| 1. |
- Balonova, Lucie, et al.
(författare)
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Characterization of protein glycosylation in Francisella tularensis subsp holarctica
- 2012
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 11:7
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Tidskriftsartikel (refereegranskat)abstract
- FTH_0069 is a previously uncharacterized strongly immunoreactive protein that has been proposed to be a novel virulence factor in Francisella tularensis. Here, the glycan structure modifying two C-terminal peptides of FTH_0069 was identified utilizing high resolution, high mass accuracy mass spectrometry, combined with in-source CID tandem MS experiments. The glycan observed at m/z 1156 was determined to be a hexasaccharide, consisting of two hexoses, three N-acetylhexosamines, and an unknown monosaccharide containing a phosphate group. The monosaccharide sequence of the glycan is tentatively proposed as X-P-HexNAc-HexNAc-Hex-Hex-HexNAc, where X denotes the unknown monosaccharide. The glycan is identical to that of DsbA glycoprotein, as well as to one of the multiple glycan structures modifying the type IV pilin PilA, suggesting a common biosynthetic pathway for the protein modification. Here, we demonstrate that the glycosylation of FTH_0069, DsbA, and PilA was affected in an isogenic mutant with a disrupted wbtDEF gene cluster encoding O-antigen synthesis and in a mutant with a deleted pglA gene encoding pilin oligosaccharyltransferase PglA. Based on our findings, we propose that PglA is involved in both pilin and general F. tularensis protein glycosylation, and we further suggest an inter-relationship between the O-antigen and the glycan synthesis in the early steps in their biosynthetic pathways. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.015016, 1-12, 2012.
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| 2. |
- Cerveny, Lukas, et al.
(författare)
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Tetratricopeptide Repeat Motifs in the World of Bacterial Pathogens : Role in Virulence Mechanisms
- 2013
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Ingår i: Infection and Immunity. - : American Society for Microbiology. - 0019-9567 .- 1098-5522. ; 81:3, s. 629-635
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Forskningsöversikt (refereegranskat)abstract
- The tetratricopeptide repeat (TPR) structural motif is known to occur in a wide variety of proteins present in prokaryotic and eukaryotic organisms. The TPR motif represents an elegant module for the assembly of various multiprotein complexes, and thus, TPR-containing proteins often play roles in vital cell processes. As the TPR profile is well defined, the complete TPR protein repertoire of a bacterium with a known genomic sequence can be predicted. This provides a tremendous opportunity for investigators to identify new TPR-containing proteins and study them in detail. In the past decade, TPR-containing proteins of bacterial pathogens have been reported to be directly related to virulence-associated functions. In this minireview, we summarize the current knowledge of the TPR-containing proteins involved in virulence mechanisms of bacterial pathogens while high-lighting the importance of TPR motifs for the proper functioning of class II chaperones of a type III secretion system in the pathogenesis of Yersinia, Pseudomonas, and Shigella.
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| 3. |
- Fabrik, Ivo, et al.
(författare)
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Application of SILAC labeling to primary bone marrow-derived dendritic cells reveals extensive GM-CSF-dependent arginine metabolism
- 2014
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 13:2, s. 752-762
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Tidskriftsartikel (refereegranskat)abstract
- Although dendritic cells (DCs) control the priming of the adaptive immunity response, a comprehensive description of their behavior at the protein level is missing. The introduction of the into the field of DC research would therefore be highly beneficial. quantitative proteomic technique of metabolic labeling (SILAC) To achieve this, we applied SILAC labeling to primary bone marow-derived DCs (BMDCs). These cells combine both biological relevance and experimental feasibility, as their in vitro generation permits the use of C-13/N-15-labeled amino acids.. Interestingly, BMDCs appear to exhibit a very active arginine metabolism. Using standard cultivation conditions, similar to 20% of all protein-incorporated proline was a byproduct of heavy arginine degradation. In addition, the dissipation of N-15 from labeled arginine to the whole proteome was observed. The latter decreased the mass accuracy in MS and affected the natural isotopic distribution of peptides. SILAC-connected metabolic issues were shown to be enhanced by GM-CSF, which is used for the differentiation of DC progenitors. Modifications of the cultivation procedure suppressed the arginine-related effects, yielding cells with a proteome labeling efficiency of >= 90%. Importantly, BMDCs generated according to the new cultivation protocol preserved their resemblance to inflammatory DCs in vivo, as evidenced by their response to LPS treatment.
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| 4. |
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| 5. |
- Havlasová, Jana, et al.
(författare)
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Proteomic analysis of anti-Franciselia tularensis LVS antibody response in murine model of tularemia
- 2005
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Ingår i: Proteomics. - Weinheim : Wiley-VCH Verlagsgesellschaft. - 1615-9853 .- 1615-9861. ; 5:8, s. 2090-2103
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Tidskriftsartikel (refereegranskat)abstract
- Francisella tularensis live vaccine strain infection of mice has been established as an experimental model of tularemia that is suitable for studies of immune mechanisms against the intracellular pathogen. In this study, the model was used to explore immunogenic repertoire of F. tularensis with the aim of identifying new molecules able to activate the host immune system, potential bacterial markers with vaccine, and diagnostic applications. Immunoproteomic approach based on the combination of two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry was applied. Globally, 36 different proteins were identified, which strongly reacted with sera from experimentally infected mice, including several putative virulence markers of intracellular pathogens as nucleoside diphosphate kinase, isocitrate dehydrogenase, RNA-binding protein Hfq, and molecular chaperone ClpB. Of them, 27 proteins are described for the first time as immunorelevant Francisella proteins. When comparing murine immunoproteome of F. tularensis with our previous data from human patients, 25 of the total of 50 identified murine sera immunoreactive spots were recognized by human sera collected from patients suffering from tularemia, as well. Immune sera from two Lps gene congenic strains of mice, C3H/HeN (Lpsn) and C3H/HeJ (Lpsd), represented murine immunoproteome in this study. The spectrum of immunoreactive spots detected by two-dimensional immunoblotting varied throughout the course of infection depending on murine strain. Nevertheless, the antibody patterns of the two strains showed significant homogeneity in being directed against almost identical subset of antigens.
