Intracellular bacteria engage a STING-TBK1-MVB12b pathway to enable paracrine cGAS-STING signalling.
ABSTRACT: 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 (EVs) in infected cells and delivered to bystander cells to stimulate the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (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 TANK-binding kinase 1 phosphorylation, which is essential for the 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 pathway for EV-mediated delivery of foreign DNA to bystander cells, and suggest that intracellular bacteria exploit this pathway to impair antibacterial defence.
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:Listeria monocytogenes is a gram-positive facultative intracellular bacterium, which replicates in the cytoplasm of myeloid cells. Interferon ? (IFN?) has been reported to play an important role in the mechanisms underlying Listeria disease. Although studies in murine cells have proposed the bacteria-derived cyclic-di-AMP to be the key bacterial immunostimulatory molecule, the mechanism for IFN? expression during L. monocytogenes infection in human myeloid cells remains unknown. Here we report that in human macrophages, Listeria DNA rather than cyclic-di-AMP is stimulating the IFN response via a pathway dependent on the DNA sensors IFI16 and cGAS as well as the signalling adaptor molecule STING. Thus, Listeria DNA is a major trigger of IFN? expression in human myeloid cells and is sensed to activate a pathway dependent on IFI16, cGAS and STING.
Project description:The vertebrate protein STING, an intracellular sensor of cyclic dinucleotides, is critical to the innate immune response and the induction of type I interferon during pathogenic infection. Here, we show that a STING ortholog (dmSTING) exists in Drosophila, which, similar to vertebrate STING, associates with cyclic dinucleotides to initiate an innate immune response. Following infection with Listeria monocytogenes, dmSTING activates an innate immune response via activation of the NF-?B transcription factor Relish, part of the immune deficiency (IMD) pathway. DmSTING-mediated activation of the immune response reduces the levels of Listeria-induced lethality and bacterial load in the host. Of significance, dmSTING triggers an innate immune response in the absence of a known functional cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) ortholog in the fly. Together, our results demonstrate that STING is an evolutionarily conserved antimicrobial effector between flies and mammals, and it comprises a key component of host defense against pathogenic infection in Drosophila.
Project description:Infection with Listeria monocytogenes strains that enter the host cell cytosol leads to a robust cytotoxic T cell response resulting in long-lived cell-mediated immunity (CMI). Upon entry into the cytosol, L. monocytogenes secretes cyclic diadenosine monophosphate (c-di-AMP) which activates the innate immune sensor STING leading to the expression of IFN-? and co-regulated genes. In this study, we examined the role of STING in the development of protective CMI to L. monocytogenes. Mice deficient for STING or its downstream effector IRF3 restricted a secondary lethal challenge with L. monocytogenes and exhibited enhanced immunity that was MyD88-independent. Conversely, enhancing STING activation during immunization by co-administration of c-di-AMP or by infection with a L. monocytogenes mutant that secretes elevated levels of c-di-AMP resulted in decreased protective immunity that was largely dependent on the type I interferon receptor. These data suggest that L. monocytogenes activation of STING downregulates CMI by induction of type I interferon.
Project description:Intracellular bacterial pathogens, such as Listeria monocytogenes, are detected in the cytosol of host immune cells. Induction of this host response is often dependent on microbial secretion systems and, in L. monocytogenes, is dependent on multidrug efflux pumps (MDRs). Using L. monocytogenes mutants that overexpressed MDRs, we identified cyclic diadenosine monophosphate (c-di-AMP) as a secreted molecule able to trigger the cytosolic host response. Overexpression of the di-adenylate cyclase, dacA (lmo2120), resulted in elevated levels of the host response during infection. c-di-AMP thus represents a putative bacterial secondary signaling molecule that triggers a cytosolic pathway of innate immunity and is predicted to be present in a wide variety of bacteria and archea.
Project description:Cells naïve to stress can display the effects of stress, such as DNA damage and apoptosis, when they are exposed to signals from stressed cells; this phenomenon is known as the bystander effect. We previously showed that bystander effect induced by ionising radiation are mediated by extracellular vesicles (EVs). Bystander effect can also be induced by other types of stress, including heat shock, but it is unclear whether EVs are involved. Here we show that EVs released from heat shocked cells are also able to induce bystander damage in unstressed populations. Naïve cells treated with media conditioned by heat shocked cells showed higher levels of DNA damage and apoptosis than cells treated with media from control cells. Treating naïve cells with EVs derived from media conditioned by heat shocked cells also induced a bystander effect when compared to control, with DNA damage and apoptosis increasing whilst the level of cell viability was reduced. We demonstrate that treatment of naïve cells with heat shocked cell-derived EVs leads to greater invasiveness in a trans-well Matrigel assay. Finally, we show that naïve cells treated with EVs from heat-shocked cells are more likely to survive a subsequent heat shock, suggesting that these EVs mediate an adaptive response. We propose that EVs released following stress mediate an intercellular response that leads to apparent stress in neighbouring cells but also greater robustness in the face of a subsequent insult.
