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Factors contributing to stress adaptation of human pathogen Bordetella pertussis
Hejnarová, Václava ; Večerek, Branislav (advisor) ; Černý, Ondřej (referee)
The role of bivalent metals as cofactors in various enzymes is essential for all domains of life. Their importance can be seen in interactions of hosts with patogens such as Bor- detella pertussis. This bacterium has several mechanisms that protect it from oxidative stress in phagosomes of immune cells. One example is Mn-dependent enzyme superoxide dismutase. Phagosome itself is an environment with limiting concentrations of several key bivalent metals. Thus, the transport of these metals must be controlled to ensure sufficent concentration for enzymatic activity and prevent from toxic cumulation and mismetallation. This study focuses on a particular locus BP3083-BP3077 that is highly expressed in B. pertussis surviving after internalization by THP-1 macrophages. The locus consists of two transporters, one of which shares homology with SitABCD Mn2` impor- ter of Salmonella enterica and is regulated by Fur family repressor. Bacterial cells were exposed to toxic concentrations of several bivalent metals and the level of gene expres- sion was studied by RT-qPCR. Deletion mutants were used for examination of bacterial phenotype. Results of the study show that the locus encodes Mn2` and Co2` transporters and becomes negatively regulated by Fur family repressor in toxic concentration of Co2` . The...
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Possibilities of use of selected entomopathogenic fungi in biological control of agricultural pest species
KONOPICKÁ, Jana
Ph.D. thesis is focused on the entomopathogenic fungi that can be used in biological control. Ph.D. thesis consists of two main parts: 1) a detailed background research, and 2) five subchapters of the experimental part and results, which contain original publications describing results of my own studies. The first study focused on the isolation and identification of new strains of entomopathogenic fungi from soil samples collected in garlic and onion fields in the Czech Republic and Israel. Furthermore, the efficacy of selected fungal strains against the bulb mite Rhizoglyphus robini was tested. A total of 5 genera of entomopathogenic fungi (Metarhizium sp., Beauveria sp., Isaria sp., Lecanicilium sp. and Purpureocillium sp.) were identified among the soil isolates from both countries. The most frequent was the genus Metarhizium sp. especially in sampling sites of the Czech Republic. The highest efficacy against Rhizoglyphus robini mites was found in Metarhizium anisopliae strains isolated from soil samples collected in the Czech Republic and in Metarhizium indigoticum strain from Israel. Mortality after 4 days of the bioassay was almost 100%. The second study investigated the efficacy of the entomopathogenic fungus Isaria fumosorosea CCM 8367 against box tree moth (Cydalima perspectalis). Fungal infection was mostly observed in pupae. However, mortality did not exceed 60%, indicating a very low susceptibility of box tree moth to Isaria fumosorosea CCM 8367. In the third subchapter, various strains of entomopathogenic fungus Beauveria bassiana were tested against the Colorado potato beetle, a serious pest of potato. First, laboratory experiments were performed and the most virulent strain BBA 08 against this pest was selected and used subsequently in further experiments. The strain efficacy against L. decemlineata adults was evaluated in pot experiments and under field conditions. The fungus was applied alone and in combination with entomopathogenic nematodes. In all experiments, the fungus reduced the number of Colorado potato beetle adults by about 30% compared to the control. The fourth study described enrichment of soil substrate with entomopathogenic fungus Isaria fumosorosea CCM 8367 for increasing its suppressivity. The results showed that the fungus successfully colonized the soil substrate and remained in it for more than 6 months at 20 °C, although the average concentration decreased slightly from 5.89×104 to 2.76×104 spores per milliliter of substrate during the experiment. The last study described dissemination of Isaria fumosorosea CCM 8367 spores by nematodes of Steinernema feltiae and Heterorhabditis bacteriophora. The results of our study revealed for the first time that the spread of conidia and blastospores of Isaria fumosorosea in soil environment is significantly enhanced by the presence of entomopathogenic nematodes.
