|
|
Bacteria genus Asaia and Wolbachia in sandflies
Sovová, Kristina ; Volf, Petr (advisor) ; Bilej, Martin (referee)
Phlebotomine sand flies are proven vectors of many pathogens including parasites of genus Leishmania. Leishmania develop in sand fly midgut which is colonized also by many others microorganisms, creating rich community known as gut microbiota. The presence and composition of gut microbiota affect sand fly mortality, but also development of transmitted pathogens. In contrast to mosquitoes, sand fly gut microbiota is not well studied. This thesis focuses on bacteria of the genera Asaia and Wolbachia and their potential impact on Leishmania in sand fly midgut. Thesis reports the first finding of Asaia sp. and Wolbachia sp. in sand flies from Balkan penninsula - hotspot for visceral leishmaniasis and phleboviruses. In 273 individuals from subgenera Larroussius were Asaia sp. and Wolbachia sp. detected with infection prevalence 2,5 % and 8,4 %, respectively. In addition, laboratory-reared sand flies were tested for presence of these bacteria: from twelve studied colonies, only Phlebotomus perniciosus was infected by Wolbachia sp. Then, we focussed on elimination of Wolbachia sp. from this laboratory colony with the aim to use Wolbachia-negative sand flies in future experiments with Leishmania. The final part of the thesis was dedicated to bacteria of the genus Asaia (specifically A. krungthepensis),...
|
|
|
Critical factors affecting pathogen development in sand flies
Jančářová, Magdaléna ; Volf, Petr (advisor) ; Ratinier, Maxime (referee) ; Bilej, Martin (referee)
This thesis deals with barriers and factors critical for development of viruses, leishmania and gregarines in sand flies. First, we focused on life cycle of sand fly-borne phleboviruses, especially possible routes of sand fly infection. As a laboratory model we chose Massilia virus (MASV), species closely related to Toscana virus, which is main causative agent of summer meningitis in Mediterranean area. We tested different ways of infection by MASV in various developmental stages of Phlebotomus perniciosus; infection of (i) first (L1) and fourth (L4) instar larvae through larval food, (ii) females by blood meal, (iii) both sexes by sugar meal. Infection of L1 and L4 by larval food and subsequent transstadial MASV transmission to adults were not efficient; from 875 adults only three were MASV-positive. Infection through bloodmeal led to high infection rate before defecation, nevertheless, post defecation the infection rate declined and only 5 out of 27 females were MASV-positive. The most efficient infection way was through the sugar meal: 72% of females (88 out of 122) and 51% of males (58 out of 113) were detected as MASV-positive. Moreover, both males and females infected by this way released MASV particles into the drop of sugar which stayed infectious for next 24 hours for other naïve sand...
|
|
| |
|
| |
|
| |
|
|
Recombinant proteins and synthetic peptides as tools for studies on antibody response against Phlebotomus orientalis
Ferencová, Blanka ; Volf, Petr (advisor) ; Bilej, Martin (referee)
Sand fly saliva contains proteins and peptides that have an important role in bloodfeeding. Some of those proteins are antigenic and repeated sand fly bites result in a specific antibody response of the bitten host. Antigenic salivary proteins of Phlebotomus orientalis, main vector of visceral leishmaniasis in Sudan and Ethiopia, were identified using immunoblot with dog sera. The 5 most promising antigens were expressed in an E. coli bacterial system. Subsequently, these proteins were tested in ELISA with sera of domestic animals from Ethiopia naturally exposed to P. orientalis, and with sera of mice bitten experimentally by this sand fly species. Salivary gland homogenate (SGH) was used as the positive control. The best antigenic properties were detected in two recombinant proteins, Yellow-related protein PorSP24 and ParSP25-like protein PorSP65, especially in tests with sheep and dog sera. However, nonspecific binding of dog sera was also detected using both antigens. In addition, we proved that sera of mice repeatedly bitten by P. papatasi and Sergentomyia schwetzi do not crossreact with SGH and the tested recombinant proteins of P. orientalis. In a second part of this thesis we designed peptides representing epitopes recognized by specific anti-saliva antibodies. Two peptides were derived from...
