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Structural mass spectrometry of Bordetella virulence factors
Jurnečka, David ; Bumba, Ladislav (advisor) ; Novák, Petr (referee) ; Řehulka, Pavel (referee)
The Bordetellae are aerobic Gram-negative coccobacilli colonizing the upper respiratory tract of mammals and thereby causing diseases with similar symptoms but different host specificity. The bacteria produce a variety of adhesins and toxins that facilitate their ability to promote infection and evade the innate immune system. Among them, the filamentous hemagglutinin (FHA) and the adenylate cyclase toxin (CyaA) are the major virulence factors providing the adherence to the host epithelial cells and the protection against bactericidal activity of phagocytic cells, respectively. Moreover, CyaA along with the Escherichia coli α-hemolysin (HlyA) and the Kingella kingae cytotoxin (RtxA) represent a prominent group of Repeats in ToXin (RTX) cytotoxins/hemolysins that undergo post-translational acylation on conserved lysine residues. Here, different mass spectrometry approaches were employed to analyze the structural features of FHA and to characterize the acylation status of the RTX toxins and their various hybrid molecules. First, the differential 16O/18O labeling revealed that the mature FHA proteins of B. pertussis (Bp-FHA) and the B. bronchiseptica (Bb-FHA) are processed at different sites, after Ala2348 and Lys2479 of the FhaB precursor, respectively. Second, the bottom-up proteomics of the...
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Regulatory mechanisms governing the virulence of the human pathogen Bordetella pertussis
Hejnarová, Václava ; Večerek, Branislav (advisor) ; Jurnečka, David (referee)
Bordetella pertussis is human pathogen, which causes severe respiratory disease called per- tussis or whooping cough. Pathogenicity of B. pertussis is mediated by a wide variety of vi- rulence factors including pertussis toxin, adenylate cyclase toxin, pertactin and filamentous haemagglutinin. Successful infection and colonization of the host depend on the precise timing of virulence factors production. For this purpose bacteria developed miscellaneous mechanisms of gene regulation. Two-component phosphotransferase systems, such as BvgAS, RisAK and PlrSR are involved in response to external stimuli. These systems of signal transduction modu- late bacterial gene expression profiles and establish consecutive phases of infection. Non-coding RNAs, particularly sRNAs and RNA chaperone Hfq provide additional level of regulation. Hfq is a post-transcriptional regulator, which mediates interaction of sRNA with target mRNA and thereby modulates their translation. Hfq affects approximately 10% of all B. pertussis genes including virulence factors such as type III secretion system, adenylate cyclase toxin, pertussis toxin and filamentous haemagglutinin. Knowledge of these regulatory mechanisms plays a key role in understanding of the pathogenesis of whooping cough and can lead to improved control over the spread...
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Virulence factors of the Trichophyton benhamiae complex
Machová, Lenka ; Čmoková, Adéla (advisor) ; Labuda, Roman (referee)
Dermatophytes are a group of fungi, some of which can cause skin diseases in humans and animals due to their ability to degrade keratinized tissue. Representatives of this group also include strains from the Trichophyton benhamiae complex, known to cause dermatophytosis especially of small rodents and rabbits. In the last decade, one of four populations of this complex has spread epidemically across Europe among guinea pigs and their breeders. To answer the question what stands behind the successful spread of this population, the gene expression and production of volatile organic compounds of epidemic and non-epidemic populations of T. benhamiae was investigated. Gene expression of three strains from each population was studied during growth in liquid medium and on ex vivo mouse skin models prepared according to a newly optimized protocol. RNAseq and RT-qPCR methods were chosen for the gene expression analysis. Based on the literature and the results of RNAseq preliminary analysis, several genes were selected for which specific primers were designed. The spectra of the produced volatile organic compounds of the same strains growing on sheep wool in vials were analyzed by GC-MS. While non-epidemic populations did not differ in gene expression and production of volatile organic compounds, the...