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Interaction of the human pathogen Bordetella pertussis with blood serum
Štipl, Daniel ; Večerek, Branislav (advisor) ; Kamanová, Jana (referee)
Bordetella pertussis is a Gram-negative strictly human pathogen and the major causative agent of whooping cough or pertussis. The incidence of this highly contagious respiratory disease in developed countries has increased in the last decades. One of the less characterized virulence factors of B. pertussis is the type three secretion system (TTSS) which is responsible for the secretion of the effector proteins into host eukaryotic cells. This diploma thesis sheds light onto factors influencing TTSS in vitro activity. Although TTSS of laboratory strain Tohama I was induced by biologically active compounds present in blood (e. g. complement proteins), TTSS of recent clinical isolate B1917 seems to be induced permanently. Furthermore, BB0302 encoding a GntR family transcription regulator in B. bronchiseptica RB50 (homologous to BP0209 of Tohama I) was studied, however, the deletion of this gene did not affect the TTSS functionality. Serum resistance is a factor that plays a key role in the pathogenesis of B pertussis. We show that Czech recent isolates (2008-2015) are significantly more resistant to serum killing in vitro than the original vaccine strains (1954-1965). This phenomenon seems to result from the adaptation of global B. pertussis population to its human host. In addition, this diploma...
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Non-conventional bacterial signaling pathways
Krupička, Jiří ; Branny, Pavel (advisor) ; Beranová, Jana (referee)
Two component systems were traditionally considered as main phosphorylation systems of bacteria involved in cell signalling. Recently, attention focuses increasingly on bacterial eukaryote-like Ser/Thr protein kinases (eSTKs). These protein kinases are structurally similar to their eukaryotic counterparts. Some eSTKs possess additional domains such as extracellular PASTA domains that were discovered in a variety of gram-positive bacteria. It has been proved that these domains can act as sensors for unlinked peptidoglycan fragments. However, majority of environmental signal molecules still remains unknown. eSTKs phosphorylate a broad spectrum of substrates including proteins involved in various cell processes such as virulence, cell wall biosynthesis, cell division, and central and secondary metabolism. Cross talk between eSTKs and two component systems also occurs. In this thesis, the current knowledge about eSTKs and their significant substrates in different bacterial species is discussed.
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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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Interaction of the human pathogen Bordetella pertussis with blood serum
Štipl, Daniel ; Večerek, Branislav (advisor) ; Kamanová, Jana (referee)
Bordetella pertussis is a Gram-negative strictly human pathogen and the major causative agent of whooping cough or pertussis. The incidence of this highly contagious respiratory disease in developed countries has increased in the last decades. One of the less characterized virulence factors of B. pertussis is the type three secretion system (TTSS) which is responsible for the secretion of the effector proteins into host eukaryotic cells. This diploma thesis sheds light onto factors influencing TTSS in vitro activity. Although TTSS of laboratory strain Tohama I was induced by biologically active compounds present in blood (e. g. complement proteins), TTSS of recent clinical isolate B1917 seems to be induced permanently. Furthermore, BB0302 encoding a GntR family transcription regulator in B. bronchiseptica RB50 (homologous to BP0209 of Tohama I) was studied, however, the deletion of this gene did not affect the TTSS functionality. Serum resistance is a factor that plays a key role in the pathogenesis of B pertussis. We show that Czech recent isolates (2008-2015) are significantly more resistant to serum killing in vitro than the original vaccine strains (1954-1965). This phenomenon seems to result from the adaptation of global B. pertussis population to its human host. In addition, this diploma...
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Role of exosomes and ectosomes in Trichomonas vaginalis virulence
Göblová, Rebeka ; Tachezy, Jan (advisor) ; Ditrich, Oleg (referee)
Trichomonas vaginalis is a causative agent of the most common non-viral sexually transmitted disease with approximately 275 mil new cases annually. Virulence of this parasitic depends on at least four factors: cell shape transformation, cytoadherence, secretion of cysteine proteases, and presence of endosymbionts. Over the past decades, extracellular vesicles appeared being another important player in the host-parasite interaction. It was discovered that T. vaginalis is one of the protists that can shed the extracellular vesicles such as exosomes and ectosomes. These vesicles are possibly involved in host-parasite communications, however limited information is available about their function. To investigate a possible role of exosomes in T. vaginalis virulence, we first selected suitable strain, which is free of endosymbionts (TV 17-2MI). Next we prepared six clones of TV 17-2MI strain to test whether the strain is homogenous concerning the virulence, or there are differences in virulence among individual cells. Mouse intraperitoneal virulence tests revealed that the clones displayed significant differences in virulence level, particularly in abscess formation and mortality of infected animals. Thus, for the first time we demonstrated heterogeneity of cells derived from a single T. vaginalis strain...
