Dokument: Click beetle luciferases as reporters of gene expression in Candida albicans
| Titel: | Click beetle luciferases as reporters of gene expression in Candida albicans | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=33248 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20150128-130037-9 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Lagadec, Quentin [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Ernst, Joachim F. [Betreuer/Doktorvater] Prof. Dr. Ernst, Joachim F. [Gutachter] Prof. Dr. Jaeger, Karl-Erich [Gutachter] | |||||||
| Stichwörter: | Filamentation, fungus, luminescence, click beetle, luciferase, reporter genes, EFG1 | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | The human fungal pathogen Candida albicans causes life-threatening infections in immunocompromised patients and is a major source of nosocomial illnesses. At present, virulence factors of C. albicans are intensely studied with the goal of identifying novel targets for antifungal drugs. The gene reporters currently employed to investigate the virulence factors of C. albicans have disadvantages including low sensitivity, high background signal or the necessity for cell lysis. In this work, we describe the use of the click beetle red and green luciferases (CBRluc and CBGluc) as versatile reporter proteins that produce light by catalysing the oxidation of beetle luciferin in the presence of oxygen and ATP.
The open reading frames for red and green click beetle luciferase were codon-adapted for C. albicans and were initially expressed in Saccharomyces cerevisiae and C. albicans strains using episomal plasmids. In both yeast species, CBRluc and CBGluc were produced and generated strong luminescence. In addition, the luciferases genes were chromosomally integrated into the C. albicans genome downstream of various promoters. All transformant strains were luminescent in the appropriate inducing conditions. In C. albicans, CBRluc and CBGluc luminescence spectra were verified and their distinct emission peaks could be measured separately using optical filters. Strong luminescence activity was observed in live cells of transformant strains, which could be improved further by a quick freeze/thaw cycle. Using the strong ACT1 promoter, in optimised conditions, the click beetle luciferase sensitivity in live cells was measured to a threshold of 50 cells. Genes implicated in the main signal transduction pathway regulating hyphal morphogenesis in C. albicans (cAMP/PKA pathway) were chosen as integration targets for CBR/CBGluc. During hyphal induction, ACT1 promoter activity increased two fold, while UME6 and HWP1 promoter activities rose drastically; the activity of the TCC1 promoter remained stable. The reactivity of the CBluc reporters was demonstrated by the detection of UME6- and HWP1-related (hypha-specific) signals only 10 min after hyphal induction. HWP1-CBluc activity, employed as a marker of hyphal growth, was used to measure the impact of different media on hypha formation. Farnesol, a quorum sensing molecule that promotes hypha formation significantly increased HWP1-CBluc related luminescence, while the hyphal inhibitor tyrosol diminished it. The capacity to accurately measure red and green signals at the same time was verified using strains carrying both luciferases under the control of two different promoters. After filtration and correction for spectra crosstalk, the luminescence of strains reflected precisely the activity of each promoter The click beetle luciferases compare favourably against other gene reporters, since no cell lysis is required for their detection and the background signal is negligible. Furthermore, as shown with the HWP1-CBluc fusion, CBluc can be used in high throughput screening for novel antifungal compounds. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Biologie » Mikrobiologie | |||||||
| Dokument erstellt am: | 28.01.2015 | |||||||
| Dateien geändert am: | 28.01.2015 | |||||||
| Promotionsantrag am: | 11.06.2014 | |||||||
| Datum der Promotion: | 02.07.2014 |
