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The characterisation of N-Acetyltransferase (NAT) in Mycobacterium tuberculosis
Thesis (PhD (Molecular Biology and Human Genetics))--University of Stellenbosch, 2005.
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| Format: | Thesis |
| Language: | English |
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Stellenbosch : University of Stellenbosch
2010
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| _version_ | 1867614005061746688 |
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| access_status_str | Open Access |
| author | Sholto-Douglas-Vernon, Carolyn |
| author2 | Van Helden, P. |
| author_browse | Sholto-Douglas-Vernon, Carolyn Van Helden, P. |
| author_facet | Van Helden, P. Sholto-Douglas-Vernon, Carolyn |
| author_sort | Sholto-Douglas-Vernon, Carolyn |
| collection | Thesis |
| dc_rights_str_mv | University of Stellenbosch |
| description | Thesis (PhD (Molecular Biology and Human Genetics))--University of Stellenbosch, 2005. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/3962 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:45:08.467Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2010 |
| publishDateRange | 2010 |
| publishDateSort | 2010 |
| publisher | Stellenbosch : University of Stellenbosch |
| publisherStr | Stellenbosch : University of Stellenbosch |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/3962 The characterisation of N-Acetyltransferase (NAT) in Mycobacterium tuberculosis Sholto-Douglas-Vernon, Carolyn Van Helden, P. Victor, T. University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Tuberculosis (TB) N-acetyltransferase Thymine Tyrosine Immune system Dissertations -- Medical biochemistry Theses -- Medical biochemistry Thesis (PhD (Molecular Biology and Human Genetics))--University of Stellenbosch, 2005. 157 leaves single sided printed, preliminary pages i-xvii and numbered pages 1-141. Includes bibliography, and abbreviations and a list of figures. ENGLISH ABSTRACT: A gene coding for Arylaminie N-acetyltransferase (NAT) has been found in Mycobacterium tuberculosis, the casual agent of tuberculosis (TB). N-acetyltransferase acetylates and inactivates isoniazid (INH), which is a front line drug used in TB therapy. A guanine to adenine SNP at basepair 619 (G619A) has previously been identified in this gene, which results in a glycine to arginine change at amino acid 207 (G207R) (Upton et al. 2001). In this study the nat gene was further characterised. The frequency of the G619A SNP was analysed in 37 M tuberculosis strain families found in the Western Cape Province of South Africa, and it was found that the G619A SNP is conserved in two strain families (strain family 3 and strain family 28). Further sequence analysis identified a new thymine to cytosine SNP at base-pair 529 (T529C) resulting in a tyrosine to histidine change at amino acid 177 (Yl77H). This SNP was found only in isolates from strain family 3. These results imply that these SNPs may be used in epidemiology studies to classify isolates into these strain families. Using Real Time PCR, the expression of nat in M bovis BCG and M tuberculosis (reference strain H37Rv) was determined over a 7 and 28 day growth cycle, respectively. Using 16S rRNA as an endogenous control, the nat gene was shown to be expressed early during the growth curve and reach its maximum expression level at approximately mid-log phase. The expression of nat was induced in drug susceptible M tuberculosis isolates (reference strain H37Rv and isolate 1430 containing both SNPs) exposed to INH at a concentration of O.Oll-lg/ml, but minimal change in expression was observed in resistant isolates (isolate 816) exposed to INH at the same concentration. Mycobacterium bovis BCG cultures exposed to INH, at a final concentration of 0.28I-lg/ml, showed an increase in protein production. The increase of nat mRNA and NAT protein in M tuberculosis and M bovis BCG, respectively, implies that INH affects the expression of NAT. The NAT protein was localised to all fractions of the cell in Mycobacterium smegmatis, M bovis BCG and M tuberculosis, using the Western blot technique. However, protein fractions from the cell envelope region showed a protein (detected with specific NAT antibodies) that ran at a higher molecular weight (MW). This implies that the cytosolic hydrophilic NAT undergoes some type of post-translational process that may make it hydrophobic, and enable it to pass into the cell envelope region. These results show for the first time how nat is expressed during the entire growth cycle of M tuberculosis and M. bovis BeG. It was shown that nat is expressed early during the growth cycle of the bacterium reaching maximum expression levels at mid-log phase. These results are in concordance with those obtained using M. smegmatis nat mutants, which taken together, show that early expression of nat is important for early growth and development of mycobacteria. The results in this study also showed that NAT appeared to be translocated into the cell envelope of the bacterium, implying that NAT may be involved in one of the pathways needed for complete formation of the cell envelope. These results suggest that NAT may be an important target for drug development, as inhibitors of NAT could result in hindered growth and hence spread of the bacterium within its host. Inhibitors may also result in the incomplete development of the cell wall, enabling the host to combat the disease using its own immune system. Doctoral 2010-08-12T12:46:26Z 2010-08-12T12:46:26Z 2005-03 Thesis http://hdl.handle.net/10019.1/3962 en University of Stellenbosch application/pdf Stellenbosch : University of Stellenbosch |
| spellingShingle | Tuberculosis (TB) N-acetyltransferase Thymine Tyrosine Immune system Dissertations -- Medical biochemistry Theses -- Medical biochemistry Sholto-Douglas-Vernon, Carolyn The characterisation of N-Acetyltransferase (NAT) in Mycobacterium tuberculosis |
| title | The characterisation of N-Acetyltransferase (NAT) in Mycobacterium tuberculosis |
| title_full | The characterisation of N-Acetyltransferase (NAT) in Mycobacterium tuberculosis |
| title_fullStr | The characterisation of N-Acetyltransferase (NAT) in Mycobacterium tuberculosis |
| title_full_unstemmed | The characterisation of N-Acetyltransferase (NAT) in Mycobacterium tuberculosis |
| title_short | The characterisation of N-Acetyltransferase (NAT) in Mycobacterium tuberculosis |
| title_sort | characterisation of n acetyltransferase nat in mycobacterium tuberculosis |
| topic | Tuberculosis (TB) N-acetyltransferase Thymine Tyrosine Immune system Dissertations -- Medical biochemistry Theses -- Medical biochemistry |
| url | http://hdl.handle.net/10019.1/3962 |
| work_keys_str_mv | AT sholtodouglasvernoncarolyn thecharacterisationofnacetyltransferasenatinmycobacteriumtuberculosis AT sholtodouglasvernoncarolyn characterisationofnacetyltransferasenatinmycobacteriumtuberculosis |