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Mechanism of action of bacterial toxins elevating the cAMP in host cells
Lepesheva, Anna ; Mašín, Jiří (advisor) ; Petráčková, Denisa (referee)
Cyclic adenosine monophosphate (cAMP) is an universal second messenger that regulates a large number of molecular mechanisms inside the eukaryotic cell. The level of synthesized cAMP is tightly regulated by endogenous adenylatecyclase (AC), and therefore this enzyme is often a target for various bacterial toxins. To manipulate intracellular cAMP levels in a target cell, bacteria have developed two different strategies for their toxins. Bordetella pertussis adenylate cyclase toxin (CyaA), Bacillus anthracis edema factor (EF) and Pseudomonas aeruginosa exotoxinY have in their structure an enzymatic AC domain which is activated by an intracellular cofactor and has several times higher activity than the eukaryotic AC enzyme itself. Other toxins, such as Bordetella pertussis pertussis toxin (PT), Vibrio cholerae cholera toxin (CT), and Escherichia coli heat labile toxin use ADP-ribosylation reaction of AC-coupled heterotrimeric G proteins to increase its activity and uncontrolled cAMP production. This work presents a literature search with accent on the molecular mechanism of interaction of these toxins with the target cell. Keywords: bacterial pathogens, virulence factors, intracellular cAMP elevation, bacterial toxins, adenylatecyclase (adenylylcyclase), Bordetella pertussis, Vibrio cholerae,...
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Cryptococcus neoformans virulence factors
Bauer, Martin ; Kuthan, Martin (advisor) ; Abrhámová, Kateřina (referee)
Cryptococcus neoformans is an opportunistic pathogenic yeast causing around 600 000 deaths annually. Its ability to cause a chronic infection is given by multiple virulence factors, which include the ability to grow in high temperature, polysaccharide capsule, oxidative stress tolerance and the expression of surface proteins. Unusual and resistant titan cells, which develop through the process of titanization, and the process of phenotypic switching can also be included. Despite intensive research, these virulence factors are yet to be fully described. Inducing factors of titan cells and participating signalling pathways are known. However, a complete model of titanization does not yet exist. So far, the mechanism of phenotypic switching is also unknown. In this work the current knowledge of virulence factors of C. neoformans is presented and summarised.
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Virulence factors of Entamoeba histolytica
Novotná, Monika ; Tachezy, Jan (advisor) ; Tůmová, Pavla (referee)
The parasitic protist Entamoeba histolytica causes intestinal disease called amoebiasis (amoebic dysentery), which is one of the most significant diseases worldwide, mainly in developing countries. The goal of this bachelor thesis is to summarize current knowledge about virulence factors of E. histolytica. It is primarly focused on adhesive lectin Gal/GalNAc, cysteine proteases, phagocytosis of apoptotic cells, amoebapore forming pores in the membranes of the target cells and trogocytosis. Keywords: virulence factors, Entamoeba histolytica, parasite, protist, amoebiasis, lectin Gal/GalNAc, cysteine proteinase, phagocytosis, trogocytosis, amoebapore
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Antibody against bacterial lectins as a tool preventing lung infections of cystic fibrosis patients
Bolshova, Arina ; Hodek, Petr (advisor) ; Kubíčková, Božena (referee)
Cystic fibrosis is an autosomal recessive disease that is caused by mutation of the CFTR protein ("cystic fibrosis transmembrane conductance regulator"). The CFTR protein is an ion channel for chloride and bicarbonate ions transmission. This defect leads to the formation of dense mucus in lungs that causing breathing problems and chronic bacterial infections. Passive immunization by administering chicken antibodies produced against pathogen can be used as a prophylaxis for patients with cystic fibrosis. Immunoglobulins Y reduce adhesion of bacteria to lung epithelial cells and prevent infection. This strategy is a good alternative to classical antibiotic therapy because it targets the pathogen and doesn't affect normal bacterial microflora. At first the chicken antibodies produced after immunization of the experimental hen by Burkholderia cenocepacia lectin (BC2L-A) were isolated and further purified on an affinity column with covalently bound lectin. The affinity purified fraction was 9 times concentrated compared to original fraction as far as the content of the specific anti-lectin antibody is concerned. The amount of affinity purificated antibodies is approx. 0.2% of the total isolated immunoglobulins. Their immunoreactivity has been verified using recombinant lectin and the lectin produced by...