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| 6. |
- Härtlova, Anetta, et al.
(författare)
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A proteomic view of the host-pathogen interaction: The host perspective.
- 2011
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Ingår i: Proteomics. - : Wiley. - 1615-9861 .- 1615-9853. ; 11:15, s. 3212-20
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Forskningsöversikt (refereegranskat)abstract
- The host-pathogen interaction represents a complex and dynamic biological system. The outcome of this interaction is dependent on the microbial pathogen properties to establish infection and the ability of the host to control infection. Although bacterial pathogens have evolved a variety of strategies to subvert host defense functions, several general mechanisms have been shown to be shared among these pathogens. As a result, host effectors that are critical for pathogen entry, survival and replication inside the host cells have become a new paradigm for antimicrobial targeting. This review focuses on the potential utility of a proteomics approach in defining the host-pathogen interaction from the host's perspective.
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| 7. |
- Härtlova, Anetta, et al.
(författare)
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Membrane rafts: a potential gateway for bacterial entry into host cells.
- 2010
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Ingår i: Microbiology and immunology. - : Wiley. - 0385-5600 .- 1348-0421. ; 54:4, s. 237-45
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Forskningsöversikt (refereegranskat)abstract
- Pathogenic bacteria have developed various mechanisms to evade host immune defense systems. Invasion of pathogenic bacteria requires interaction of the pathogen with host receptors, followed by activation of signal transduction pathways and rearrangement of the cytoskeleton to facilitate bacterial entry. Numerous bacteria exploit specialized plasma membrane microdomains, commonly called membrane rafts, which are rich in cholesterol, sphingolipids and a special set of signaling molecules which allow entry to host cells and establishment of a protected niche within the host. This review focuses on the current understanding of the raft hypothesis and the means by which pathogenic bacteria subvert membrane microdomains to promote infection.
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| 8. |
- Härtlova, Anetta, et al.
(författare)
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Quantitative proteomics analysis of macrophage-derived lipid rafts reveals induction of autophagy pathway at the early time of Francisella tularensis LVS infection
- 2014
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 13:2, s. 796-804
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Tidskriftsartikel (refereegranskat)abstract
- Francisella tularensis is a highly infectious intracellular pathogen that has evolved an efficient strategy to subvert host defense response to survive inside the host. The molecular mechanisms regulating these host-pathogen interactions and especially those that are initiated at the time of the bacterial entry via its attachment to the host plasma membrane likely predetermine the intracellular fate of pathogen. Here, we provide the evidence that infection of macrophages with F. tularensis leads to changes in protein composition of macrophage-derived lipid rafts, isolated as detergent-resistant membranes (DRMs). Using SILAC-based quantitative proteomic approach, we observed the accumulation of autophagic adaptor protein p62 at the early, stages of microbe-host cell interaction. We confirmed the colocalization of the p62 with ubiquitinated and LC3-decorated intracellular F. tularensis microbes with its maximum at 1 h postinfection. Furthermore, the infection of p62-knockdown host cells led to the transient increase in the intracellular number of microbes up to 4 h after in vitro infection. Together, these data suggest that the activation of the autophagy pathway in F. tularensis infected macrophages, which impacts the early phase of microbial proliferation, is subsequently circumvented by ongoing infection.
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| 9. |
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| 10. |
- Krocova, Zuzana, et al.
(författare)
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Interaction of B cells with intracellular pathogen Francisella tularensis.
- 2008
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Ingår i: Microbial pathogenesis. - : Elsevier BV. - 0882-4010. ; 45:2, s. 79-85
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Tidskriftsartikel (refereegranskat)abstract
- Immunity to Francisella tularensis is largely mediated by T lymphocytes but an important role of B lymphocytes in early stage of infection was previously uncovered. We wanted to find out if F. tularensis is able to infect B cells and/or influence them by direct contact. To investigate this possibility we infected B cell lines from mouse (A20) or humans (Ramos RA-1), or primary mouse spleen cells, with F. tularensis LVS and F. tularensis FSC200 in vitro. In all cases, we detected bacteria on the cell surface and inside the B cells using transmission electron microscopy. More than 20% cells were infected by microbes after 24h. The number of bacteria, determined by CFU, increased about 1 log during 24h. Infection with live bacteria led to apoptosis of Ramos cells and mouse CD19(+) spleen cells. Approximately 30% of cells were apoptotic after 24h and 70% after 48 h, independently of the F. tularensis strain, while only 10% of non-infected cell were apoptotic at either time point. Apoptosis was confirmed by Western blot using anti-PARP antibodies. Thus, this study demonstrates unique phenomenon - namely, the ability of the intracellular pathogen F. tularensis to invade and induce apoptosis in B cells.
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