|
|
Bacterial toxins acting on biological membranes
Benešová, Anna ; Fišer, Radovan (advisor) ; Sýkora, Jan (referee)
Colicin E1 is a bacterial toxin, which has been researched for a long time and belongs to a group of pore-forming colicins. Pore-forming colicins act on cytoplasmic membranes of sensitive cells. They form ionophoric channels, which can depolarize the membrane and kill the cell. Colicins in general can be considered as a tool used by related bacterial strains of Escherichia coli and other Enterobacteriaceae to compete for resources. Recently, a hypothesis emerged that colicin E1 is not a tool for killing related bacterial strains but rather a tool for killing human cells by uropathogenic strains of Escherichia coli. So far, colicin E1 was described inaccurately, together with its different sequence variant - newly described colicin E11. Colicin E11 could have evolved as a weapon against other bacteria of gut microbiota. The aim of this thesis was to compare the pore-forming abilities of colicins E1 and E11 by measurements on planar lipid membranes and liposomes of various lipid compositions. Based on results it seems that both colicins form channels of comparable conductance and they are both mildly cation-selective. Measurements on liposomes showed higher activity for colicin E1 compared to colicin E11 on membranes mimicking both mammalian and Gram-negative bacterial cytoplasmic membrane. Results...
|
|
|
Hypothetical protein Spr1962 as a new substrate of the signalling pathway of the Ser/Thr protein kinase StkP and phosphatase PhpP.
Mašková, Kateřina ; Ulrych, Aleš (advisor) ; Fišer, Radovan (referee)
Hypothetical protein Spr1962 as a new substrate of the signalling pathway of the Ser/Thr protein kinase StkP and phosphatase PhpP The extracellular human pathogen Streptococcus pneumoniae encodes in its genome only a eukaryotic-type serine/threonine protein kinase (StkP) and the corresponding PP2C phosphatase (PhpP) in its genome. This makes it a unique model organism for the studying of signaling pathways in bacteria. To date, several substrates of this signaling protein have been identified and characterized, including DivIVA, KhpB, FtsA, FtsZ, MacP, GlmM, GpsB, ComE and others. These proteins are involved in various cellular processes, including cell division and peptidoglycan biosynthesis. In a global phosphoproteome study based on the LC- MS approach, one of the potential new substrates was the hypothetical protein Spr1962, whose phosphorylation was detected only in the hyperphosphorylated strain with deleted PhpP phosphatase. The aim of this work was to characterize this new substrate Spr1962 and to clarify its possible function. Based on the significant structural similarity to Spr1962 the FloT flotillin protein of Bacillus subtilis, it was suggested that it might be a protein with an analogous function in pneumococci. Deletion of this gene in different genetic backrounds was found to cause an...
|
|
|
Elucidation of the properties and structure of the pore-forming domain of colicin U produced by bacterium Shigella boydii.
Dolejšová, Tereza ; Fišer, Radovan (advisor) ; Krůšek, Jan (referee) ; Osička, Radim (referee)
Colicin U is a protein produced by bacterium Shigella boydii. It belongs to the group of pore-forming colicins. These colicins interact with receptors in the outer membrane of bacteria closely related to a producing colicinogenic strain. After interaction with the receptor, colicin is translocated across the outer membrane and periplasm to the cytoplasmic membrane where it forms pores. Consequently, the pore formation leads to membrane depolarization and cell death. In this thesis I decided to study the pore-forming properties of colicin U and its membrane topology. It is shown that colicin U pores are formed by only one colicin molecule and they are voltage dependent. Using measurements with nonelectrolytes we estimated a theoretical inner profile of the pore and its inner diameter to be between 0.7 and 1 nm. Above that, a membrane topology of colicin U pore-forming domain (PFD) is studied. BLM measurements with biotinylated colicin U showed that a significant part of colicin's PFD was translocated to the opposite side of the membrane after the pore opening. The segment between substituted amino acids F463 and D486 was evidenced to be on the trans side of the membrane after the pore opening. Additionally, properties of peptide H1, which reflects a significant part of the first α- helix of colicin...
|
|
|
Relationship between the structure of synthetic organic molecules, their behavior in the phospholipid membrane and their effect on bacteria
Brzobohatá, Hana ; Fišer, Radovan (advisor) ; Mašín, Jiří (referee)
Lipophoshopnoxins (LPPOs) are organic compounds with antimicrobial activity, which take place on the baterial membrane by forming pores. LPPOs are promising bactericidal molecules with low MIC (minimal inhibition concentration) and high HC50 (hemolytic concentration) values. Structurally, LPPOs belong to the so-called synthetic antimicrobial peptidomimetics, which do not contain any peptidic bonds, but imitate the structure and function of membrane active oligopeptides (e.g. by copying the distribution of the surface charge of the molecule). The aim of this study was to describe the antimicrobial properties of the molecules DR 7072, DR 34P1, DR 362 and DR 236, which represent LEGO-LPPOs as a new generation of this antimicrobials. The selected molecules differ in the length of linker module, in the structure of hydrophobic module or both. All tested molecules have comparable antimicrobial activity with MIC between 2 and 16 μg/ml and have the ability to permeabilize bacterial membrane of both Gram negatives (G- ; Pseudomonas aeruginosa CCM 3955) and Gram positives (G+ ; Staphylococcus aureus CCM 4223). LEGO-LPPO have different mechanism of action on S. aureus and P. aeruginosa. The molecules show different mechanism of action against the mentioned bacteria. The bactericidal effect on G+ is faster and...
