Investigating the localisation of the ESX-3 secretion system in Mycobacterium smegmatis

[摘要] ENGLISH ABSTRACT: Mycobacterium tuberculosis is a pathogenic organism that infects a third of the world's population andcauses approximately 2 million deaths per year. Extensive research has been done on this pathogen,however our knowledge of the mechanisms of pathogenicity remain limited. The M. tuberculosis genomecontains five ESAT-6 gene cluster regions, ESX-1 to 5, which encode specialized type VII secretionsystems. These secretion systems are known to secrete members of the ESAT-6/CFP-10 and PE/PPEprotein families, some of which contribute to the pathogenicity and phagosomal escape of the pathogen.ESX-3 has been shown to be essential for in vitro growth and survival of M. tuberculosis. The expressionof ESX-3 in M. tuberculosis is regulated by IdeR and Zur, in response to intracellular iron and zincconcentrations, respectively. Interestingly, ESX-3 is not essential for the growth and survival of thesaprophytic organism M. smegmatis.In this study, we aimed to identify the subcellular localisation of the individual components of the ESX-3secretion system in the non-pathogenic, fast-growing organism M. smegmatis. The esx conservedcomponent (ecc) genes from ESX-3 were expressed from the episomal expression vector pDMNI asfusion proteins with green fluorescent protein (GFP). MSMEG_0615 (eccA3), MSMEG_0616 (eccB3),MSMEG_0623 (eccD3) and MSMEG_0626 (eccE3) were successfully cloned into pDMNI and expressionof fusion proteins was confirmed by Western blotting for MSMEG_0615-GFP, MSMEG_0616-GFP andMSMEG_0626-GFP in M. smegmatis. In the M. smegmatis ESX-3 knock-out (with MSMEG_0615 toMSMEG_0626 deleted) expression was confirmed for MSMEG_0615-GFP and MSMEG0626-GFP.Fluorescent microscopy determined that MSMEG_0615-GFP localised to a single mycobacterial pole inboth strains. MSMEG_0616-GFP and MSMEG_0626-GFP were found to be membrane associated in M.smegmatis, while MSMEG_0626-GFP was found to be membrane associated in the M. smegmatis ESX-3knock-out.The unipolar localisation of MSMEG_0615-GFP suggests that the assembled ESX-3 secretion systemapparatus is situated at a single pole in M. smegmatis. Therefore, we hypothesize that MSMEG_0615might act as a recruiter protein that is involved in the assembly of ESX-3 at the mycobacterial pole.

[发布机构] Stellenbosch University