Thesis (MScMed (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2007. / The expansion of the Mycobacterium tuberculosis PE and PPE gene families seems to be linked to that
of the immunologically-important ESAT-6 (esx) gene clusters secretion system, as the ancestral
members of these families are found only within the ESAT-6 gene cluster regions. These ancestral
members are also the only copies in the earlier mycobacteria like M. smegmatis. The later duplications
of the PE and PPE families belonging to the PGRS and MPTR subgroups, have been implicated in
virulence and are only found within the genomes of the pathogenic mycobacteria closely related to the
M. tuberculosis complex. The aim of this study was to compare the subcellular localization of the later
duplications of the PE and PPE gene families belonging to the PGRS and MPTR subgroups with that of
the ancestral PE and PPE proteins found in M. smegmatis and to investigate whether the ESX secretion
apparatus is involved in the trafficking of these proteins. The PE (Rv3872) and PPE (Rv3873) genes
from M. smegmatis were PCR amplified with a C-terminal HA tag using M. smegmatis genomic DNA as
template. Two PPE-MPTR genes, Rv0442c and Rv0878c, and one PE_PGRS gene, Rv2615c, were
also PCR amplified with a C-terminal HA tag using M. tuberculosis genomic DNA as template. All
genes were cloned into the mycobacterial expression vector p19Kpro. Expression and localization was
investigated using SDS-PAGE and Western blotting. The PE and PPE genes expressed in M.
smegmatis were found to be present within the cell wall, membrane, and cytosol fractions, but not in the
culture filtrate, indicating no secretion. The PPE-MPTR and PE_PGRS genes expressed in M.
smegmatis, were also found to be present within the cell wall, membrane and cytosol fractions, but not
in the culture filtrate, indicating that they are also not secreted. We hypothesize that their secretion is
dependent on ESAT-6 gene cluster region 5, which is absent from the genome of M. smegmatis.
Ancestral PE and PPE proteins are secreted efficiently in M. tuberculosis. The ESAT-6 gene cluster
Region 3 and Region 4 of M. smegmatis were knocked out, and these knockout mutants could be used
in future studies to investigate if the ESAT-6 gene cluster region 1 is involved in the secretion of the
ancestral and recent PE and PPE proteins.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/1862 |
| Date | 12 1900 |
| Creators | Mahasha, Phetole Walter |
| Contributors | Gey van Pittius, N. C., Warren, R. M., University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 2483227 bytes, application/pdf |
| Rights | University of Stellenbosch |
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