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Genomic analysis of Paenibacillus larvae and its relation to virulence
Vlková, Kateřina ; Hrabák, Jaroslav (advisor) ; Fišer, Radovan (referee)
Paenibacillus larvae is a Gram-positive sporulating bacterium that causes American foulbrood (AFB). It is one of the most dangerous bacterial pathogens of the honeybee (Apis mellifera). P. larvae spores are highly infectious to bee larvae and resist physicochemical influences. P. larvae is subtyped using repPCR with ERIC primers (Enterobacterial Repetitive Integrance Consensus) into five genotypes (ERIC I-V), which possess different colony morphology, metabolism and especially virulence. There is a significant genetic variability among isolates of P. larvae, which may contribute to differences in virulence. P. larvae isolates used in this work were obtained from clinical cases of American foulbrood as well as from a debris collected from bee hives with no American foulbrood symptoms from all over the Czech Republic in cooperation with the Beekeeping Research Institute, s.r.o., Dol. The isolates were obtained from larvae and hive debris. Both virulet and avirulet strains were sequenced using the SMRT (single molecule real time) method on the Sequel platform (PacBio). This method is suitable for Whole Genome Sequencing (WGS), because it allows sequencing of long reads with high accuracy, eliminating the effect of a large number of repetitive sequences during the genome assembly. Furthermore,...
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The function of ClpX chaperone in bacteria
Kýr, Jan ; Balíková Novotná, Gabriela (advisor) ; Šiková, Michaela (referee)
Intracellular proteolysis is an essential regulatory process that affects cellular physiology. Since proteolysis destroys proteins irreversibly, this process must be strictly controlled. The AAA+ proteins are the key factors in regulated proteolysis in bacteria. These proteins consist of two functional domains, the AAA+ chaperone domain and the protease domain. One particular group of these AAA+ protein is the Clp protein family. Functional domains of the Clp family are formed by seperate proteins. The hexameric unfoldase ClpX is a member of this protein family. This unfoldase can interact with the highly conserved ClpP protease to form a ClpXP proteolytic complex. This proteolytic complex utilizes the energy of ATP binding and hydrolysis to unfold and translocate the specifically tagged substrate into the ClpP degradation chamber. Substrate recognition is mediated by the binding of ClpX to short unstructured sequences called degradation tags. ClpX recognizes several degradation tags, but the most important one is recognition of the ssrA degradation tag, which is the output of the tmRNA ribosome rescue system. Although ClpX interacts with ClpP, it affects a variety of cellular processes such as the expression of virulence factors or the adaptation to stress factors, ClpX can work independently of...
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Molecular mechanisms of virulence regulation in Staphylococcus aureus
Kekrt, Lukáš ; Tkadlec, Jan (advisor) ; Botka, Tibor (referee)
Staphylococcus aureus is one of the most common causes of bacterial infection in human. The bacterium is equipped by the broad spectrum of virulence factors, including modulators of immune response and also invasion factors able to destroy human cells and tissues. S. aureus is also able to asymptomatically colonize healthy individuals. Precise regulation of virulence factor expression is crucial for S. aureus survival. The aim of this thesis is to describe in detail molecular mechanisms regulating S. aureus virulence factor expression. Among these mechanisms belongs quorum sensing dependent agr system, sigma B general stress response, Sar family of transcription factors and metabolism- dependent regulators CodY and CcpA. Keywords: S. aureus, virulence, quorum sensing, agr systém, sar, sigma B, CodY, alpha-hemolysin.
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