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Adenylate cyclase toxin of Bordetella pertussis, its conformation and ion balance in host cell.
Motlová, Lucia ; Konopásek, Ivo (advisor) ; Krůšek, Jan (referee)
Adenylate cyclase (CyaA, ACT) toxin is one of the major virulence factors of Bordetella pertussis. Although CyaA binds to many types of membranes, it is assumed that the integrin CD11b/CD18 is its receptor which is expressed on the surface of myeloid cells. CyaA belongs to the family of RTX toxin-hemolysins. CyaA acts on the host cells by two independent activities. One of them is the conversion of ATP to cyclic AMP, which is catalyzed by adenylate cyclase (AC) domain after its translocation into the cytosol of the host cell, which leads to the entry of calcium cations into the host cell. Translocation is probably initiated by interaction of CyaA monomer with the target membrane. The second activity is the formation of CyaA channel selective for cations, which probably causes colloid osmotic lysis of target cells. The channel forming activity is provided by RTX hemolysin domain which most probably forms oligomers, although it was found that CyaA as a monomer causes leakage of potassium cations from the host cell. It is also not clear whether the oligomerization of CyaA would occur in solution, or after interaction with the host membrane. The aim of this study was to examine the flow of sodium ions on the membrane of murine macrophages J774A.1, which express integrin CD11b/CD18 on their surface....
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Supported Phospholipid Bilayers and their Interactions with Proteins Studied by Ellipsometry, Atomic Force Microscopy and Confocal Fluorescence Microscopy
Macháň, Radek ; Hof, Martin (advisor) ; Fidler, Vlastimil (referee) ; Konopásek, Ivo (referee)
Supported lipid bilayers have been used as an artificial model of biological membranes and their interaction with 5 selected antimicrobial peptides was studied by several experimental techniques, mainly ellipsometry, laser scanning microscopy and fluorescence correlation spectroscopy. The thesis explains basic principles of the applied techniques focusing on their aspects relevant to characterization of lipid bilayers. The biological significance of antimicrobial peptides, their modes of interaction with membranes and the basic characteristics of the selected peptides are briefly discussed. The following text describes the main types of experimental studies performed and the interpretation of their results. Peptide-induced changes in lipid bilayer morphology were characterized by ellipsometry and laser scanning microscopy. Most interesting effects were observed in the case of melittin, which induced formation of long lipid tubules protruding from the bilayer. Lipid lateral diffusion measured by fluorescence correlation spectroscopy can provide information on bilayer organization on length-scales below resolution of optical microscopy.
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Structural and functional characterization of yeast plasma membrane domains
Strádalová, Vendula ; Malínský, Jan (advisor) ; Palková, Zdena (referee) ; Konopásek, Ivo (referee)
Plasma membrane (PM) of living cells hosts variety of important cellular functions that must be precisely coordinated in space and time. Recent research shows that the plasma membrane is organized into specific domains to accomplish all these tasks. Our laboratory is focused on the organization of the plasma membrane in Saccharomyces cerevisiae where several distinct lateral compartments were identified at the fluorescence microscopy level. One of them is the Membrane Compartment occupied by arginine transporter Can1 (MCC) which consists of isolated, highly stable, ergosterol enriched, 300nm patches containing specific proton symporters and proteins of unknown function (Sur7- and Nce102-like). These membrane domains are organized by cytosolic protein complexes called eisosomes, composed mainly of proteins Pil1 and Lsp1. This work is a continuation of studies that tried to elucidate the composition, structure and function of MCC. In the first section of this work we concentrated on ultrastructural characterization of MCC domains. Foremost, we developed a protocol preserving the plasma membrane ultrastructure. The comparison of cryofixed and chemically crosslinked cells clearly showed that cryofixation by high pressure freezing together with freeze substitution and low temperature resin embedding...
