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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/3962 |
| Date | 03 1900 |
| Creators | Sholto-Douglas-Vernon, Carolyn |
| Contributors | Van Helden, P., Victor, T., University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | University of Stellenbosch |
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