Project description:Engineered bacteriophages provide powerful tools for biotechnology, diagnostics, pathogen control, and therapy. However, current techniques for phage editing are experimentally challenging and limited to few phages and host organisms. Viruses that target Gram-positive bacteria are particularly difficult to modify. Here, we present a platform technology that enables rapid, accurate, and selection-free construction of synthetic, tailor-made phages that infect Gram-positive bacteria. To this end, custom-designed, synthetic phage genomes were assembled in vitro from smaller DNA fragments. We show that replicating, cell wall-deficient Listeria monocytogenes L-form bacteria can reboot synthetic phage genomes upon transfection, i.e., produce virus particles from naked, synthetic DNA. Surprisingly, Listeria L-form cells not only support rebooting of native and synthetic Listeria phage genomes but also enable cross-genus reactivation of Bacillus and Staphylococcus phages from their DNA, thereby broadening the approach to phages that infect other important Gram-positive pathogens. We then used this platform to generate virulent phages by targeted modification of temperate phage genomes and demonstrated their superior killing efficacy. These synthetic, virulent phages were further armed by incorporation of enzybiotics into their genomes as a genetic payload, which allowed targeting of phage-resistant bystander cells. In conclusion, this straightforward and robust synthetic biology approach redefines the possibilities for the development of improved and completely new phage applications, including phage therapy.
Project description:Radiation-induced bystander effects refer to the induction of biological changes in cells not directly hit by radiation implying that the number of cells affected by radiation is larger than the actual number of irradiated cells. Recent in vitro studies suggest the role of extracellular vesicles (EVs) in mediating radiation-induced bystander signals, but in vivo investigations are still lacking. Here, we report an in vivo study investigating the role of EVs in mediating radiation effects. C57BL/6 mice were total-body irradiated with X-rays (0.1, 0.25, 2?Gy), and 24?h later, EVs were isolated from the bone marrow (BM) and were intravenously injected into unirradiated (so-called bystander) animals. EV-induced systemic effects were compared to radiation effects in the directly irradiated animals. Similar to direct radiation, EVs from irradiated mice induced complex DNA damage in EV-recipient animals, manifested in an increased level of chromosomal aberrations and the activation of the DNA damage response. However, while DNA damage after direct irradiation increased with the dose, EV-induced effects peaked at lower doses. A significantly reduced hematopoietic stem cell pool in the BM as well as CD4+ and CD8+ lymphocyte pool in the spleen was detected in mice injected with EVs isolated from animals irradiated with 2?Gy. These EV-induced alterations were comparable to changes present in the directly irradiated mice. The pool of TLR4-expressing dendritic cells was different in the directly irradiated mice, where it increased after 2?Gy and in the EV-recipient animals, where it strongly decreased in a dose-independent manner. A panel of eight differentially expressed microRNAs (miRNA) was identified in the EVs originating from both low- and high-dose-irradiated mice, with a predicted involvement in pathways related to DNA damage repair, hematopoietic, and immune system regulation, suggesting a direct involvement of these pathways in mediating radiation-induced systemic effects. In conclusion, we proved the role of EVs in transmitting certain radiation effects, identified miRNAs carried by EVs potentially responsible for these effects, and showed that the pattern of changes was often different in the directly irradiated and EV-recipient bystander mice, suggesting different mechanisms.
Project description:The innate immune system is critical for the early detection of invading pathogens and for initiating cellular host defence countermeasures, which include the production of type I interferon (IFN). However, little is known about how the innate immune system is galvanized to respond to DNA-based microbes. Here we show that STING (stimulator of interferon genes) is critical for the induction of IFN by non-CpG intracellular DNA species produced by various DNA pathogens after infection. Murine embryonic fibroblasts, as well as antigen presenting cells such as macrophages and dendritic cells (exposed to intracellular B-form DNA, the DNA virus herpes simplex virus 1 (HSV-1) or bacteria Listeria monocytogenes), were found to require STING to initiate effective IFN production. Accordingly, Sting-knockout mice were susceptible to lethal infection after exposure to HSV-1. The importance of STING in facilitating DNA-mediated innate immune responses was further evident because cytotoxic T-cell responses induced by plasmid DNA vaccination were reduced in Sting-deficient animals. In the presence of intracellular DNA, STING relocalized with TANK-binding kinase 1 (TBK1) from the endoplasmic reticulum to perinuclear vesicles containing the exocyst component Sec5 (also known as EXOC2). Collectively, our studies indicate that STING is essential for host defence against DNA pathogens such as HSV-1 and facilitates the adjuvant activity of DNA-based vaccines.
Project description:One of the hallmarks of adaptive immunity is the development of a long-term pathogen specific memory response. While persistent memory T cells certainly impact the immune response during a secondary challenge, their role in unrelated infections is less clear. To address this issue, we utilized lymphocytic choriomeningitis virus (LCMV) and Listeria monocytogenes immune mice to investigate whether bystander memory T cells influence Leishmania major infection. Despite similar parasite burdens, LCMV and Listeria immune mice exhibited a significant increase in leishmanial lesion size compared to mice infected with L. major alone. This increased lesion size was due to a severe inflammatory response, consisting not only of monocytes and neutrophils, but also significantly more CD8 T cells. Many of the CD8 T cells were LCMV specific and expressed gzmB and NKG2D, but unexpectedly expressed very little IFN-?. Moreover, if CD8 T cells were depleted in LCMV immune mice prior to challenge with L. major, the increase in lesion size was lost. Strikingly, treating with NKG2D blocking antibodies abrogated the increased immunopathology observed in LCMV immune mice, showing that NKG2D engagement on LCMV specific memory CD8 T cells was required for the observed phenotype. These results indicate that bystander memory CD8 T cells can participate in an unrelated immune response and induce immunopathology through an NKG2D dependent mechanism without providing increased protection.