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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...
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Effect of surfactin on the lipid moiety of Bacillus subtilis cytoplasmic membrane
Sklenářová, Petra ; Seydlová, Gabriela (advisor) ; Lichá, Irena (referee)
Surfactin, a secondary metabolite produced by Bacillus subtilis, is a surface active compound and antibiotic permeabilizing membrane bilayer. The aim of this study was to reveal the self-resistance strategy at the level of the lipid moiety of cytoplasmic membrane, which B. subtilis employs to combat surfactin in concentrations that are lethal for other bacterial species. Non-producing strain B. subtilis 168 was cultivated in the presence of two different sublethal concentrations of surfactin (350 a 650 µg/ml), which was isolated from the culture broth of B. subtilis ATCC 21332. Presence of surfactin in the medium resulted in a concentration dependent lag phase, which took 40 min (350 µg/ml) and 3 h (650 µg/ml), respectively. Afterwards, the culture grew with the altered doubling time of 44 min (350 µg/ml) and 126 min (650 µg/ml), respectively. Surfactin induced substantial changes in the phospholipid composition of the cytoplasmic membrane. The proportion of the major phospholipid component phosphatidylglycerol decreased and inversely, the level of phosphatidylethanolamine increased. Interestingly, the content of phosphatidic acid rose considerably in the presence of surfactin concentration causing stimulation of B. subtilis growth (350 µg/ml). Liposome leakage assay using phospholipids mimicking...
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Cytoplasmic membrane and tonoplast dynamics during closing and opening of stomata
Röder, Matěj ; Žárský, Viktor (advisor) ; Albrechtová, Jana (referee)
Stomata are epidermal structures mediating regulated contact of plant apoplast with surrounding environment via stomatal opening. Change of turgor plays crucial part in initiation of stomatal opening or closure. During stomatal movement, guard cell undergo considerable and repetitive changes in cell volume and consequently surface area over a period of minutes. Alteration in the surface of membrane must occurs due to limited stretching capability of the plasma membrane. It can be achieved through membrane invaginations and endocytosis of small vesicles. Microscopy and electrophysiology techniques have proven that both processes are happening in guard cells. These processes are controlled and managed by complex web of signal pathways in which actin and microtubular cytoskeletons, SNARE proteins, ion channels and others molecules have crucial parts. The aim of this work is to summarize current knowledge on the processes and mechanisms of these membranes surface changes and their molecular principle.
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Mode of daptomycin action and mechanism of resistance
Helusová, Michaela ; Mikušová, Gabriela (advisor) ; Lichá, Irena (referee)
Daptomycin is one of antimicrobial peptides. These molecules are part of immune system of all organisms. Daptomycin consists of a cyclic peptide core and a lipophilic tail. Daptomycin is produced by Streptomyces roseosporus and is used to treat serious gram-positive infections. Daptomycin is active also against methicillin-resistant Staphylococcus aureus. Its antimicrobial effect depends on the presence of calcium ions and phosphatidylglycerol. Daptomycin targets bacterial cytoplasmic membrane, where it forms oligomers. Mode of action of daptomycin probably includes pore-forming effect leading to membrane damage. This disturbance causes ion efflux from cytoplasm which leads to membrane potential disruption, which results in inhibition of macromolecular synthesis and cell death. Daptomycin also causes changes in cell morphology. Despite its unusual mode of action, several mechanisms of resistance have emerged in some pathogenic strains. These are for example decrease in the amount of phosphatidylglycerol in the membrane, increase in the amount of lysylphosphatidylglycerol, release of membrane phospholipids or mutations in genes which control peptidoglycan synthesis.
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Role of cytoplasmic membrane and cell wall in bacterial resistance to cationic antimicrobial peptides
Havlová, Noemi ; Seydlová, Gabriela (advisor) ; Vopálenská, Irena (referee)
Cationic antimicrobial peptides are part of the innate immune system of all organisms. Their properties such as structure, charge, amphipathicity and hydrophobicity make them promising agents with the potential for use not only in treatment of bacterial infections but also against some viruses, parasites, fungi and cancer cells. One of their possible targets is the cytoplasmic membrane, which they permeabilize. This mode of action has several advantages. The important feature of antimicrobial peptides is their selectivity for bacterial membranes, which makes them harmless to eukaryotic host cells. Another advantage is that the development of bacterial resistance against these peptides is more difficult since the action of antimicrobial peptides is rapid. Nevertheless, there appeared some bacterial strains that are insensitive to already used antimicrobial peptides. By using target modification resistant bacteria are able to prevent the bactericidal effects of the antimicrobial peptides. At the level of cytoplasmic membrane bacteria can alter its electrostatic or structural properties of membrane lipids and thus the antimicrobial peptides lose their ability to interact with the membrane and permeabilize it. Understanding the mode of action of antimicrobial peptides and mechanisms by which bacteria...
