Glykosylace proteinů hraje významnou ulohu v patogenitě některých bakterii. V předloženem přispěvku uvadime jednak přehled soucasných analytických metod, ktere se využivaji v analýze glykosylaci eukaryotických proteinů, a dale pak metody s experimentalně ověřenými modifikacemi, ktere mohou být aplikovany při studiu prokaryotických glykoproteinů.
Summary Intracellular bacterial pathogens generally express chaperones such as Hsp100s during multiplication in host cells, allowing them to survive potentially hostile conditions. Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularaemia. The ability of F. tularensis to multiply and survive in macrophages is considered essential for its virulence. Although previous mutant screens in Francisella have identified the Hsp100 chaperone ClpB as important for intracellular survival, no detailed study has been performed. We demonstrate here that ClpB of F. tularensis live vaccine strain (LVS) is important for resistance to cellular stress. Promoter analysis shows that the transcriptional start is preceded by a σ 32 ‐like promoter sequence and we demonstrate that expression of clpB is induced by heat shock. This indicates that expression of clpB is dependent on the heat‐shock response mediated by σ 32 , the only alternative σ‐factor present in Francisella . Our studies demonstrate that ClpB contributes to intracellular multiplication in vitro , but is not essential. However, ClpB is absolutely required for Francisella to replicate in target organs and induce disease in mice. Proteomic analysis of membrane‐enriched fractions shows that five proteins are recovered at lower levels in the mutant strain. The crucial role of ClpB for in vivo persistence of Francisella may be linked to its assumed function in reactivation of aggregated proteins under in vivo stress conditions.
The expression of calcium-binding protein S100A9 was investigated in 23 matched sets of colorectal carcinoma and normal colon mucosa using two-dimensional gel electrophoresis. We found that, from a group of 23 patients, the level of S100A9 protein, in comparison with matched normal colon mucosa, was significantly increased in malignant tissues of 16 patients (70%). Furthermore, an additional protein, identified by matrix-assisted laser desorption/ionization - mass spectrometry (MALDI-MS) as S100A8, exhibited an increased expression in the same specimens of malignant tissues as the S100A9 protein. The immunohistological analysis revealed the accumulation of S100A9 positive cells, macrophages and polymorphonuclear leukocytes along the invasive margin of colorectal carcinoma. The S100A8 protein was found to be produced in the same location. The possible participation of both proteins and, especially, its heterodimeric complex calprotectin in colorectal carcinoma regression could be taken into account.
In-depth proteome discovery analysis represents new strategy in an effort to identify novel reliable specific protein markers for hypertrophic cardiomyopathy and other life threatening cardiovascular diseases. To systematically identify novel protein biomarkers of cardiovascular diseases with high mortality we employed an isobaric tag for relative and absolute quantitation (iTRAQ) proteome technology to make comparative analysis of plasma samples obtained from patients suffering from non-obstructive hypertrophic cardiomyopathy, stable dilated cardiomyopathy, aortic valve stenosis, chronic stable coronary artery disease and stable arterial hypertension. We found 128 plasma proteins whose abundances were uniquely regulated among the analyzed cardiovascular pathologies. 49 of them have not been described yet. Additionally, application of statistical exploratory analyses of the measured protein profiles indicated the relationship in pathophysiology of the examined cardiovascular pathologies.
The history of national tularemia research started in 1936 when the first outbreak was recognized in south-east Moravia.Since then in average about one hundred cases have been recorded annually.As tularemia was endemic in former Czechoslovakia, three research groups which concentrated on this disease were formed during decades.The first two groups have worked from sixties and were associated with Jiri Libich (Prague) and Darina Gurycova (Bratislava).The third group which concentrated on the research of natural foci started during late seventies in Valtice (Zdenek Hubalek).The experimental research was, and still is, mainly associated with military research, recently with the Proteomic Center (Faculty of Military Health Sciences, University of Defence) in Hradec Kralove.This center opens molecular approaches to the analysis of Francisella tularensis microbes on one side and the studies on mutual host-pathogen interaction on the other side.One of the significant aims of the research is searching for the new typing and diagnostic markers of Francisella tularensis for the military and medical practice.Thus, scientists from former Czechoslovakia and the Czech Republic contributed significantly to current knowledge on Francisella pathogenesis and their results were highly appraised by international scientific community.
Francisella tularensis (F. tularensis) is highly infectious for humans via aerosol route and untreated infections with the highly virulent subsp. tularensis can be fatal. Our knowledge regarding key virulence determinants has increased recently but is still somewhat limited. Surface proteins are potential virulence factors and therapeutic targets, and in this study, we decided to target three genes encoding putative membrane lipoproteins in F. tularensis LVS. One of the genes encoded a protein with high homology to the protein family of disulfide oxidoreductases DsbA. The two other genes encoded proteins with homology to the VacJ, a virulence determinant of Shigella flexneri. The gene encoding the DsbA homologue was verified to be required for survival and replication in macrophages and importantly also for in vivo virulence in the mouse infection model for tularemia. Using a combination of classical and shotgun proteome analyses, we were able to identify several proteins that accumulated in fractions enriched for membrane-associated proteins in the dsbA mutant. These proteins are substrate candidates for the DsbA disulfide oxidoreductase as well as being responsible for the virulence attenuation of the dsbA mutant.
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