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Optimization of methods for evaluation of antimicrobial activity of selected active matters against chosen spectrum of bacterial pathogens
BRZÁKOVÁ, Michaela
The bachelor thesis deals with optimalization of selected methods (e.g. evaluation of bacterial suspension concentration, Resazurin antibacterial assay) needed for evaluation of active substances antibacterial activity against pathogens Pectobacterium carotovorum, Pseudomonas syringae and Clavibacter michiganensis subsp. michiganensis. Literature review contains information about potato tubers proteins, especially proteins with antibacterial activities, as well as a brief overview of mechanisms of action of plant defense system against pathogenic microorganisms and the effect of antimicrobial substances on bacterial pathogens. The experimental part deals with optimalization of methods needed for evaluation of antibacterial activities of selected substances. Optimized were methods of McFarland Turbidity Standards, Most-probable number method and Resazurin antibacterial assay. It was found that increase of optical density, measured by spectrometer at 620 nm, is in direct correlation with increase of bacterial suspension concentration (CFU/ml). It was identified proteins of patatin complex and protease inhibitors by using SDS-PAGE method to evaluated potato tuber proteins of variety Bella and Westamyl and also changes of molar mass of the patatin proteins in relation of their modification were evaluated. Antimicrobial activity of these proteins was verified by resazurin antibacterial assay and ability of modified proteins of cultivars Bella and Westamyl in higher concentration (10 mg/ml) to inhibit growth of bacteria Pseudomonas syringae.
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Larvae of flesh fly Neobellieria bullata as a source for novel antimicrobial peptides
Neubauerová, Tereza ; Macková, Martina ; Macek, Tomáš ; Šanda, Miloslav ; Voburka, Zdeněk
The antibiotic resistant strains of microorganisms are increasing threat for public health. Antimicrobial peptides from natural sources display a wide spectrum of activity. In our laboratory, several peptide fractions with antimicrobial acivity against gram-positive and gramnegative bacteria and fungi were isolated from larvae of flesh fly Neobellieria bullata. The presence of peptides was proved by tricine electrophoresis, molecular weights and sequences determination by MS and Edman sequencing, respectively. In comparison with database sequences we obtained structures present mainly in insect and amphibian antimicrobial peptides, but total matches were not higher than in 36%. Upon the obtained sequence motifs we designed structures of several synthetic peptides. These were synthetised on the Rink amide MBHA resin. Synthesis was performed according Fmoc/PyBOP-DIEA protocol. Antimicrobial activity of these peptides was determined.
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Identification and characterization of histidine-rich peptides from hard ticks \kur{Ixodes ricinus} and \kur{Ixodes scapularis}.
DORŇÁKOVÁ, Veronika
Antimicrobial (cationic) proteins play an important role in innate imunity. Such proteins can possess antibacterial, antiendotoxic or fungicidal abilities. The rising resistence of microbes to common antibiotics evokes acute need of studying more endogenous proteins to reveal new potential antibiotics. Ticks, the blood-feeding ectoparasites with effectual defense system, present an endless source of newly described and unknown antimicrobial peptides/proteins with significant theurapeutic potential. This study represents identification of histidine-rich proteins detected in Ixodes ricinus and Ixodes scapularis, that are related to recently described new family of proteins isolated from Rhipicephalus microplus (protein microplusin) and Amblyomma hebraeum (protein heraein). Analysis and characterization of newly identified histidine rich proteins, study of their antimicrobial and protease inhibitory effect are the main goals of this study.
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Ricinusin {--} a new antimicrobial peptide isolated from the hard tick \kur{Ixodes ricinus}: the third member of the tick histidine-rich defense protein family.
DORŇÁKOVÁ, Veronika
New gene encoding antimicrobial protein (ricinusin) was isolated from I. ricinus. The 405 nt cDNA fragment contained a 135 aa ORF, encoding a 14.5 kDa protein with 19 aa signal peptide. Gen Bank comparison of ricinusin showed weak similarity to a new type of histidine rich antimicrobial protein with unique HEAHEAHEA repeat from Boophilus microplus and Amblyomma hebraeum. The differential gene expression was strongly induced by blood feeding in larva, nymph and adults. The site of differential expression in salivary glands and gut indicates that ricinusin is induced as a part of broad-spectrum immune response. To study the amtimicrobial capacity of ricinusin and its capatibility in clearing Borrelia burgdorferi infections, it was expressed as a His-tagged fusion protein in bacterial expression system. Analysis of the genomic organization of ricinusin gene showed that it is intronless.
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