|
| |
|
|
Ser/Thr protein kinases in mycobacteria
Borovcová, Taťána ; Doubravová, Linda (advisor) ; Konopásek, Ivo (referee)
The Mycobacterium tuberculosis genome encodes 11 Ser/Thr protein kinases. These protein kinases are structurally related to eukaryotic protein kinases. The phosphoproteome contains hundreds of proteins phosphorylated on Ser/Thr residues that influence all aspects of cell biology, which supports the critical role of phosphorylation in the regulation of physiology. Particularly important role in regulation belongs to protein kinases PknA, PknB, PknG and PknL, these protein kinases occur in all species of mycobacteria. Although only PknA and PknB are essencial for the M. tuberculosis, they regulate cell shape through the regulation of cell wall synthesis and cell division. Another important protein kinase is PknG, although not essential for growth it is necessary for virulence, because it promotes the survival of pathogen inside macrophages of the host. As a result, Ser/Thr protein kinases represent an interesting target for inhibitor development that could be used as drugs against tuberculosis.
|
|
|
Membrane vesicles in bacteria and their role in antibiotic resistance
Benešová, Anna ; Konopásek, Ivo (advisor) ; Pospíšil, Jiří (referee)
Membrane vesicles are produced by both Gram-positive and Gram-negative bacteria. Mechanism of their formation differs between these two groups of bacteria. It is caused by the different structure of their cell envelope. Gram-negative bacteria contain outer membrane and membrane vesicles can originate from this membrane. Membrane vesicles of Gram-positive bacteria are derived from the cytoplasmic membrane. They have to cross the barrier of the thick layer of peptidoglycan. Membrane vesicles contain cellular components whose properties enable vesicles to fulfil various functions. Antibiotic resistance can be counted as one of these functions. This thesis discusses three ways used by the membrane vesicles to protect the cells from antibiotics: transport of enzymes that degrade antibiotics, removal of antibiotics from cell's surroundings and role of membrane vesicles in horizontal gene transfer.
|
|
|
Model membranes studied by advanced fluorescence techniques and molecular dynamics simulations
Melcrová, Adéla ; Hof, Martin (advisor) ; Heyda, Jan (referee) ; Konopásek, Ivo (referee)
In this thesis, we start with the description of the biophysical properties of the plasma membrane models upon signaling processess such as the increased cytoso- lic concentration of calcium ions, or posttranslational modifications of membrane proteins. Calcium signaling is characterized by a rapid increase of its cytosolic concentration. We identify calcium binding sites and characterize the binding in the plasma membrane models of increasing complexity from pure phospholipid bilayers, through cholesterol and peptide rich lipid membranes, to membranes ex- tracted from HEK293 cells. We use Time-Dependent Fluorescent Shift method, which provides direct information on hydration and mobility in defined regions of a lipid bilayer, accompanied with molecular dynamic (MD) simulations, which give molecular details of the studied interactions. The initial step of signaling mediated by PAG protein is its double palmi- toylation. We investigate changes of the biophysical properties of both the lipid membrane and the peptide itself upon the incorporation of the palmitoyls. Em- ploying all atom MD simulations, we study inter- and intramolecular interactions as well as changes in membrane hydration, thickness, or lipid ordering. The second part of the thesis, realized in a direct collaboration with a phar- macological...
|
|
|
Identification of new substrates of Ser/Thr protein kinase StkP
Kleinová, Simona ; Ulrych, Aleš (advisor) ; Konopásek, Ivo (referee)
Streptococcus pneumoniae encodes single serine/threonine protein kinase StkP and its cognate protein phosphatase PhpP. This signalling couple phosphorylates/dephosphorylates many target proteins involved in various cellular processes. So far, only few ot them was characterized in detail. Global phosphoproteomic analysis in the ∆stkP mutant strain background resulted in the identification of protein Spr0175 as phosphorylated on threonine 7. The main aim of this work was to characterize this new substrate. The ∆spr0175 mutant strains were prepared in the wild type genetic background Rx and R6 and then monitored for their growth and cell morphology. Mutant strains exhibited morphological defects revealing potential involvement of Spr0175 in the process of cell division. In the wild type D39 the deletion was unsuccesful, which may entail possible essentiality of Spr0175 in D39 strain. The results obtained also confirmed that the Spr0175 is modified in in vitro and in vivo conditions at threonine 7. In vitro study also confirmed minor phosphorylation at T4 residue. By using co-immunoprecipitation assay we demonstrated that Spr0175 protein can form oligomeric structures. Another aim of this work was cellular localization of Spr0175. By using fluorescent microscopy we showed that GFP-Spr0175 fusion...
|
|
|
Bacterial RTX toxins and their calcium-binding sites
Lišková, Petra ; Konopásek, Ivo (advisor) ; Holoubek, Aleš (referee) ; Hof, Martin (referee)
FrpC protein produced by Neisseria meningitidis in a human host belongs to the family of bacterial RTX toxins due to the presence of RTX domain. FrpC possesses a calcium-dependent auto-catalytic cleavage activity which is localized within its 177 amino-acids long segment Self-Processing Module (SPM). As the SPM is naturally intrinsically disordered protein without bound Ca2+, the calcium binding is crucial for SPM folding which is followed by the auto-catalytic processing. The elucidation of the SPM structure may be the key step for understanding of enzymatic and biological function. The structure of folded SPM itself can be characterized only with difficulties due to the presence of flexible loop according to preliminary NMR data. The subject of this work is the description of SPM using fluorescence methods, characterization of ions binding to SPM and structural changes occurring during Ca2+ binding. In this work, the ion binding properties of SPM segment and its ion-induced folding was characterized. It was found that the dissociation constant kD of 17 μM coincided with the folding of SPM into the native calcium-bound state which occurs in the concentration range between 1 and 20 μM Ca2+. In the attempt to characterize the structure of ion binding site, the fully active single tryptophan mutants...
|
|
|
Complex characterization of subgingival plaque - use of modern sequencing methods in diagnostics and monitoring of treatment of periodontal diseases
Těšínská, Barbora ; Najmanová, Lucie (advisor) ; Konopásek, Ivo (referee)
Periodontitis is a multifactorial inflammatory disease which can result in a complete loss of teeth. Its main cause is the accumulation of bacteria from the dental plaque followed by massive reaction of the host immune system. It was proved that the composition of oral microbiome (OM) differs in periodontally healthy individuals and patients with periodontitis. This work aims to solve specific parts of the long-term project concerning the taxonomic composition of the OM of periodontally healthy individuals and patients with chronic and aggressive form of the disease. The OM was characterized based on 16s rDNA sequencing. It is evident from the results that the shift in the OM composition occurs prior the development of clincal signs of the disease and that the precise dental care can significantly postpone or even avoid the onset of the disease. When comparing the OM composition in individuals with chronic and aggressive periodontitis, no remarkable differences were detected to explain the faster progress of the aggressive form of the disease. This work also aimed to compare the results obtained by 454 pyrosequencing and Illumina. Both sequencing methods were found to provide statistically comparable results. Illumina MiSeq thus can be employed to build on the former results of the long-term study...
|
|
| |
|
| |
|
| |
guest ::
