineered Lm strains that lack components of the MEP pathway. Direct infection of Mo-DC with these bacteria were unchanged in their ability to activate CD107a expression in Vγ9Vδ2 T cells despite an inability to synthesize HMBPP. Importantly, functional BTN3A1 was essential for this activation. Unexpectedly, we found that cytoplasmic entry of Lm into human dendritic cells resulted in upregulation of cholesterol metabolism in these cells, and the effect of pathway regulatory drugs suggest this occurs via increased synthesis of the alternative endogenous Vγ9Vδ2 ligand isoprenyl pyrophosphate (IPP) and/or its isomer dimethylallyl pyrophosphate (DMAPP). Thus, following direct infection, host pathways regulated by cytoplasmic entry of Lm can trigger Vγ9Vδ2 T cell recognition of infected cells without production of the unique bacterial ligand HMBPP.

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Decision letter for "Essential role of IκB NS for in vivo CD4 + T cell activation, proliferation and Th1 cell differentiation during Listeria monocytogenes infection in mice"

10.1002/eji.201847961/v1/decision1 ◽

2018 ◽

Keyword(s):

Cell Differentiation ◽

Listeria Monocytogenes ◽

T Cell ◽

T Cell Activation ◽

Essential Role ◽

Cell Activation ◽

Th1 Cell

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Molecular serotyping and virulence potential of Listeria monocytogenes isolated from bovine, swine and human in the province of Quebec

10.31274/safepork-180809-254 ◽

2015 ◽

Author(s):

P. Fravalo ◽

T. Cherifi ◽

K. Neira ◽

A. Letellier ◽

J.-H. Fairbrother ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Molecular Serotyping ◽

Virulence Potential

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The Antitumor Effects of Britanin on Hepatocellular Carcinoma Cells and its Real-Time Evaluation by In Vivo Bioluminescence Imaging

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200227092623 ◽

2020 ◽

Vol 20 (9) ◽

pp. 1147-1156

Author(s):

Hanrui Li ◽

GeTao Du ◽

Lu Yang ◽

Liaojun Pang ◽

Yonghua Zhan

Keyword(s):

Hepatocellular Carcinoma ◽

Western Blot ◽

Blot Analysis ◽

Tumor Size ◽

Hepg2 Cells ◽

Western Blot Analysis ◽

Bioluminescence Imaging ◽

Antitumor Effects ◽

In Vivo Bioluminescence Imaging

Background: Hepatocellular carcinoma is cancer with many new cases and the highest mortality rate. Chemotherapy is the most commonly used method for the clinical treatment of hepatocellular carcinoma. Natural products have become clinically important chemotherapeutic drugs due to their great potential for pharmacological development. Many sesquiterpene lactone compounds have been proven to have antitumor effects on hepatocellular carcinoma. Objective: Britanin is a sesquiterpene lactone compound that can be considered for the treatment of hepatocellular carcinoma. The present study aimed to investigate the antitumor effect of britanin. Methods: BEL 7402 and HepG2 cells were used to study the cytotoxicity and antitumor effects of britanin. Preliminary studies on the nuclear factor kappa B pathway were conducted by western blot analysis. A BEL 7402-luc subcutaneous tumor model was established for the in vivo antitumor studies of britanin. In vivo bioluminescence imaging was conducted to monitor changes in tumor size. Results: The results of the cytotoxicity analysis showed that the IC50 values for britanin in BEL 7402 and HepG2 cells were 2.702μM and 6.006μM, respectively. The results of the colony formation demonstrated that the number of cells in a colony was reduced significantly after britanin treatment. And the results of transwell migration assays showed that the migration ability of tumor cells was significantly weakened after treatment with britanin. Tumor size measurements and staining results showed that tumor size was inhibited after britanin treatment. The western blot analysis results showed the inhibition of p65 protein expression and reduced the ratio of Bcl-2/Bax after treatment. Conclusion: A series of in vitro and in vivo experiments demonstrated that britanin had good antitumor effects and provided an option for hepatocellular carcinoma treatment.

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Characterisation of Listeria monocytogenes food-associated isolates to assess environmental fitness and virulence potential

International Journal of Food Microbiology ◽

10.1016/j.ijfoodmicro.2021.109247 ◽

2021 ◽

pp. 109247

Author(s):

Jessica A. Gray ◽

P. Scott Chandry ◽

Mandeep Kaur ◽

Chawalit Kocharunchitt ◽

John P. Bowman ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Virulence Potential ◽

Environmental Fitness

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Detection and Potential Virulence of Viable but Non-Culturable (VBNC) Listeria monocytogenes: A Review

Microorganisms ◽

10.3390/microorganisms9010194 ◽

2021 ◽

Vol 9 (1) ◽

pp. 194

Author(s):

Nathan E. Wideman ◽

James D. Oliver ◽

Philip Glen Crandall ◽

Nathan A. Jarvis

Keyword(s):

Public Health ◽

Listeria Monocytogenes ◽

Viable Count ◽

Vbnc State ◽

Testing Methods ◽

Tetrazolium Chloride ◽

Definitive Evidence ◽

Virulence Potential ◽

Vbnc Bacteria ◽

The Impact

The detection, enumeration, and virulence potential of viable but non-culturable (VBNC) pathogens continues to be a topic of discussion. While there is a lack of definitive evidence that VBNC Listeria monocytogenes (Lm) pose a public health risk, recent studies suggest that Lm in its VBNC state remains virulent. VBNC bacteria cannot be enumerated by traditional plating methods, so the results from routine Lm testing may not demonstrate a sample’s true hazard to public health. We suggest that supplementing routine Lm testing methods with methods designed to enumerate VBNC cells may more accurately represent the true level of risk. This review summarizes five methods for enumerating VNBC Lm: Live/Dead BacLightTM staining, ethidium monoazide and propidium monoazide-stained real-time polymerase chain reaction (EMA- and PMA-PCR), direct viable count (DVC), 5-cyano-2,3-ditolyl tetrazolium chloride-4′,6-diamidino-2-phenylindole (CTC-DAPI) double staining, and carboxy-fluorescein diacetate (CDFA) staining. Of these five supplementary methods, the Live/Dead BacLightTM staining and CFDA-DVC staining currently appear to be the most accurate for VBNC Lm enumeration. In addition, the impact of the VBNC state on the virulence of Lm is reviewed. Widespread use of these supplemental methods would provide supporting data to identify the conditions under which Lm can revert from its VBNC state into an actively multiplying state and help identify the environmental triggers that can cause Lm to become virulent. Highlights: Rationale for testing for all viable Listeria (Lm) is presented. Routine environmental sampling and plating methods may miss viable Lm cells. An overview and comparison of available VBNC testing methods is given. There is a need for resuscitation techniques to recover Lm from VBNC. A review of testing results for post VBNC virulence is compared

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