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Construction of a genetically detoxified Bordetella pertussis strain to develope a new generation of whole-cell vaccine
Bočková, Barbora ; Holubová, Jana (advisor) ; Seydlová, Gabriela (referee)
Bordetella pertussis is a strictly human pathogen colonizing the upper respiratory tract, causing a respiratory disease known as whooping cough or pertussis. The introduction of whole-cell vaccines and acellular vaccines, resulted in a significant reduction in the incidence of disease and reduce the fatalities associated with infection. However, epidemiological data show a significant increase in the incidence of the disease in recent decades. The increasing incidence is mainly attributed to the transition from the whole- cell vaccine to an acellular vaccine. Based on research from recent years has shown that acellular vaccines have many drawbacks, and it is therefore necessary to change the vaccination strategy. One possible solution to the situation is the development of a new generation of whole-cell vaccines with reduced reactogenicity. The new whole-cell vaccine was prepared by a genetically modified B. pertussis strain. B. pertussis was modified using allelic exchange to develop strain encoding enzymatically inactive pertussis toxin, modified lipid A and lacking dermonecrotic toxin. This combination of genetic modifications in mice led to a decrease in reactogenicity test vaccine in vivo. In case of intranasal infection whole-cell vaccine containing genetically modified strain is providing...
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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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Bordetella pertussis and whooping cough: Bacterium and its virulence factors, epidemiology of disease and vaccination strategy.
Bočková, Barbora ; Holubová, Jana (advisor) ; Seydlová, Gabriela (referee)
Bordetella pertussis, a gram-negative bacterium, is a human pathogen which affects the upper respiratory tract. It is the causative agent of whooping cough or pertussis. B. pertussis produces several virulence factors consisting of toxins and adhesins. Whole cell vaccine and subsequent acellular vaccine were developed against pertussis in the past. However, a gradual increase of pertussis incidences has been reported in the last twenty years. This thesis provides basic information about B. pertussis and whooping cough. The main aim of the herein presented work is to summarize the contemporary epidemiologic situation along with determining reasons for increased pertussis cases. In addition, possible solutions for the present situation are proposed.
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The phenomenon of persistence in bacteria - the role of toxin-antitoxin systems.
Váchal, Martin ; Lichá, Irena (advisor) ; Seydlová, Gabriela (referee)
Most bacterial species currently studied are able to generate a small fraction of heterogeneous persister cells which are tolerant to antibiotics or other antimicrobials and still genetically identical to the susceptible parental population. Bacterial persisters emerge as a result of the stochastic regulation of cellular processes. Persistence can be triggered by stressful environmental stimuli or emerge spontaneously under favourable growth conditions. According to their origin, persistent subpopulations were divided into type I and type II persisters. Many recent studies indicate that toxin-antitoxin (TA) systems increase persistence. TA systems are ubiquitous genetic elements in prokaryotes and consist of a stable toxin, inhibiting essential cellular functions in persister cells, and an unstable antitoxin, which counteracts the activity of its toxin. Overexpression of toxin parts in excess of their corresponding antitoxin leads to multidrug tolerance (MDT). This work summarizes causes of persister formation and their hypothetical survival strategies and deals primarily with TA systems, controlling bacterial persistence of model organism Escherichia coli. The emphasis is put on the description of type I TA system TisB/IstR-1, type II TA systems HipBA, RelBE, MazEF, DinJ-YafQ, MqsRA, type V TA...
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Nucleotide metabolism in the development of Saccharomyces cerevisiae colony
Dvořáková, Markéta ; Palková, Zdena (advisor) ; Seydlová, Gabriela (referee)
Yeasts are unicellular microorganisms which are able to form colonies. The morphology of colonies is characteristic for the strain and differs according to particular growth conditions. Saccharomyces cerevisiae colonies are able to produce volatile ammonia which functions as a signalling molecule alarming nutrient depletion (PALKOVÁ et al. 1997). Changes in gene expression, metabolism and ammonia production occur during the development of giant S. cerevisiae colonies. Genes with changes in expression have been identified to be involved in ammonia transport, amino acid metabolism and also in nucleotide metabolism. Genes, whose deletion may affect the induction of ammonia production have been described (ČÁP et al., 2010), (VÁCHOVÁ a PALKOVÁ 2005), (PALKOVÁ et al. 2002). This work is focused on studying of an impact of deletions of selected genes involved in nucleotide metabolism, as well as of changes in extracellular concentrations of different bases on growth, morphology, ammonia production, cell morphology and differentiation of giant S. cerevisiae colonies. S. cerevisiae strains producing Gfp-tagged proteins (Ade4p-Gfp, Adk1p-Gfp, Urk1p-Gfp, Fcy2p-Gfp, Fur4p-Gfp, Fcy22p-Gfp) have been constructed and analyzed. Development of S. cerevisiae giant colonies with deletions of selected genes (ADE5,7,...
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Function of LmbW protein in biosynthesis of antibiotic lincomycin
Steiningerová, Lucie ; Janata, Jiří (advisor) ; Seydlová, Gabriela (referee)
4-Alkyl-L-proline derivatives (APD) are specialized precursors involved in the biosynthesis of at least three groups of different natural compounds: some pyrrolo-1,4-benzodiazepines with antitumor activity, bacterial hormone hormaomycin and clinically used lincosamide antibiotic lincomycin. These compounds share a biosynthetic pathway encoded by 5 or 6 homologous genes present in the biosynthetic gene clusters of the producing organisms. Similarities in biosynthesis and differences between APD structures of these compounds could be used to prepare a hybrid producing strain of biologically more effective lincomycin derivative. Unusual amino acid 4-propyl-L-proline (PPL) is the APD precursor of lincomycin. The originally proposed scheme of the PPL pathway does not comply with our current knowledge. Therefore, it was necessary to revise this scheme according to new results. The first two steps of the PPL pathway are functionally proved. Probing the next step was the main aim of this work. The protein LmbW was overproduced and its methyltransferase activity was confirmed in vitro. LmbW is able to directly methylate intermediate of second step of the pathway while the originally scheme proposed methylation at a later stage of biosynthesis. LmbW is also able to attach a longer alkyl chain to its substrate. This...