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Characterization of 32,33-didehydroroflamycoin - secondary metabolite from Streptomyces durmitorensis
Koukalová, Alena ; Černý, Jan (advisor) ; Konopásek, Ivo (referee)
Streptomycetes are soil filamentous Gram-positive bacteria that produce wide variety of pigments and biologically active substances including macrolides. Some of them are used as very efficient antibiotics and strong antifungal agents in medicine, others have became useful tools for staining biomembranes and detecting cholesterol via their internal fluorescence. Actinomycete Streptomyces durmitorensis (wild type strain MS405T ) is a bacteria isolated from Durmitor National Park in Montenegro soil samples. It produces secondary metabolite that has been identified as 32,33-didehydroroflamycoin (DDHR) closely related to the macrolides roflamycoin and generaly used filipin. DDHR exhibits cytototoxic activity against mammalian cells and yeast Saccharomyces cerevisiae strain EGY48. In addition it has interesting fluorescence properties allowing visualization of some membrane components. DDHR interacts with biomembranes, causes their disintegration leading to changes of the actin and tubulin cytoskeleton organization and in higher concentrations it causes cells necrosis. DDHR-sterol interaction in cell membranes decreases fluorescence intensity of DDHR. The compound is able to fluorescently stain aberrant lysosomes and could be therefore potentially used in diagnostics of some lysosomal storage disease.
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Spr0334, new protein of cell division in Streptococcus pneumoniae.
Štekerová, Nela ; Doubravová, Linda (advisor) ; Konopásek, Ivo (referee)
Spr0334, new protein of cell division in Streptococcus pneumoniae Streptococcus pneumoniae is an important human pathogen. The geonome of this bacteria encodes a single gene for eukaryotic-like serine / threonine protein kinase called StkP. StkP regulates many physiological processes such as pathogenesis, competence for genetic transformation, resistance to various stresses and resistance to antibiotics. It also affects the transcription of many genes involved in cell wall biosynthesis, pyrimidine metabolism, DNA repair and iron uptake. Recent studies have shown that StkP is located in the cell division septum and significantly regulates cell division and morphology. Its substrates include, among others, cell division protein DivIVA, FtsZ and FtsA. Analysis of phosphoproteome maps of wild type and ΔstkP mutant strain of S. pneumoniae showed that in vivo StkP phosphorylates several putative substrates including the protein Spr0334. Mass spectrometry analysis identified phosphorylation sites of the protein Spr0334: threonine 67 and threonine 78. Furthermore, it was found that the protein Spr0334 is located in the cell division septum, which led to the hypothesis that it could be newly identified cell division protein in S. pneumoniae. The main aim of this thesis was to describe the function of the...
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Fluorescence studies of bacterial membrane proteins and cell signalling.
Fišer, Radovan ; Konopásek, Ivo (advisor) ; Hof, Martin (referee) ; Forstová, Jitka (referee)
(English) This work is based on five publications studying mostly adenylate cyclase toxin (CyaA) from Bordetella pertussis and its interaction with biological membranes. CyaA permeabilizes cell membranes by forming small cationselective pores and subverts cellular signaling by delivering an adenylate cyclase (AC) enzyme that converts ATP to cAMP into host cells. First study clarifies the membrane disruption mechanisms of CyaA and another bacterial RTX toxin; αhemolysin (HlyA) from Escherichia coli. For this purpose, we employed a fluorescence requenching method using liposomes as target membranes. We showed that both toxins induced a graded leakage of liposome content with different ion selectivities (Fišer a Konopásek 2009). Both AC delivery and pore formation were previously shown to involve a predicted amphipathic αhelix(502522). In the second publication we investigated another predicted transmembrane αhelix(565591) that comprises a Glu(570) and Glu(581) pair. We examined the roles of these glutamates in the activity of CyaA, mostly on planar lipid membranes end erythrocytes. Negative charge at position 570, but not at position 581, was found to be essential for cation selectivity of the pore, suggesting a role of Glu(570) in...
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Cytoplasmic membrane of Bacillus subtilis Regulation of the physical parameters
Beranová, Jana ; Konopásek, Ivo (advisor) ; Branny, Pavel (referee) ; Holoubek, Aleš (referee)
Bacillus subtilis, a model Gram-positive soil bacterium, employs two distinct mechanisms in its membrane adaptation to low temperature: 1) Long-term adaptation to suboptimal temperature is accomplished by increasing the ratio of anteiso- to iso-branched fatty acids in the membrane lipids. 2) After a sudden temperature decrease, the oxygen-dependent fatty acid desaturase (Des) is induced which desaturates fatty-acyl chains incorporated in membrane lipids. The transcription of the gene encoding desaturase, des, is activated by the decrease of the membrane order, via two- component system DesK-DesR. In this work, I studied the influence of cultivation conditions on the mechanisms of B. subtilis membrane adjustments for a low temperature employing fatty acid analysis, fluorescence spectroscopy, differential scanning calorimetry and methods of molecular biology. In the first part of this work, I examined the impact of the cultivation medium on the composition and biophysical features of the B. subtilis cytoplasmic membrane during growth under the optimal (40 řC) and suboptimal (20 řC) cultivation temperature. I compared the nutrient-rich complex medium containing glucose and the mineral medium supplemented with either glucose or glycerol. The results obtained showed the crucial importance of medium...
