Project description:The innate immune system is crucial for eventual control of infections, but may also contribute to pathology. Listeria monocytogenes is an intracellular gram-positive bacteria and a major cause of food-borne disease. However, important knowledge on the interactions between L. monocytogenes and the immune system is still missing. Here we report that Listeria DNA is sorted into extracellular vesicles (EV)s in infected cells and delivered to bystander cells to stimulate the cGAS-STING pathway. This was also observed during infections with Francisella tularensis and Legionella pneumophila. We identify the multivesicular body protein MVB12b as a target for TBK1 phosphorylation, which is essential for sorting of DNA into EVs and stimulation of bystander cells. EVs from Listeria-infected cells inhibited T cell proliferation, and primed T cells for apoptosis. Collectively, we describe a novel pathway for EV-mediated delivery of foreign DNA to bystander cells, and suggest that intracellular bacteria exploit this pathway to impair anti-bacterial defense.
Project description:HEK-Blue™ ISG Cells and HEK-Blue™ ISG-KO-STING Cells (Invivogen) were transfected with dacA (Listeria monocytogenes) for 12 and 24 hours. Through this approach, we described the STING-dependent and STING-independent transcriptional response to dacA. Our analysis reveals a STING-dependent enrichment of interferon stimulated genes beginning 12 hours post-transfection, and a STING-independent enrichment of genes associated with protein translation and oxidative phosphorylation.
Project description:Listeria monocytogenes is a food-borne pathogen and the causative agent of listeriosis, an infection which typically arises through the consumption of contaminated foodstuffs. L. monocytogenes is a psychotrophic and facultatively anaerobic; properties which permit growth under refrigeration conditions and within modified atmosphere packaging. Through transcriptional changes L. monocytogenes is able to mount adaptive responses against stressors. Such responses typically cross protect against subsequent stresses.
Project description:We report positional cloning and characterization of a novel Sting allele that fails to activate IFN production in the wild-derived mice of MOLF/Ei strain in response to HSV (Herpes Simplex Virus) and Listeria monocytogenes both in vitro and in vivo. We show that previously uncharacterized mutations in the N-terminal of STING are responsible for low levels of IFN due to failure of MOLF STING to respond to cytosolic STING agonists such as 2â3âcGAMP, 5,6-Dimethylxanthenone-4-acetic acid (DMXAA) or poly(deoxyadenylic-deoxythymidylic) acid (dAdT). Using Next-Generation Sequencing (NGS) for the analysis of DNA-responses in congenic C57BL6.StingMOLF/MOLF (MOLF STING) mouse macrophages, we show that these mutations in MOLF/Ei discriminate in responses between different STING agonists. Macrophages from C57BL6.StingB6/B6¬ (B6 STING), or B6 STING expressing littermates were stimulated as a positive control for STING activation, and STING -/- (STING KO) macrophages served as a negative control. To examine differential gene regulation downstream of murine Tmem173 (STING) allelic variants. We stimulated macrophages from B6 mice congenic for MOLF STING; macrophages from B6 STING expressing littermates were stimulated as a positive control for STING activation, and STING -/- (STING KO) macrophages served as a negative control. One replicate of RNA-seq reads after each stimulation provided. Conditions are peritoneal macrophages from B6 congenic mice expressing: B6 STING, MOLF STING, or STING KO stimulated with 2â3âcGAMP, 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), poly(deoxyadenylic-deoxythymidylic) acid (dAdT), or no treatment. This constitutes a total of 12 reads analyzed in this data set.
Project description:We report positional cloning and characterization of a novel Sting allele that fails to activate IFN production in the wild-derived mice of MOLF/Ei strain in response to HSV (Herpes Simplex Virus) and Listeria monocytogenes both in vitro and in vivo. We show that previously uncharacterized mutations in the N-terminal of STING are responsible for low levels of IFN due to failure of MOLF STING to respond to cytosolic STING agonists such as 2’3’cGAMP, 5,6-Dimethylxanthenone-4-acetic acid (DMXAA) or poly(deoxyadenylic-deoxythymidylic) acid (dAdT). Using Next-Generation Sequencing (NGS) for the analysis of DNA-responses in congenic C57BL6.StingMOLF/MOLF (MOLF STING) mouse macrophages, we show that these mutations in MOLF/Ei discriminate in responses between different STING agonists. Macrophages from C57BL6.StingB6/B6¬ (B6 STING), or B6 STING expressing littermates were stimulated as a positive control for STING activation, and STING -/- (STING KO) macrophages served as a negative control.
Project description:DNA damage response kinase ATM regulates the genetic program of lymphocytes with phsiologically induced DNA DSBs. In bone marrow-derived macrophages, related kinase DNAPKcs is also responsible for activating DNA damage responses after infection with Listeria monocytogenes. Here we show that both ATM and DNA-PKcs regulate the genetic program of Listeria monocytogenes-infected macrophages.
Project description:Several Toll-like receptors are activated by Listeria monocytogenes infection, resulting in the activation of MyD88 dependent signaling pathway. However, the negative role of MyD88 in gene expresson is unclear. To address this, we performed microarray analysis of mRNAs from WT or MyD88-/- peritoneal macrophages infected with Listeria monocytogenes.
Project description:DNA damage response kinase ATM regulates the genetic program of lymphocytes with phsiologically induced DNA DSBs. In bone marrow-derived macrophages, related kinase DNAPKcs is also responsible for activating DNA damage responses after infection with Listeria monocytogenes. Here we show that both ATM and DNA-PKcs regulate the genetic program of Listeria monocytogenes-infected macrophages. Two independent bone marrow-derived macrophage cultures for each genotype (LysMcre/+ and Scid: Atmc/c: LysMcre/+) were infected with Listeria monocytogenes for 24 hrs at an MOI of 5. RNA was isolated using RNeasy (Qiagen). Gene expression profiling was performed using Illumina MouseRef-8 expression microarrays.
Project description:This study will evaluate the safety and tolerability of a personalized live, attenuated, double-deleted Listeria monocytogenes (pLADD) treatment in adults with metastatic colorectal cancer.
Project description:The foodborne pathogen Listeria monocytogenes uses a number of transcriptional regulators, including the negative regulator HrcA, to control gene expression under different environmental conditions and in response to stress. Gene expression patterns of DhrcA stationary phase cells were compared to wt to identify hrcA-dependent genes. We identified 61 HrcA-dependent genes that showed significant expression ratios (adj. P < 0.05), with ≥ 1.5-fold differential expression between ΔhrcA and wt. Combined with microarray analysis, Hidden Markov Model searches show HrcA directly repress at least 8 genes. Keywords: Listeria monocytogenes, HrcA regulon, stationary phase