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Effect of surfactin on the lipid moiety of Bacillus subtilis cytoplasmic membrane
Sklenářová, Petra ; Seydlová, Gabriela (advisor) ; Lichá, Irena (referee)
Surfactin, a secondary metabolite produced by Bacillus subtilis, is a surface active compound and antibiotic permeabilizing membrane bilayer. The aim of this study was to reveal the self-resistance strategy at the level of the lipid moiety of cytoplasmic membrane, which B. subtilis employs to combat surfactin in concentrations that are lethal for other bacterial species. Non-producing strain B. subtilis 168 was cultivated in the presence of two different sublethal concentrations of surfactin (350 a 650 µg/ml), which was isolated from the culture broth of B. subtilis ATCC 21332. Presence of surfactin in the medium resulted in a concentration dependent lag phase, which took 40 min (350 µg/ml) and 3 h (650 µg/ml), respectively. Afterwards, the culture grew with the altered doubling time of 44 min (350 µg/ml) and 126 min (650 µg/ml), respectively. Surfactin induced substantial changes in the phospholipid composition of the cytoplasmic membrane. The proportion of the major phospholipid component phosphatidylglycerol decreased and inversely, the level of phosphatidylethanolamine increased. Interestingly, the content of phosphatidic acid rose considerably in the presence of surfactin concentration causing stimulation of B. subtilis growth (350 µg/ml). Liposome leakage assay using phospholipids mimicking...
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Cytoplasmic membrane and tonoplast dynamics during closing and opening of stomata
Röder, Matěj ; Žárský, Viktor (advisor) ; Albrechtová, Jana (referee)
Stomata are epidermal structures mediating regulated contact of plant apoplast with surrounding environment via stomatal opening. Change of turgor plays crucial part in initiation of stomatal opening or closure. During stomatal movement, guard cell undergo considerable and repetitive changes in cell volume and consequently surface area over a period of minutes. Alteration in the surface of membrane must occurs due to limited stretching capability of the plasma membrane. It can be achieved through membrane invaginations and endocytosis of small vesicles. Microscopy and electrophysiology techniques have proven that both processes are happening in guard cells. These processes are controlled and managed by complex web of signal pathways in which actin and microtubular cytoskeletons, SNARE proteins, ion channels and others molecules have crucial parts. The aim of this work is to summarize current knowledge on the processes and mechanisms of these membranes surface changes and their molecular principle.
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Mechanism of surfactin self-resistance in the Bacillus subtilis cytoplasmatic membrane
Seydlová, Gabriela ; Svobodová, Jaroslava (advisor) ; Spížek, Jaroslav (referee) ; Julák, Alois (referee)
Surfactin, a lipopeptide surfactant and antibiotic produced by Bacillus subtilis, exhibits a strong membrane perturbation. One of the drawbacks hindering its commercial applications is the unknown mechanism of surfactin self-resistance in the producer. Therefore, the aim of this study was to examine the self-protective mechanisms of the cytoplasmic membrane of B. subtilis against the deleterious effect of surfactin. In order to reach this task, two isogenic pairs of strains differing only in surfactin production were constructed. It was found out that the early response of the producer is realized by increasing the amount of total lipid content in the membrane already with the onset of surfactin synthesis. This process leads to lowering the surfactin-lipid ratio in the membrane. In parallel with the growing surfactin concentration the content of anionic phospholipids with cardiolipin as the major representative rises up to 24 % of the total. Together with the fall of phosphatidylethanolamine these changes promote the membrane stabilization and protect it against the interaction with surfactin. These alterations result in higher rigidity both of the polar head and hydrophobic chain region of the membrane as the steady state anisotropy of DPH and TMA-DPH showed. After 24 h of cultivation induction of...
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Cytoplasmic membrane adaptation to surfactin in Bacillus subtilis non-producing strain
Stružinská, Olga ; Svobodová, Jaroslava (advisor) ; Fišer, Radovan (referee)
Cytoplasmic membrane adaptation to surfactin in Bacillus subtilis 168 non-producing strain Surfactin, the most potent surface active compound and antibiotic is produced by bacteria of the genus Bacillus. Surfactin interacts with membrane bilayers, that results in destabilization and permeabilization of this structure. However mechanism of surfactin self-resistance in the producer's membrane is not understood. The aim of this study was to characterize the adaptive processes occurring at the level of cytoplasmic membrane of surfactin non-producing strain B. subtilis 168, which was exposed to exogenously added surfactin during the exponential phase of growth. The cultivation protocol of B. subtilis growth on agar media plates supplemented with surfactin was developed. Two surfactin concentrations that inhibit (400 g/ml) and even stimulate (300 g/ml) the growth of B. subtilis 168 strain were assessed. Surfactin brought about the growth arrest for 3 hours and the restored growth rate decreased in the case of inhibitory concentration, whereas the stimulatory concentration increased the growth rate and resulted in higher final density of the population. TLC was performed to analyze the polar head groups of membrane phospholipids. The portion of phosphatidylserine was found to increase at both surfactin...
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The Demembranation as a Tool to Study a Movement and Physiology of Activation of Fish Spermatozoa
BLAŽKOVÁ, Jaroslava
The aim of the thesis was the summarization of information about the demembranation method as a means of study of the physiology of activation and motility of fish spermatozoa. A simple demonstration of the method is also presented. The demembranation of fish sperm is a method, in which cytoplasmic membrane (the phospholipid bilayer, which separate intercellular and extracellular environments) is destroyed. A mild non-ionic detergent, TrintonX-100, was used in the study. Common carp (Cyprinus carpio) as a representative member of Teleost fish and sterlet as a member of Chondrostean (elasmobranch) fish were studied in the thesis.
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