Project description:ISG15 is primarily documented as an interferon-stimulated, ubiquitin-like protein (ubl), which has anti-viral activity. Although ISG15 was the founding member of the ubl protein family, very little is known about its function. We have found that ISG15 expression in non-phagocytic cells is dramatically induced upon Listeria infection and that surprisingly this induction can be Type I Interferon independent. Listeria-mediated ISG15 induction depends on the cytosolic surveillance pathway, which senses bacterial DNA and signals through STING, TBK1, IRF3 and IRF7. Most importantly, we observed that ISG15 expression restricts Listeria infection both in vitro and in vivo. We then made use of Stable Isotope Labeling in tissue culture (SILAC) to identify the ISGylated proteins that could be responsible for the ISG15-mediated protective effect. Our SILAC analysis revealed that overexpression of ISG15 leads to a striking ISGylation of integral membrane proteins of the endoplasmic reticulum and Golgi apparatus, which correlates with increased canonical secretion of cytokines. Taken together, our data reveal a previously uncharacterized signaling pathway that restricts Listeria infection and acts via ISGylation, reinforcing the view that ISG15 is a key component of the innate immune arsenal of the mammalian host.
Project description:ISG15 is primarily documented as an interferon-stimulated, ubiquitin-like protein (ubl), which has anti-viral activity. Although ISG15 was the founding member of the ubl protein family, very little is known about its function. We have found that ISG15 expression in non-phagocytic cells is dramatically induced upon Listeria infection and that surprisingly this induction can be Type I Interferon independent. Listeria-mediated ISG15 induction depends on the cytosolic surveillance pathway, which senses bacterial DNA and signals through STING, TBK1, IRF3 and IRF7. Most importantly, we observed that ISG15 expression restricts Listeria infection both in vitro and in vivo. We then made use of Stable Isotope Labeling in tissue culture (SILAC) to identify the ISGylated proteins that could be responsible for the ISG15-mediated protective effect. Our SILAC analysis revealed that overexpression of ISG15 leads to a striking ISGylation of integral membrane proteins of the endoplasmic reticulum and Golgi apparatus, which correlates with increased canonical secretion of cytokines. Taken together, our data reveal a previously uncharacterized signaling pathway that restricts Listeria infection and acts via ISGylation, reinforcing the view that ISG15 is a key component of the innate immune arsenal of the mammalian host.
Project description:ISG15 is an interferon-stimulated, ubiquitin-like protein, with anti-viral and anti-bacterial activity, however its precise mechanism of action during viral or bacterial infection remains elusive. We previously found that ISG15 restricts Listeria infection both in vitro and in vivo and identified ISGylated proteins that could be responsible for the protective effect. Here, we endeavored to map the in vivo ISGylome following Listeria infection to mechanistically elucidate the function of ISG15 in host defense. To do so we combined a genetic approach employing a murine model of deregulated ISGylation with quantitative proteomics of immune-enriched endogenous ISG15 modification sites. In this way, we mapped 930 ISG15 sites in 438 proteins. In addition, we also quantified protein level changes in the host proteome following infection. Gene ontology analysis revealed that ISGylated proteins were mostly enriched in proteins which alter cellular metabolic processes, including four upstream modulators of the catabolic and antibacterial pathway of autophagy. Structural analysis further revealed that a number of ISG15 modifications can occur at catalytic sites or dimerization interfaces of enzymes. Finally, we showed that animals and cells with deregulated ISGylation have increased infection-induced autophagy through the modification of mTOR, WIPI2, and AMBRA1. Taken together, these findings ascribe a putative role of ISGylation to temporarily reprogram organismal metabolism following infection through direct modification of a variety of enzymes in the liver.
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:Time course study of the mouse infection by comparing the genomic transcriptional patterns of Listeria monocytogenes EGDe grown under laboratory conditions (exponential growth phase) with that of in vivo-grown bacteria (in mouse spleens) over three days of infection. Time course study of the mouse infection by comparing the genomic transcriptional patterns of Listeria monocytogenes EGDe grown under laboratory conditions (exponential growth phase) with that of in vivo-grown bacteria (in mouse spleens) over three days of infection.
Project description:These studies were designed to examine the transcription of Listeria monocytogenes strains 10403S and LO28 during intracellular replication in mammalian macrophages. Duplicate WT Listeria monocytogenes (strains 10403S and LO28) were used to infect mouse bone marrow-derived macrophages (BMMs). Bacterial RNA was harvested at 4 hours post-infection.
Project description:Time course study of the mouse infection by comparing the genomic transcriptional patterns of Listeria monocytogenes EGDe grown under laboratory conditions (exponential growth phase) with that of in vivo-grown bacteria (in mouse spleens) over three days of infection.