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: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 is a foodborne intracellular bacterial pathogen leading to human listeriosis. Despite a high mortality rate and increasing antibiotic resistance no clinically approved vaccine against Listeria is available. To identify antigens for this bacterial pathogen that can be encoded in mRNA vaccine formulations, we screened for Listeria epitopes presented on the surface of infected human cell lines by mass spectrometry-based immunopeptidomics. In between more than 15,000 human self-peptides, we detected 68 Listeria epitopes from 42 different bacterial proteins, including several known antigens. Peptide epitopes presented on different cell lines were often derived from the same bacterial surface proteins, classifying these antigens as potential vaccine candidates. Encoding these highly presented antigens in lipid nanoparticle mRNA vaccine formulations resulted in high levels of protection in vaccination challenge experiments in mice. Our results pave the way for the development of a clinical mRNA vaccine against Listeria and demonstrate the power of immunopeptidomics for next-generation bacterial vaccine development.
Project description:Listeria monocytogenes is a foodborne intracellular bacterial model pathogen. Protective immunity against Listeria depends on an effective CD8 T-cell responses, but very few T cell epitopes are known in mice as most common animal infection model for listeriosis. To identify epitopes we screened for Listeria epitopes presented in the spleen of infected mice by mass spectrometry-based immunopeptidomics. In between more than 6,000 mouse self-peptides presented on MHC Class I molecules, we detected 26 Listeria peptides from 25 different bacterial proteins, including previously reported antigens. Bacterial immunopeptides with confirmed fragmentation spectra were further tested for their potential to CD8 T cells, revealing VTYNYINI from the putative cell wall surface anchor family protein LMON_0576 as a novel bona fide peptide epitope. Despite its high biological potency in a prime boost model, this epitope did not protect against challenge infection but can be used as a research tool to probe CD8 T cell responses in mouse models of Listeria infection. Our results demonstrate the power of immunopeptidomics for bacterial antigen identification but highlight the need for in-depth immune characterization for vaccine candidate selection.
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:Lipocalin 24p3 (24p3) is a neutrophil secondary granule protein. 24p3 is also a siderocalin, which binds several bacterial siderophores. It was therefore proposed that synthesis and secretion of 24p3 by stimulated macrophages or release of 24p3 upon neutrophil degranulation sequesters iron-laden siderophores to attenuate bacterial growth. Accordingly, 24p3-deficient mice are susceptible to bacterial pathogens whose siderophores would normally be chelated by 24p3. Specific granule deficiency (SGD) is a rare congenital disorder characterized by complete absence of proteins in secondary granules. Neutrophils from SGD patients, who are prone to bacterial infections, lack normal functions but the potential role of 24p3 in neutrophil dysfunction in SGD is not known. Here we show that neutrophils from 24p3-deficient mice are defective in many neutrophil functions. Specifically, neutrophils in 24p3-deficient mice do not extravasate to sites of infection and are defective for chemotaxis. A transcriptome analysis revealed that genes that control cytoskeletal reorganization are selectively suppressed in 24p3-deficient neutrophils. Additionally, small regulatory RNAs (miRNAs) that control upstream regulators of cytoskeletal proteins are also increased in 24p3-deficient neutrophils. Further, 24p3-deficient neutrophils failed to phagocytose bacteria, which may account for the enhanced sensitivity of 24p3-deficient mice to both intracellular (Listeria monocytogenes) and extracellular (Candida albicans, Staphylococcus aureus) pathogens. Interestingly, Listeria does not secrete siderophores and additionally, the siderophore secreted by Candida is not sequestered by 24p3. Therefore, the heightened sensitivity of 24p3-deficient mice to these pathogens is not due to sequestration of siderophores limiting iron availability, but is a consequence of impaired neutrophil function. Key words: Lipocalin, 24p3, neutrophils, cell motility, chemotaxis, MIRNA-362-3p
Project description:Transcriptional profling of a Listeria monocytogenes under nisin treatment comparing ctsR mutant and wild type one condition (nisin treament 20ug/ml, 24 hours) experiment, ctsR mutant vs. wild type Listeria monocytogenes Scott A, 2 biological replicates, 4 technical replicates
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.