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Lyme borreliosis diagnostics using in vitro cellular immune response testing
Prokopová, Tereza ; Drbal, Karel (advisor) ; Melter, Oto (referee)
Lyme borreliosis is a multisystemic disease affecting skin, joints, heart and central nervous system. The disease is caused by spirochetes of Borrelia burgdorferi sensu lato complex. These bacteria are spread by ticks of Ixodes genus. In 2016 there were almost 4,000 newly infected individuals reported in the Czech Republic. Contemporary serological diagnostics of Lyme borreliosis is not sensitive nor specific enough and does not even correlate with the pathology of the disease in the early or late phases. For the correct diagnosis of the disease it is necessary to detect the pathogen and its genotype. For this reason we had aimed at two goals. Through the digital droplet PCR (ddPCR) method we detected Borrelia-specific DNA and its genotype. The detection limit of borrelial DNA was set on gDNA samples isolated from the tick. Detection threshold for the initial amount of 1 ng of tick gDNA is at the range of 10-17 g of specific borrelial DNA. Borrelia spp. coinfection was detected in 5 out of 12 tested samples. The most frequent type was B. garinii which was detected in 5 samples. On the basis of published sequences for virulent factors we have designed specific primers in conserved regions of the genes flanking their variable segments to be PCR amplified. Gene variability will be monitored through...
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The structure and role of type III secretion system and other virulence factors in pathogenesis of pertussis
Štipl, Daniel ; Večerek, Branislav (advisor) ; Pinkas, Dominik (referee)
Bordetella pertussis is a significant human pathogen which colonises a respiratory tract. The infection with B. pertussis results in serious and highly contagious disease called pertussis or whooping cough. B. pertussis produces wide range of virulence factors such as pertussis toxin, adenylate cyclase toxin, dermonecrotic toxin, tracheal cytotoxin, adhesins and type III secretion system (T3SS). The BvgAS is two-component signal transduction system that provides the complex regulation of B. pertussis virulence. The virulence factor T3SS is used by some Gram-negative bacteria to colonise the host and is responsible for pathogenesis of the infection. T3SS takes a role in virulence of mammalian pathogen B. bronchiseptica, closely related to B. pertussis. The importance of T3SS in virulence of B. pertussis remains to be investigated. Significant advance in structure, function and regulation of the most of virulence factors have been accomplished in last few decades. The causative agents of pathogenesis in that infection remain unknown. Key words: Bordetella, T3SS, gene expresssion regulation, virulence factor, pathogenesis
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Production and secretion of virulence factors in Bordetella pertussis
Držmíšek, Jakub ; Večerek, Branislav (advisor) ; Petráčková, Denisa (referee)
Bordetella pertussis is a strictly human pathogen and causative agent of infectious respiratory disease called whooping cough. In order to establish successful infection and colonization of the host, B. pertussis uses a broad spectrum of virulence factors such as adhesins (filamentous hemagglutinin, pertactin, and fimbriae) and toxins (adenylate cyclase and pertussis toxins). In addition, the type 3 secretion system (T3SS) was also found in the genus Bordetella. In connection to our previous characterisation of B. pertussis strain lacking the gene encoding RNA chaperone Hfq (Δhfq), which proved that Hfq is required for T3SS functionality, the recombinant T3SS proteins BopB, BopD, BopC and BopN were purified to homogeneity. Next, the specific antibodies were obtained using purified recombinant proteins in order to study the production of the T3SS components in B. pertussis. Using refined anti- BopC antibodies it was for the first time shown that laboratory-adapted B. pertussis strain secretes BopC protein into medium. The recombinant translocators BopB and BopD were also used to examine their pore-forming activity using planar black lipid membranes. Based on the characterisation of hfq deletion mutant, having impaired production of membrane proteins when compared to the wild type, mass spectrometry...
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