Project description:Listeria monocytogenes is an opportunistic foodborne pathogen responsible for listeriosis, the third most common foodborne disease. Many different Listeria strains and seroptypes exist, however a proteogenomic resource which would provide a basis for bridging the gap in the molecular understanding between the Listeria genotype and phenotypes via proteotypes is still missing. Here we devised a next-generation proteogenomics strategy which enables the community now to rapidly proteotype Listeria strains and relate the information back to the genotype. Based on sequencing and de novo assembly of the two most commonly used Listeria strain model systems, EGD-e and ScottA, we established a comprehensive Listeria proteogenomic database. A genome comparison established core and strain-specific genes with potential relevance for virulence differences. Next we established a DIA/SWATH-based proteotyping strategy, including a new and robust sample preparation workflow, enabling the reproducible, sensitive and relative quantitative measurement of Listeria proteotypes. This re-usable DIA/SWATH library and new public resource covers 70% of the potentially expressed ORFs of Listeria and represents the most extensive spectral library for Listeria proteotype analysis to date. We used these two new resources to investigate the Listeria proteotype in three states mimicking the upper gastrointestinal passage. Exposure of Listeria to bile salts at 37 °C, mimicking conditions encountered in the duodenum, showed significant proteotype perturbations including an increase of FlaA, the structural protein of flagella. Given that Listeria is known to lose its flagella above 30 °C, this was an unexpected finding. The formation of flagella, which might have implications within the infectivity cycle, was validated by parallel reaction monitoring, light and scanning electron microscopy. QPCR data of flaA transcripts showed no significant differences suggesting a regulation at the post-transcriptional level. Together, we provide a comprehensive proteogenomic resource and toolbox for the Listeria community enabling the analysis of Listeria genotype-proteotype-phenotype relationships.
Project description:Listeria monocytogenes causes severe foodborne illness in pregnant women and immunocompromised individuals. After the intestinal phase of infection, the liver plays a central role in the clearance of this pathogen through its important functions in immunity. However, recent evidence suggests that subpopulations of L. monocytogenes may escape eradication after prolonged infection of hepatocytes, by entering a persistence phase in vacuoles. Here, we examine whether this long-term infection alters hepatocyte defense pathways, which may be instrumental for bacterial persistence. We first established models of Listeria infection in human hepatocyte cell lines HepG2 and Huh7 and in primary mouse hepatocytes (PMH). In these cells, Listeria efficiently enters the persistence stage after a 3-day infection, while inducing a type I (PMH) or type I/III (HepG2) or no (Huh7) interferon response. RNA-seq analysis identified a common signature of long-term Listeria infection on the hepatocyte transcriptome, characterized by overexpression of a set of genes involved in antiviral immunity and under-expression of many acute phase protein (APP) genes, particularly involved in the complement and coagulation systems. The decrease in APP transcript amounts correlated with lower protein abundance in the secretome of infected cells, as shown by proteomics, and also occurred in the presence of APP inducers (IL-6 or IL-1b). The results also suggest that long-term Listeria infection affects lipid metabolism pathways. Collectively, these results reveal that long-term infection with L. monocytogenes profoundly deregulates the innate immune functions of hepatocytes, which could generate an environment favorable to the establishment of persistent infection.
Project description:Phosphopeptides were identified in Listeria monocytogesn strain constitutivally expressing PrfA. Also, the phosphoproteins and proteins were identified that are overexpressed/underextressed in response to PrfA.
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:Adaptation of Listeria weihenstephanensis to anaerobiosis. Trascriptional profiling at two different temperatures of L. weihenstephanensis