|
|
| |
|
|
Effects of microbiota on defense system of earthworms
Dvořák, Jiří ; Bilej, Martin (advisor) ; Kopečný, Jan (referee) ; Horák, Petr (referee)
Earthworms are important soil invertebrate organisms that participate in nutrient cycling in terrestrial ecosystems and in the formation of the soil profile from the physical, chemical and also microbial point of view. Soils are considered the most microbially diverse environments on earth. All invertebrates living in soil therefore need to possess a complex immune system. Earthworms are used as a model organism in immunology for decades. Their simple body plan consists of two main body cavities: true coelom and digestive tube. Both coelomic cavity and digestive tract represent open systems with permanent contact with soil microorganisms. Eisenia andrei species is used as a standard immunological model in our laboratory for many years. E andrei earthworms live in compost, microbially abundant environment, which is reflected in their well-developed immune system. Some new mechanisms of E. andrei defense system are described in this work. Two novel pattern recognition receptors (PRRs), Toll-like receptor (TLR) and lipopolysaccharide binding protein/bactericidal permeability-increasing protein (LBP/BPI) were characterized in earthworms. These molecules are expressed in coelomocytes and their production is upregulated after microbial challenge. Moreover, both receptors were detected in digestive tract...
|
|
|
Molecular mechanisms of regulation of FcɛRI signaling in mast cells
Bambousková, Monika ; Dráber, Petr (advisor) ; Černý, Jan (referee) ; Bilej, Martin (referee)
Mast cells are critical component of the immune system. In pathological situations, they are activated and are responsible for allergic reaction. Therefore, detail understanding of mast cell activation at molecular level is important for design of new therapies of allergic diseases. Principal transmembrane receptor of mast cells is the high-affinity Fc receptor for IgE (FcεRI). FcεRI anchors IgE on mast cell surface and upon cross-linking with multivalent antigen it becomes phosphorylated at its intracellular immunoreceptor tyrosine-based activation motifs (ITAMs). This triggers signaling cascade leading to cell degranulation and cytokine production. The antigen- mediated signaling through the FcεRI is critically dependent on interplay with intracellular protein- tyrosine kinases that phosphorylate the ITAM motifs and many other components of the signaling pathway. This study was focused on better understanding of signaling events leading to mast cell activation; emphasis was put on early activation events. First, we examined the role of protein- tyrosine phosphatases (PTP) in FcεRI phosphorylation. We found that upon antigen triggering of FcεRI, PTPs undergo inhibition by oxidation of their active site located tyrosine. Studies of plasma membrane topography of inactivated PTPs showed their...
|
|
|
Recognition of microbial patterns in earthworms
Škanta, František ; Bilej, Martin (advisor) ; Kopečný, Jan (referee) ; Šimek, Miloslav (referee)
Survival of earthworms in the environment depends on their ability to recognize and eliminate potential pathogens. Two closely related earthworm species Eisenia andrei and Eisenia fetida inhabit different environment with specific microbiota. Both species can be reliably determined using of species-specific primers for cytochrome c oxidase I (COI) and stringent PCR conditions. Whereas, we did not observed any substantial differences in the expression and activity of CCF and lysozyme upon microbial challenge, the expression as well as the hemolytic activity of fetidin/lysenins was considerably higher in E. andrei as compared to E. fetida. Genomic DNA analyses revealed significantly higher level of fetidin/lysenins in E. andrei compared to E. fetida suggesting hypothetical gene duplication. Earthworms live in permanent close contact with microbial environment. Coelom cavity as well as the gut of E. andrei earthvworm differs in the number of bacteria. The number of bacteria in the gut is more than six time higher than in coelomic fluid. High microbial load of E. coli O55, B. subtilis W23, and S. cerevisiae S288 in the earthworm environment, resulted in an increase of microorganisms in both, the coelom and the gut. The changes in mRNA levels of defense molecules (pattern recognition receptors CCF,...
|
guest ::
