The trafficking of the Mycobacterium tuberculosis PE and PPE proteins

[摘要] The expansion of the Mycobacterium tuberculosis PE and PPE gene families seems to be linked to thatof the immunologically-important ESAT-6 (esx) gene clusters secretion system, as the ancestralmembers of these families are found only within the ESAT-6 gene cluster regions. These ancestralmembers are also the only copies in the earlier mycobacteria like M. smegmatis. The later duplicationsof the PE and PPE families belonging to the PGRS and MPTR subgroups, have been implicated invirulence and are only found within the genomes of the pathogenic mycobacteria closely related to theM. tuberculosis complex. The aim of this study was to compare the subcellular localization of the laterduplications of the PE and PPE gene families belonging to the PGRS and MPTR subgroups with that ofthe ancestral PE and PPE proteins found in M. smegmatis and to investigate whether the ESX secretionapparatus is involved in the trafficking of these proteins. The PE (Rv3872) and PPE (Rv3873) genesfrom M. smegmatis were PCR amplified with a C-terminal HA tag using M. smegmatis genomic DNA astemplate. Two PPE-MPTR genes, Rv0442c and Rv0878c, and one PE_PGRS gene, Rv2615c, werealso PCR amplified with a C-terminal HA tag using M. tuberculosis genomic DNA as template. Allgenes were cloned into the mycobacterial expression vector p19Kpro. Expression and localization wasinvestigated 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 theculture 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 notin the culture filtrate, indicating that they are also not secreted. We hypothesize that their secretion isdependent 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 clusterRegion 3 and Region 4 of M. smegmatis were knocked out, and these knockout mutants could be usedin future studies to investigate if the ESAT-6 gene cluster region 1 is involved in the secretion of theancestral and recent PE and PPE proteins.

[发布机构] Stellenbosch University