Showing papers by "Jiri Stulik published in 2005"

Proteomic analysis of anti-Francisella tularensis LVS antibody response in murine model of tularemia.

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.

Insights into the oxidative stress response in Francisella tularensis LVS and its mutant ΔiglC1 + 2 by proteomics analysis

Abstract: Francisella tularensis is a facultative intracellular pathogen. Its capacity to induce disease depends on the ability to invade and multiply within a wide range of eukaryotic cells, such as professional phagocytes. The comparative disinterest in tularemia in the past relative to other human bacterial pathogens is reflected in the paucity of information concerning the mechanisms of pathogenesis. Only a few genes and gene products associated with Francisella virulence are known to date. The aim of this study was to find and identify proteins of F. tularensis live vaccine strain induced in the presence of hydrogen peroxide, and to investigate the role of the IglC protein in the regulation of genes expressed upon peroxide stress. The [35S]-radiolabelled protein patterns were examined for both the wild live vaccine strain and its ΔiglC1 + 2 mutant defective in synthesis of the IglC protein that was found to be strongly up-regulated during intracellular growth in murine macrophages in vitro and upon exposure to hydrogen peroxide. Globally, we found 21 protein spots whose levels were significantly altered in the presence of hydrogen peroxide in both the wild-type and mutant strains.

Coxiella burnetii Whole Cell Lysate Protein Identification by Mass Spectrometry and Tandem Mass Spectrometry

Abstract: The whole cell lysate of Coxiella burnetii strain RSA 493 was separated by two-dimensional electrophoresis and more than 500 protein spots were found on silver-stained reference map. Spots from the gels were subjected to identification based on peptide mass fingerprinting (PMF). In order to identify additional proteins, tandem mass spectrometry (MS/MS) using electrospray and matrix-assisted laser desorption/ionization techniques was applied. The three independent approaches resulted in the identification of 197 open reading frames (ORFs). Fifty-two proteins were identified by PMF and at least with one of the MS/MS methods, 37 proteins with both MS/MS instruments, and 19 proteins with all three techniques applied. All predicted C. burnetii ORFs were compared with the Clusters of Orthologous Groups database. The data related to identified proteins were stored and indexed in a file that can be read and searched using Microsoft Access.

Proteome alterations in gamma-irradiated human T-lymphocyte leukemia cells.

Abstract: Szkanderova, S., Vavrova, J., Hernychova, L., Neubauerova, V., Lenco, J. and Stulik, J. Proteome Alterations in Gamma-Irradiated Human T-Lymphocyte Leukemia Cells. Radiat. Res. 163, 307–315 (2005). Analyses of the protein expression profiles of irradiated cells may be beneficial for identification of new biomolecules of radiation-induced cell damage. Therefore, in this study we exploited the proteomic approach to identify proteins whose expression is significantly altered in γ-irradiated human T-lymphocyte leukemia cells. MOLT-4 cells were irradiated with 7.5 Gy and the cell lysates were collected at different times after irradiation (2, 5 and 12 h). The proteins were separated by two-dimensional electrophoresis and quantified using an image evaluation system. Proteins exhibiting significant radiation-induced alterations in abundance were identified by peptide mass fingerprinting. We identified 14 proteins that were either up- or down-regulated. Cellular levels of four of the proteins (Rho GDP d...