The trafficking of the Mycobacterium tuberculosis PE and PPE proteins

Mahasha, Phetole Walter (2007-12)

Thesis (MScMed (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2007.

Thesis

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.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/1862
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