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Influence of environment in host cells on heterogeneity of virulence factors expression in Salmonella
Mathéová, Paulína ; Černý, Ondřej (advisor) ; Konopásek, Ivo (referee)
Bacteria of the genus Salmonella are one of the most important human pathogens. Salmonella expresses several virulence factors, including the type three secretion system encoded on the Salmonella pathogenicity island 2 (SPI-2 T3SS) and its effector proteins. SPI- 2 T3SS effector proteins allow the bacteria to colonize host cells, persist there and use them as a Trojan horse for migration throughout the host organism. Although the function of the SPI- 2 T3SS is essential for the bacterial population during systemic infection, some bacteria do not express this virulence factor and benefit from its expression by other members of the population. Similar heterogeneity in expression of other virulence factors has been shown to be crucial for their function. This work focuses on the heterogeneity of expression of selected SPI-2 T3SS effector proteins under the influence of host factors (pH, ROS, RNS) affecting bacteria during intracellular infection of macrophages. Using a fluorescent reporter system suitable for flow cytometry the expression of selected effector proteins was detected at the level of individual bacteria. The obtained data show, that individual bacteria respond differently to the decrease in pH in their vicinity. Part of the bacterial population starts to express the studied effector...
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Application of human monocytic cell line THP-1 for study of pathogenesis in whooping cough agent Bordetella pertussis
Čurnová, Ivana ; Petráčková, Denisa (advisor) ; Mašín, Jiří (referee)
Bordetella pertussis is strictly human pathogen that causes severe infection of the respiratory tract known as whooping cough, which is currently on the rise. B. pertussis was considered as an extracellular pathogen for a very long time. Recently it was shown the ability of B. pertussis to survive inside early endosomes of macrophages. This ability is studied in the human monocytic cell line THP-1 and also in primary macrophages from human donors. This diploma thesis is focused on THP-1 infectious model and mainly for the early phase of infection. A previously performed transcriptomic study showed significantly affected genes of B. pertussis during intracellular survival in THP-1 macrophages. In this study, we selected genes that are in some way related to intracellular survival inside human macrophages or have significantly effect for intracellular survival. The effect of the mutation in these genes was tested both on the level of cytotoxicity to THP-1 cells and the related number of surviving bacteria inside the macrophages. The deletion strain in two genes for cysteine dioxygenase (BP2871 and BP3011) and the mutant strain allocated in the Bvg+ phase were less cytotoxic than the control strain. Monitoring the effect of opsonization to intracellular survival have not such clear results. The effect...
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Analysis of flagellar proteins in Clostridium difficile isolates of clinically relevant PCR-ribotypes
Houdková, Kateřina ; Konečná, Klára (advisor) ; Tichý, Aleš (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Kateřina Houdková MPharm Supervisor: RNDr. Klára Konečná, Ph.D. Consultant: PharmDr. Jiří Dresler, Ph.D. Title of rigorous thesis: Analysis of flagellar proteins in C. difficile isolates of clinically relevant PCR-ribotypes Background: Strains of C. difficile of known human epidemiologic importance are associated with severe clinical features of C. difficile infection (CDI). In this study, a panel of eight different PCR-ribotypes (RTs) with their proteins released in vitro were subjected to analysis. The aim of this work is to monitor the relationship between secretions of individual proteins associated with flagellar formation and function in C. difficile strains of variable virulence. Methods: Within our research, a combination of tandem mass spectrometry with liquid chromatography was used. The semi-quantitative analysis employed label free quantification (LFQ) approach. Results: From the quantifiable proteins, 17 were significantly increased in functional annotations. Among them, several known factors connected with flagellar assembly and other functions were identified. Higher expression of selected flagellar proteins clearly distinguished RTs 027, 176, 005 and 012, confirming the pathogenic...
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Secondary metabolites in fungal pathogenesis
Veselý, Martin ; Čmoková, Adéla (advisor) ; Machová, Lenka (referee)
The ability of fungal pathogens to induce infection and later survive in its host is dependent on virulence factors. Often these factors are based on primary metabolites (hydrophobins, proteases, phospholipases, catalases etc.). Nonetheless many pathogen produced secondary metabolites are also involved in the infection process. Their true role during infection used to be rather undervalued. First part of this bachelor degree thesis aims to describe host-pathogen relation and afterwards introduce reader with some basic, commonly accepted virulence factors of fungi. In the second part of this thesis known roles of secondary metabolites are described in each stages of progressing infection. In the first stage a pathogen needs to create a suitable habitat on host tissues to ensure growth. This is in part done by antimicrobial substances (...). In later stages pathogen prevents phagocytosis of its spores by host (melanins). Successful infection is often accompanied with host tissue damage that is induced by production of cytotoxic substances (xanthomegnin, riboflavin) and immunoregulation of host immune system (gliotoxin, pseurotin). Last but not least there are virulence factors that ensure prolonged survival in host (siderophores). Fungal pathogens of poikiloterm animals are mostly not primary...
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Structure and function of RTX toxins of Gram-negative bacteria
Zhuk, Karyna ; Osička, Radim (advisor) ; Šulc, Miroslav (referee)
RTX toxins (Repeats in ToXin) are produced by Gram-negative bacteria, most of which are important human or animal pathogens. The polypeptide chain of each RTX toxin consists of four conserved regions. An N-terminal hydrophobic domain, which is important for insertion of the RTX toxin into the host cell membrane and pore formation. The hydrophobic domain is followed by an acylated segment containing conserved lysine residues, at which the toxin is acylated and thus activated. The C-terminal portion of each RTX toxin contains a repeat domain to which calcium ions bind. The C-terminus of the toxin contains a secretion signal that is recognized by the type I secretion system, which transports the toxin from the bacterial cytosol to the external environment. After secretion, RTX toxins interact with the cell surface via specific β2 integrins and/or glycosylated structures such as glycoproteins and gangliosides or membrane components such as sphingomyelins and cholesterol. Once bound to the cell, RTX toxin monomers insert into the membrane and oligomerize to form pores. The uncontrolled flow of ions through these pores can lead to disruption of bactericidal functions of myeloid phagocytes, stimulation or suppression of the release of pro-inflammatory cytokines, modulation of various signaling and...
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Virulence factors of Bordetella pertussis
Držmíšek, Jakub ; Večerek, Branislav (advisor) ; Vopálenská, Irena (referee)
Bordetella pertusis is a Gram-negative, aerobic, non-spore-forming coccobacillus. Although it's strictly human pathogen, it's possible to infect other mammals at laboratory conditions. Transmission among hosts is mediated via respiratory tract droplets. Infection could be direct, host to host, alternatively by contaminated environment. Bordetella colonizes upper respiratory tract, wherefrom descends into lungs and causes disease known as whooping cough or pertussis leading to 195 000 deaths of 16 mil. incidences per year (according to WHO report from 2010). More than twenty years before, respectively to found pertussis toxin, that time intensively under examination, pertussis was marked as toxin-mediated disease. In the course of time, more other virulence factors were revealed, that could be divided into groups of adhesins, toxins and others. Adhesins are filamentous haemagglutinin, pertactin and fimbriae. Toxins include pertussis toxin, adenylate cyclase, tracheal cytotoxin, dermonecrotic toxin and lipopolysaccharide. Most of virulence factors are regulated by two component system Bvg. However, it is needed lots of other factors for successful infection as for example autotransporters or so called siderophores serving as iron acquisition from environment. Secretion of virulence factors is mediated by its...
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Structural mass spectrometry of Bordetella virulence factors
Jurnečka, David
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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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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Structural mass spectrometry of Bordetella virulence factors
Jurnečka, David
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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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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