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Localization of Lyme disease spirochetes \kur{Borrelia burgdorferi} in ticks \kur{Ixodes ricinus}
STRNAD, Martin
Lyme disease is the most common vector-borne infection in the Western world with an annual incidence usually in excess of 100 cases per 100 000 people in temperate areas of the United States and Europe. Same as other infectious diseases, Lyme borreliosis wreaks havoc on the host they have invaded. B. burgdorferi, the causative agent of this disease, circulates among wildlife vertebrate hosts and Ixodes tick vectors but may sometimes infect humans. Its natural enzootic cycle usually occurs as follows: The larval/nymphal stage tick feeds on an infected host. During this engorgement, the spirochetes reach the tick gut and stay confined to it. After the tick molts into the next developmental stage, it finds a second host. The new bloodmeal triggers the spirochetes to multiply within the gut and traverse the gut endothelium in a highly organized manner. They finally disseminate through the hemocoel up to the tick salivary glands and into the new host. We studied whether B. burgdorferi is capable of reaching the tick salivary glands during the first infective feeding period in uninfected ticks.
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Molecular characterization of NADPH oxidase in the gut of the tick \kur{Ixodes ricinus}
KUČERA, Matěj
This thesis focuses on characterization of one member of NADPH oxidases - dual oxidase (DUOX) which has been described as the main factor of epithelial immunity in the gut of model organism Drosophila melanogaster. We have identified an orthologous gene coding for DUOX in the tick I. ricinus and described its tissue expression profile. The DUOX is mainly expressed in the gut of unfed ticks and seems to be downregulated upon artificial microbial infection. A fragment of tick DUOX was prepared as recombinant protein and used for preparation of specific antibodies to be used for further characterization of the enzyme. Our main aim is to highlight the importance of tick DUOX producing the reactive oxygen species and their role in the defence against pathogenic organisms within the tick gut.
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Preparation of recombinant inhibitor of serine proteases from the tick \kur{Ixodes ricinus}
VLNOVÁ, Ivana
Tick serine protease inhibitors could be important anti-tick vaccines targets because of their properties and functions. The aim of this work was to prepare recombinant inhibitor of serine proteases from the tick Ixodes ricinus in baculovirus expression system. Two tick saliva proteins of the serpine superfamily were selected for this purpose and transformed into plasmids. One recombinant protein was expressed in baculovirus expression system, purified and its biochemical analyses were done.
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Identification and characterization of newly found antimicrobial peptide (IRAMP) from hard tick \kur{Ixodes ricinus}
OUŘEDNÍKOVÁ, Lucie
Antimicrobial proteins (AMPs) are effector molecules and an important part of the innate immune system. AMPs have a broad antimicrobial spectrum and lyse microbial cells by interaction with biomembranes. Besides direct impact in host defence, AMPs are mediators of inflammation with impact on epithelial and inflammatory cells influencing diverse processes such as cytokine release, cell proliferation, angiogenesis, wound healing, chemotaxis, immune induction, and protease-antiprotease balance. AMPs could replace antibiotics which efficiency has decreased due to extensive clinical use. Therefore knowledge of mechanism of action of antimicrobial peptides, their properties and possible usage is essential for their further use as therapeutics. Ticks are blood-feeding ectoparisites that serve as extremely effective vectors of pathogens. Analysis of the ticks molecules that are involved in immune response to the pathogens invasion represent one of the strategies in searching for new compounds that might be used in future as theurapeutic agents. This study represents analysis of newly identified antimicrobial peptide form in the hard tick Ixodes ricinus (IRAMP). IRAMP revealed the high similarity to the recently described antimicrobial peptide isolated from hard tick Ixodes scapularis (protein ISAMP). Analysis and characterization of novel AMP, testing its antimicrobial potential and expression pattern are the main objectives of this study
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Dynamics of digestive enzymes in the gut of ticks \kur{Ixodes ricinus} during blood feeding on the host.
KONVIČKOVÁ, Jitka
Proteolysis of ingested blood proteins is a crucial physiological process in ticks. This thesis focuses on profiling of dynamics of digestive enzymes in the ticks Ixodes ricinus during the course of blood-feeding on the host and during digestive phase after detachment from the host. We have measured the activity of digestive peptidases (using specific fluorescent substrates and inhibitors). The enzyme activities were negligible at the early stage of bloodfeeding than increased dramatically towards the end of the feeding period and dropped down immediately after detachment from the host. The activity of most enzymes increased again during the digestive phase before oviposition of females or metamorphosis of nymphs. The protein level of each peptidase was confirmed by immunolocalization using Western blot analyses.
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Isolation of the antimicrobial peptide gene (defensin) from the hard tick \kur{Ixodes ricinus}, challenged by the pathogen infection
SKLADANÁ, Veronika
Antimicrobial peptides are major components of the innate immune response of epithelial cells. In hematophagous organisms, which acts as vectors of parasitic diseases, in particular Lyme borreliosis, the gut pathogen induce the expression of defensin, that provide the first barier of host defence. This raises the posibility that defensin may play a key role in the development of parasitic infection. The gene expressed in midgut was isolated from cDNA of the hard tick, Ixodes ricinus. The gene is coding the protein, that is produced and secreted during tick infection, and is known as defensin. Expression of the gene for defensin (224 bp) was induced by the pathogen. The gene was cloned into bacterial expression system.
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IrAM4: Partial characterisation of a molecule similar to \recke{alpha}\dindex{2}-macroglobulin from a tick \kur{Ixodes ricinus}
ABSOLONOVÁ, Markéta
Ixodes ricinus is a hard tick that can transmit several diseases that are capable of affecting humans. Among those are tick-borne encephalitis and Lyme disease. The focus of this work is on IrAM4, a member of tick ?2-macroglobulin family (?2M-F) of proteins which belong to the evolutionarily oldest constituents of the innate immune system. ?2-macroglobulins are protease inhibitors and act primarily in inactivation of proteases secreted by invading pathogens within their infection cycle. The aim of this study was to identify ?2-M of Ixodes ricinus (IrAM4) from corresponding ortholog of protein ?2-M named IsAM4 present in the genome of closely related Ixodes scapularis. The partial sequence was determined by amplification of cDNA and subsequent sequencing of PCR products. RT-PCR tissue profiling revealed that IrAM4 is present in ovaries and salivary glands but not in the tick gut. The recombinant fragment of IrAM4 was afterwards prepared for immunization of a rabbit and the obtained polyclonal antibodies were used for Western blot analysis. The results showed that IrAM4 is mainly present in the hemolymph and probably in salivary glands and ovaries but it is not expressed in the gut. The native IrAM4 seems to be composed of two disulfide bound subunits. However, the exact structure of the molecule was not analyzed in this work.
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