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Analysis of protein cargo of extracellualr vesicles isolated from the yeast Candida albicans
Hlubučková, Lucie ; Konečná, Klára (advisor) ; Janďourek, Ondřej (referee)
Charles University Faculty of Pharmacy in Hradec Králové Study program: Pharmacy Candidate: Lucie Hlubučková Consultant: RNDr. Klára Konečná, Ph.D. Title of thesis: Analysis of protein cargo of extracellular vesicles isolated from the yeast Candida albicans Backgroung: The aim of this diploma thesis was to analyze the protein cargo carried in extracellular vesicles released from the yeast Candida albicans (C. albicans), which is one of the most important mycotic agens. Extracellular vesicles (EVs) are utilized as "transport vehicles", for the delivery of effector molecules into extracellular milieu. These molecules and primarilly proteins can play different roles in host-pathogen interactions. Proteins isolated from EVs and identified by proteomic approach were sorted into categories according to their molecular function and localization for the purpose of finding out, which proteins are predominantly distributed via extracellular vesicles into extracellular space after induction of nutrition starvation. Analysis of EVs protein cargo with focus on virulence factors could extend the knowledge about extracellular vesicles and their potential role in pathogenesis. Methods: Chosen C. albicans yeast strain was a clinical isolate strain isolated from a premenopausal women suffering from recurrent...
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Study of bacterial adherence on lung epithelia of Cystic fibrosis patients
Vašková, Lucie ; Hodek, Petr (advisor) ; Nosková, Libuše (referee)
A b stra ct C ystic fibrosis is an autosom alrecessive disease that is one of the m ost com m on hereditary disorders. T he disease is caused by the m utation in the gene encoding C F T R chloride channe l w hich leads to the failure of ion transportand a significant increase in viscosity o f m ucus,affecting especially the respiratory system .T he increase of saltand the presence of thick m ucus in the lungs suppress the antibacterialeffects of the im m une system .B acterial infections are the m ost com m on cause of death in patients w ith C F, especially the P seudom onas aerugino sa infection. A ntibiotics are used for treatm ent of the infection by this pathogen,butthe treatm ent is frequently com plicated by developing resistance.D ue to this fact, new w ays of treatm ent have been searched for. T he m ethod of passive im m unization of patients w ith yolk IgY antibodies seem s to be prom ising. T o exam ine the effect of these antibodies in in vivo m odel system , lung epithelial cells of a healthy subject (N uL i-1) and of a C F patient (C uF i-1) w ere exposed to 3 strains of P seudom onas aerugino sa. A ntibodies against the lectin of P seudom onas aerugino sa P A - IIL , w hich is an im portant adhesion structure of this bacterium , prepared from tw o im m unized anim als w ere com pared. T o...
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The detection of factors virulence and resistance genes bacterial pathogens in patients with cystic fibrosis
Jandová, Oldřiška ; Melter, Oto (advisor) ; Nyč, Otakar (referee)
Cystic fibrosis is an autosomal recessive genetic disorder, which is caused by mutation in CFTR gene (Cystic Fibrosis Transmembrane Conductance Regulator). This gene encodes protein with the same name, which is responsible for pathogenesis of CF. Cystic fibrosis is characteristic for frequent infection of respiratory system, which causes, destruction of lung tissue. These infections are characterized by occurrence of typical bacterial pathogenes, for example: S. aureus, P. aeruginosa etc. S. aureus is one of the most typical opportunistic pathogens, which causes serious difficulties in patients with the cystic fibrosis. Strains of S. aureus are characterized by production of multiple virulence factors and resistance to broad spectrum of antibiotics. Besides common mechanisms of resistance there is also possibility of emergence of so called Small Colony Variants in chronically infected patients. These resistant subpopulation is relatively common among S. aureus isolates of patients with CF. The aim of this work was to study isolates from three patients with cystic fibrosis, who are chronically infected by S. aureus. Our goal was to determine changes in the pattern of the antibiotic resistance and occurrence of virulence factors together with description of SCV strains. Powered by TCPDF (www.tcpdf.org)
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