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Differences in physiology between r and K bacterial strategists.
Moserová, Andrea ; Konopásek, Ivo (advisor) ; Krištůfek, Václav (referee)
Differences in physiology between r and K-bacterial strategists. The definition of bacterial r/K-strategists is currently based on the time interval they need to form a colony on agar plate. Also, their growth rate which is often used to identify r/K-position within a pair of bacterial strains. To date it was evidenced that also other physiological characteristics relate to bacterial r/K-status, for example their different ability to 1) adapt for changing conditions 2) utilize complex or very diluted substrates, 3) use secondary metabolites to cope with other strains and possibly also others. The intersection of macro- and microbiological r/K-conceptions lies in time distribution of r/K-strategists during succession. The aim of this study was to verify the basic r/K-characteristics on nine chosen strains and to correlate them with their physiological differences that are implicitly regarded as characteristic for r/K-groups. The study deals with growth rates measurements on both liquid and solid media, identification of fatty acid composition and membrane fluidity of strains cultivated at near-optimum and cold temperature in order to track the differences in cold adaptation. The study also deals with the description of possible new K-strategist characteristic: the lack of correlation between the colony...
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Cold adaptation in stationary phase in Bacillus subtilis
Beranová, Anna ; Konopásek, Ivo (advisor) ; Svobodová, Jaroslava (referee)
Cold adaptation in stationary phase in Bacillus subtilis One of the most important abiotic factor which influences life of bacterial cells is the ambient temperature. A decrease of this temperature is usually accompanied usually with the loss of the fluidity of bacterial cytoplasmatic membrane. While the mechanisms of the responses to the cold shock during the exponential phase of growth are well known for Bacillus subtilis, the responses of stationary phase cells had not been studied yet (despite the stationary phase is the most common state of microorganism in the nature). There are two independent mechanisms which restores much needed fluidity in Bacillus subtilis - short-term adaptation and long-term adaptation. Short-term adaptation is based on the function of fatty acid desaturase coded by des gene. Long-term adaptation relies on the change in ratio of iso- and anteiso- branched fatty acids. In this work we examinated membrane adaptation during stationary phase under two different conditions, namely under cultivation at stable low temperature and after cold shock. The highest activity of Pdes was observed for cultivation at 25 řC and for the cold shock applied from cultivation in 37 řC to 25 řC. Anisotropy measurements and fatty acids analysis were also performed. Results indicated, that the...
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Z-scan Fluorescence Correlation Spectroscopy Applied in Studies of Model Phospholipid Membranes
Kulakowska, Anna ; Hof, Martin (advisor) ; Konopásek, Ivo (referee) ; Dejneka, Alexander (referee)
6. Summary First of all, the issue whether and to what extent lipid mobility in the bilayer is altered when the bilayer is being deposited on the solid support was addressed. We designed a method that allows a direct comparison of the diffusion coefficients of labelled-lipids in the free-standing lipid bilayer of GUVs and that of the bilayer interacting with the mica surface (SPBs) in the glucose solution. The lateral diffusion analysis strongly indicate that the observed phenomenon is an unrestricted 2D diffusion. The results clearly show that the diffusion is slowed by more than 2 times for the interaction with the support. Moreover, we believe that the quantitative comparison of lipid diffusion in two frequently used model membranes can be helpful when comparing data reported in the literature. Additionally, the obtained data clearly indicate the existence of an inter-leaflet coupling. The lateral diffusion of investigated membrane probes and phospholipid analogues depend on the character and structure of the used fluorescent probe. The obtained lateral diffusion of the flavone probe, F2N12S is significantly faster than that of the fluorescent lipid analogues under study. Moreover, flavone probe exhibits approximately an order of magnitude faster flip-flop in comparison to the Atto633-DOPE lipid analogue...
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