Dokument: Key Roles of Essential Genes in Mycobacterial Glycoconjugate Biosynthesis: otsB2 and Rv0225
| Titel: | Key Roles of Essential Genes in Mycobacterial Glycoconjugate Biosynthesis: otsB2 and Rv0225 | |||||||
| Weiterer Titel: | Schlüsselfunktionen Essentieller Gene in der Mykobakteriellen Glykokonjugat-Biosynthese: otsB2 und Rv0225 | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=53612 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20200716-103427-1 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Dipl.-Biol. Korte, Jan [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Kalscheuer, Rainer [Gutachter] Prof. Dr. Jaeger, Karl-Erich [Gutachter] | |||||||
| Stichwörter: | Antibiotika, Tuberkulose, M. tuberculosis, Trehalose, MGLP | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | (Parts of this summary related to OtsB2 has been published in PLoS Pathog 12(12): e1006043)
On a global perspective, Mycobacterium tuberculosis, the etiologic agent of tuberculosis, represents the most devastating bacterial pathogen. Multi-drug resistance is a major force causing the exacerbation of the global tuberculosis pandemic. Strains are emerging worldwide at an alarming rate which are resistant to most available antitubercular drugs and which are very difficult to control. The whole structure of the mycobacterial cell wall is of considerable complexity and thickness, which confers high intrinsic resistance against antibiotics. First-line drugs such as isoniazid and ethambutol target the mycobacterial cell wall and have been used for the clinical treatment since many decades, justifiying the ongoing research on the mycobacterial cell wall assembly to identify novel drug targets. In order to contribute to Mycobacterium tuberculosis research, two projects were in focus of this doctoral thesis investigating key roles of the essential mycobacterial cell wall associated genes otsB2 and Rv0225. Both genes are likely involved in a direct or indirect manner in the mycolic acid metabolism in mycobacteria. Mycolic acids are the major component of the mycobacterial cell wall and occur in different forms of gycoconjugates that together form a highly hydrophobic layer. Project 1 „Trehalose-6-Phosphate-Mediated Toxicity Determines Essentiality of OtsB2 in Mycobacterium tuberculosis in Vitro and in Mice“ deals with mycolic acid-containing trehalose glycoconjugates, which are essential for mycomembrane formation. Generally, trehalose biosynthesis is considered an attractive target for the development of antimicrobials against fungal, helminthic and bacterial pathogens including Mycobacterium tuberculosis. The most common biosynthetic route involves the trehalose-6-phosphate (T6P) synthase OtsA and T6P phosphatase OtsB2 that generate trehalose from ADP/UDP-glucose and glucose-6-phosphate. In order to assess the drug target potential of T6P phosphatase, we generated a conditional mutant of Mycobacterium tuberculosis allowing the regulated gene silencing of the T6P phosphatase gene otsB2. We found that otsB2 is essential for growth of Mycoacterium tuberculosis in vitro as well as for the acute infection phase in mice following aerosol infection. By contrast, otsB2 is not essential for the chronic infection phase in mice, highlighting the substantial remodelling of trehalose metabolism during infection by Mycobacerium tuberculosis. Blocking OtsB2 resulted in the accumulation of its substrate T6P, which appears to be toxic, leading to the self-poisoning of cells. Accordingly, blocking T6P production in a ΔotsA mutant abrogated otsB2 essentiality. T6P accumulation elicited a global upregulation of more than 800 genes, which might result from an increase in RNA stability implied by the enhanced neutralization of toxins exhibiting ribonuclease activity. Surprisingly, overlap with the stress response caused by the accumulation of another toxic sugar phosphate molecule, maltose-1-phosphate, was minimal. A genome-wide screen for synthetic lethal interactions with otsA identified numerous genes, revealing additional potential drug targets synergistic with OtsB2 suitable for combination therapies that would minimize the emergence of resistance to OtsB2 inhibitors. Project 2 „Functional Analysis of the Putative Essential Glycosyltransferase Rv0225 in a Cell Wall Associated Gene Cluster in Mycobacterium Species“ describes the functional analysis of the annotated glycosyltransferase Rv0225, which might play a role in synthesis of methylglucose lipopolysaccharides (MGLPs), which in turn are supposed to affect mycolic acid metabolism in the mycobacterial cell. Since MGLPs are only found in slow-growing mycobacteria, they likely play an essential role in bacterial growth for Mycobacterium tuberculosis. Studies suggest that the polymer has a regulatory function for the fatty acid synthase I (FAS-I). Its biological function and structure are not completely understood. We hypothesized that Rv0225 might elongate the proximal part of the MGLP glucan backbone. Our results based on conditional gene silencing demonstrate that Rv0225 is strictly essential for in vitro growth in Mycobacterium smegmatis strain mc2155, Mycobacterium bovis BCG-Pasteur and, more important, also for the virulent Mycobacterium tuberculosis strain H37Rv. In vitro killing kinetics revealed that inactivation of Rv0225 is bactericidal, resulting in rapid loss of viability upon gene silencing. Partially silenced conditional Mycobacterium bovis and Mycobcaterium smegmatis mutants exhibited a different colony morphology in comparison to the fully induced mutants, indicative of an altered cell wall structure. A transcriptome profiling of the conditional Mycobcaterium tuberculosis Rv0225 mutant using RNAseq points towards cell wall stress in partially silenced cells, which is in agreement with the mentioned altered colony morphology. High-resolution mass spectroscopy ESI-MS (electron spray ionisation-mass spectrometry) revealed a subtle but significant loss of prominent α-mycolic acids under partial silenced gene expression conditions, highlighting a cell wall associated function of Rv0225. Further gene editing experiments on the Rv0225 conserved mycobacterial gene cluster Rv0224c -Rv0228, showed the dispensability of the putative methyltransferase Rv0224c for in vitro growth. On the contrary, the conditional Mycobcaterium tuberculosis Rv0226c mutant showed impaired growth on solid medium under completely silenced gene expression conditions, suggesting a partial essential role of the putative transmembrane protein for in vitro growth. Strikingly, when the orthologue of Rv0226c was partially silenced in Mycobacterium bovis, the mutant exhibited the same alteration in cellular morphology compared to the partial silenced Rv0225 mutant. This also suggests a cell wall associated function for Rv0226c, possibly in the same pathway with Rv0225. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Pharmazie » Pharmazeutische Biologie und Biotechnologie | |||||||
| Dokument erstellt am: | 16.07.2020 | |||||||
| Dateien geändert am: | 16.07.2020 | |||||||
| Promotionsantrag am: | 06.11.2019 | |||||||
| Datum der Promotion: | 26.05.2020 |