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Ecological roles of bacteria associated with plant litter
Tláskal, Vojtěch ; Baldrian, Petr (advisor) ; Seydlová, Gabriela (referee)
Leaf litter in temperate forests represents an important input of carbon into the soil. Main players in the decomposition of leaf litter are fungi and bacteria. While the role of fungi in litter decomposition was repeatedly addressed, there are just a few field studies where litter-associated bacteria were also considered. The aim of this work was to summarize available literature studying the structure and function of bacterial community during litter degradation. The nature of different possible substrates is discussed. Genetic approach is briefly outlined. Factors such as diversity, spatially distribution and abiotic factors that can influence community are also considered. It can be supposed that community composition changes with the change of litter chemistry and nutrient availability. The development of bacterial community might be driven by the decreasing availability of nutrients in litter. Labile compounds are utilised at the beginning of decomposition, while recalcitrant substrates are utilised later by specialist taxa. Members of the phyla Actinobacteria, Acidobacteria, Bacteroidetes and Proteobacteria seem to be dominant during decomposition. Different types of relationships between fungi and bacteria also most likely influence the composition of community. This review shows that the bacterial...
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Biosynthesis of lower polyketide chains in manumycin antibiotics - the length-affecting factors
Kolek, Jan ; Petříček, Miroslav (advisor) ; Seydlová, Gabriela (referee)
Manumycin antibiotics represent an important class of secondary metabolites produced by Streptomyces bacteria. They belong to a big class of polyketide metabolites and posses significant antimicrobial, anti-inflammatory, antitumor, and many other biological activities. They are characterized by two short polyketide chains, which are attached to a central subunit. Polyketide chains are synthesized by enzymes of the iterative type II polyketide-synthase. Mechanism of regulation of the polyketide chains length has not been known yet. Understanding mechanism can lead to biosynthesis of novel manumycin antibiotics with predetermined chain lengths what may improve their biological activities in favour of a practical use of these compounds. We prepared a mutant strain of asukamycin producer Streptomyces nodosus ssp. asukaensis with deletion of genes coding for type I/II β-ketoacylsynthase and protein AsuC14, which is a potential factor affecting lower polyketide chain length, for the identification of the chain length factor in manumycin antibiotics producers. Next, the genes for type I/II β-ketoacylsynthase and potential chain length-affecting factor C14 from strains producing manumycins with variable length of the lower polyketide chains were expressed in this mutant strain. Our results demonstrate...
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Cell wall integrity signalling pathway and yeast colony morphology
Reslová, Gabriela ; Schierová, Michaela (advisor) ; Seydlová, Gabriela (referee)
In the yeast Saccharomyces cerevisiae, stress on the cell wall is caused by various external influences (e.g. exposure to chemicals, oxidative stress, osmotic changes, pH changes or heat shock) which trigger the cell wall integrity signalling pathway (CWI). The aim of my work was to investigate the effect of the CWI pathway on yeast colony morphogenesis. Using strains with deletions in genes of the CWI pathway derived from two parental strains BR-F-Flo11p-GFP and PORT, I have found that differences in genetic background influences the process and activation of this pathway. Among the strains derived from BR-F-Flo11p-GFP, only the strain with the deletion of MID2 affects the appearance of colonies. MID2 encodes a cell-surface sensor of CWI pathway. In all deletion strains derived from PORT, the disruption of the CWI pathway causes a slower development of colonies growing on glycerol medium supplemented with 0,05 mM selenate inducing fluffy colony morphology. The largest effect has deletion of gene MTL1 which also encodes a cell-surface sensor with homology to Mid2. I have confirmed that strains with deletions in genes of CWI pathway have altered sensitivity to inhibitors disrupting cell wall integrity (Calcofluor white, Congo red, zymolyase). By means of zymolyase assay, I have confirmed the...
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Mode of action of antimicrobial lipopeptides produced by Bacillus subtilis
Pinkas, Dominik ; Seydlová, Gabriela (advisor) ; Žíla, Vojtěch (referee)
Increasing bacterial resistance to classical antibiotics and emergence of multi-resistant strains impose a constant threat. Antimicrobial compounds of bacterial origin are an important source of new antibacterial therapeutic agents needed to answer this issue. Three families of lipopeptides produced by Bacillus subtilis - surfactins, fengycins and iturins represent an interesting class of such compounds. They exert a wide range of biological activities and possess a good potential for modifications and improvement of their structure and function. Lipopeptides produced by B. subtilis are surface active compounds capable of reducing surface/interface tension. The variety of their biological activities stems from their ability to insert into lipid membranes leading to disruption and permeabilization of the membrane. Specific mode of action differs between the three families but the common feature is that it is concentration dependent. First, lipopeptides induce ion leakage, pore formation and then the increasing concentration eventually causes complete solubilisation of the membrane in a detergent-like manner. In addition, surfactin can inhibit some enzymes by chelating divalent cations required for their activity. These properties make the B. subtilis lipopeptides promising compounds for commercial applications.
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