|
|
|
Non-covalent interactions of tryptophan in protein structure
Sokol, Albert ; Fišer, Radovan (advisor) ; Jurkiewicz, Piotr (referee)
A thorough knowledge of non-covalent amino acid interactions within a protein structure is essential for a complete understanding of its conformation, stability and function. Among all the amino acids that usually make up a protein, tryptophan is distinguished both by its rarity and size of its side chain formed by an indole group. It is able to provide various types of indispensable interactions within the protein and between different polypeptide chains, but also between the protein and a biological membrane. In addition, it is the most commonly used natural fluorophore. Databases of solved protein structures are commonly used to study amino acid interactions and allow more or less complex analyzes of the issue. Thus many non-covalent interactions that may occur between tryptophan and other amino acids have been found. However, most of these analyzes focus on specific interactions and do not follow up the tryptophan's environment as a whole, where all amino acids interact. Some newly developed methods have been used in this Thesis, specifically the occurrence profiles of the individual amino acids around the indole group of tryptophan and the results were compared with an available literature. The amino acid that has the greatest preference for tryptophan turned out to be tryptophan again, and...
|
|
| |
|
| |
|
|
Bacterial toxins translocating across the membrane of eucaryotic cells
Poledňák, Jan ; Fišer, Radovan (advisor) ; Žáčková Suchanová, Jiřina (referee)
The bacterial protein toxins endowed with the ability to translocate accross the plasmatic membrane are often crucial virulence factors of pathogenic bacteria invading eucaryotic organisms. These toxins translocate either their own protein domains carrying toxic activity or can form pores transfering other substances like small ions, DNA, RNA or proteins. By observing the translocation of these molecules together with others artificially prepared agens on synthetic membranes allows detailed understanding of mode of action of individual pore- forming toxins. Some of the toxins were actually described in such a detail, that can serve as investigation tools for characterization of new translocated molecules. One of such example is the transfer of nucleotides or whole nucleic acid molecules accross the membrane pore of α- hemolysine of S. aureus. This applications is in recent days commercially used for DNA sequencing.
|
|
|
Structure and function of IRES elements in yeast
Černý, Jiří ; Pospíšek, Martin (advisor) ; Fišer, Radovan (referee)
Translation initiation is an important step in control of gene expression. Cellular mRNAs are usually translated via classical cap-dependent pathway, but some of them can also be translated by an alternative pathway like IRES mediated one. In the first part of my work I studied a possibility of IRES dependent translation initiation of RRN3 and BAS1 mRNAs from yeast Saccharomyces cerevisiae. I inserted BAS1 and RRN3 5'UTR into the intercistronic spacer of pFGAL4h and its promotreless version and measured the activity of secondary reporters in enegineered yeast strains. My data indicates presence of cryptic promoter in tested regions. To confirm this results I first inserted the RRN3 5'UTR into the intercistronic region of another bicistronic plasmid - pRFh and measured expression of second cistron. The results were similar like in the case of pFGAL4h system. The presence of shorter RNAs transcribed from cryptic promoter within the intercistronic region was shown using real time PCR. My quest in the second part of this work was to prepare RNAs useful for next investigation of translation control of lariat capped RNAs produced by GIR1 ribozyme in a slime mold Didymium iridis. I have prepared a wide set of DNA molecules coding different versions of GIR1 in front of the firefly luciferase ORF which served as...
|
|
|
Effect of Genotoxic 2-Nitrofluorene and Its Metabolites on DNA In Vivo and Possibilities of Investigation of this Effect Using Electrochemical DNA Biosensors In Vitro
Stávková, Klára ; Vyskočil, Vlastimil (advisor) ; Fišer, Radovan (referee)
Presented Bachelor Thesis is targeted on the topic of hazardous 2-nitrofluorene (NF), which is known as one of the markers of the presence of nitrated polycyclic aromatic hydrocarbons. This Thesis summarizes the current knowledge about the formation of this xenobiotic, its occurrence and transport in living and working environment, and about its negative effects on living organisms, especially on their DNA. These findings obtained upon the in vivo investigations are compared in this Thesis with information obtained using electrochemical DNA biosensors, which represent very promising in vitro alternative to the study of processes proceeding in living organisms upon the interaction of their DNA with the xenobiotic. NF is mainly released to the atmosphere through the exhaust gases of diesel and gasoline engines. It is adsorbed onto solid particulate matters, through which it is spread over long distances and can enter the air passages of animals. Through an atmospheric fall-out, NF contaminates waters and soils, where it is transported together with other nutrients and food to plants and animals; by consummation of such organisms, it can finally enter the body of higher animals and the human. It has been shown that NF is a chemically stable substance. On the other hand, it is metabolized intensively...
|
guest ::
RSS feed.