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Riboswitch regulation of methionine metabolism and vitamin B12 uptake in mycobacteria – implications for drug susceptibility and pathogenesis
Alterations in the genetic capacity for cobamide biosynthesis have been identified as potentially critical in the evolution of Mycobacterium tuberculosis from a putative environmental ancestor. Moreover, recent studies have implicated cobamide biosynthesis pathway genes in the adaptation of the baci...
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| Format: | Thesis |
| Language: | English |
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Department of Pathology
2020
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| _version_ | 1867613452806127617 |
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| access_status_str | Open Access |
| author | Kipkorir, Terry |
| author2 | Warner, Digby |
| author_browse | Kipkorir, Terry Warner, Digby |
| author_facet | Warner, Digby Kipkorir, Terry |
| author_sort | Kipkorir, Terry |
| collection | Thesis |
| description | Alterations in the genetic capacity for cobamide biosynthesis have been identified as potentially critical in the evolution of Mycobacterium tuberculosis from a putative environmental ancestor. Moreover, recent studies have implicated cobamide biosynthesis pathway genes in the adaptation of the bacillus to intracellular pathogenesis. Although mycobacteria retain essential biochemical reactions that require cobamides, the specific role of these co-factors during tuberculosis (TB) disease remains unresolved. This thesis aimed to examine the production, uptake, and utilization of cobamides in mycobacteria using M. smegmatis as a model. To this end, the genetic capacity for de novo production and uptake of cobamide in host-associated and environmental mycobacteria was assessed, followed by direct validation in M. smegmatis. A combination of genetics, gene expression analysis, live-cell time-lapse microscopy and targeted metabolite and protein analysis via mass spectrometry (MS) was then employed to investigate cobamide riboswitch-dependent regulation of methionine biosynthesis in M. smegmatis. Results indicated that, in wild-type M. smegmatis, de novo cobamide biosynthesis ensured constitutive repression of metE, the gene encoding the mycobacterial cobalamin-independent methionine synthase. Owing to this repression, metH, a gene encoding the cobalamin-dependent methionine synthase, was found to be conditionally essential for bacillary replication in vitro. Drug susceptibility testing to investigate the link between cobamides and the intrinsic resistance to anti-folate antibiotics confirmed novel mycobacterial vulnerabilities in cobamide-related methionine metabolism, indicating that the outcomes of cobamidedependent regulation may have relevance to mycobacterial pathogenesis and drug discovery. In contrast to M. tuberculosis, which was previously shown to transport exogenous CNCbl readily, M. smegmatis poorly assimilated exogenous co-factor despite the presence of multiple putative cobamide transporters. However, uptake was enhanced in a mutant requiring CNCbl for growth. Elucidating the factors which regulate cobamide biosynthesis and co-factor utilization in M. smegmatis, an environmental mycobacterium, might provide a lens through which to consider the differential regulation and utilization of cobamides in M. tuberculosis, an obligate pathogen with a limited host range. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/31134 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:36:22.916Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2020 |
| publishDateRange | 2020 |
| publishDateSort | 2020 |
| publisher | Department of Pathology |
| publisherStr | Department of Pathology |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/31134 Riboswitch regulation of methionine metabolism and vitamin B12 uptake in mycobacteria – implications for drug susceptibility and pathogenesis Kipkorir, Terry Warner, Digby Mizrahi, Valerie Molecular Mycobacteriology Alterations in the genetic capacity for cobamide biosynthesis have been identified as potentially critical in the evolution of Mycobacterium tuberculosis from a putative environmental ancestor. Moreover, recent studies have implicated cobamide biosynthesis pathway genes in the adaptation of the bacillus to intracellular pathogenesis. Although mycobacteria retain essential biochemical reactions that require cobamides, the specific role of these co-factors during tuberculosis (TB) disease remains unresolved. This thesis aimed to examine the production, uptake, and utilization of cobamides in mycobacteria using M. smegmatis as a model. To this end, the genetic capacity for de novo production and uptake of cobamide in host-associated and environmental mycobacteria was assessed, followed by direct validation in M. smegmatis. A combination of genetics, gene expression analysis, live-cell time-lapse microscopy and targeted metabolite and protein analysis via mass spectrometry (MS) was then employed to investigate cobamide riboswitch-dependent regulation of methionine biosynthesis in M. smegmatis. Results indicated that, in wild-type M. smegmatis, de novo cobamide biosynthesis ensured constitutive repression of metE, the gene encoding the mycobacterial cobalamin-independent methionine synthase. Owing to this repression, metH, a gene encoding the cobalamin-dependent methionine synthase, was found to be conditionally essential for bacillary replication in vitro. Drug susceptibility testing to investigate the link between cobamides and the intrinsic resistance to anti-folate antibiotics confirmed novel mycobacterial vulnerabilities in cobamide-related methionine metabolism, indicating that the outcomes of cobamidedependent regulation may have relevance to mycobacterial pathogenesis and drug discovery. In contrast to M. tuberculosis, which was previously shown to transport exogenous CNCbl readily, M. smegmatis poorly assimilated exogenous co-factor despite the presence of multiple putative cobamide transporters. However, uptake was enhanced in a mutant requiring CNCbl for growth. Elucidating the factors which regulate cobamide biosynthesis and co-factor utilization in M. smegmatis, an environmental mycobacterium, might provide a lens through which to consider the differential regulation and utilization of cobamides in M. tuberculosis, an obligate pathogen with a limited host range. 2020-02-17T11:04:39Z 2020-02-17T11:04:39Z 2019 2020-02-17T10:06:10Z Doctoral Thesis Doctoral PhD http://hdl.handle.net/11427/31134 eng application/pdf Department of Pathology Faculty of Health Sciences |
| spellingShingle | Molecular Mycobacteriology Kipkorir, Terry Riboswitch regulation of methionine metabolism and vitamin B12 uptake in mycobacteria – implications for drug susceptibility and pathogenesis |
| thesis_degree_str | Doctoral |
| title | Riboswitch regulation of methionine metabolism and vitamin B12 uptake in mycobacteria – implications for drug susceptibility and pathogenesis |
| title_full | Riboswitch regulation of methionine metabolism and vitamin B12 uptake in mycobacteria – implications for drug susceptibility and pathogenesis |
| title_fullStr | Riboswitch regulation of methionine metabolism and vitamin B12 uptake in mycobacteria – implications for drug susceptibility and pathogenesis |
| title_full_unstemmed | Riboswitch regulation of methionine metabolism and vitamin B12 uptake in mycobacteria – implications for drug susceptibility and pathogenesis |
| title_short | Riboswitch regulation of methionine metabolism and vitamin B12 uptake in mycobacteria – implications for drug susceptibility and pathogenesis |
| title_sort | riboswitch regulation of methionine metabolism and vitamin b12 uptake in mycobacteria implications for drug susceptibility and pathogenesis |
| topic | Molecular Mycobacteriology |
| url | http://hdl.handle.net/11427/31134 |
| work_keys_str_mv | AT kipkorirterry riboswitchregulationofmethioninemetabolismandvitaminb12uptakeinmycobacteriaimplicationsfordrugsusceptibilityandpathogenesis |