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

Steyn, Natassja Lise (2012-12)

Thesis (MScMedSc)--Stellenbosch University, 2012.

Thesis

ENGLISH ABSTRACT: Mycobacterium tuberculosis is a pathogenic organism that infects a third of the world’s population and causes 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 genome contains five ESAT-6 gene cluster regions, ESX-1 to 5, which encode specialized type VII secretion systems. These secretion systems are known to secrete members of the ESAT-6/CFP-10 and PE/PPE protein 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 expression of ESX-3 in M. tuberculosis is regulated by IdeR and Zur, in response to intracellular iron and zinc concentrations, respectively. Interestingly, ESX-3 is not essential for the growth and survival of the saprophytic organism M. smegmatis. In this study, we aimed to identify the subcellular localisation of the individual components of the ESX-3 secretion system in the non-pathogenic, fast-growing organism M. smegmatis. The esx conserved component (ecc) genes from ESX-3 were expressed from the episomal expression vector pDMNI as fusion 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 expression of fusion proteins was confirmed by Western blotting for MSMEG_0615-GFP, MSMEG_0616-GFP and MSMEG_0626-GFP in M. smegmatis. In the M. smegmatis ESX-3 knock-out (with MSMEG_0615 to MSMEG_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 in both 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-3 knock-out. The unipolar localisation of MSMEG_0615-GFP suggests that the assembled ESX-3 secretion system apparatus is situated at a single pole in M. smegmatis. Therefore, we hypothesize that MSMEG_0615 might act as a recruiter protein that is involved in the assembly of ESX-3 at the mycobacterial pole.

AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis is ‘n patogene organisme wat ‘n derde van die wêreld se bevolking infekteer en eis jaarliks 2 miljoen lewens deur tuberkulose. Ten spyte van uitgebreide navorsing, is daar min kennis oor die meganismes van patogenisiteit van hierdie organisme. Die M. tuberculosis genoom bevat vyf duplikasies van die ESAT-6 geen groep gebiede, ESX-1 tot 5, wat kodeer vir gespesialiseerde Tipe VII sekresie sisteme. Hierdie sekresie sisteme is bekend vir die sekresie van lede van die ESAT-6/CFP-10 en PE/PPE proteïen families, waarvan sommige bydra tot die patogenisieit en fagosomale ontsnapping van hierdie organisme. ESX-3 is noodsaaklik vir die in vitro groei en oorlewing van M. tuberculosis. Die uitdrukking van ESX-3 in M. tuberculosis word gereguleer deur IdeR en Zur in reaksie op intrasellulêre yster en sink konsentrasies, onderskeidelik. ESX-3 word nie benodig vir die groei en oorlewing van die saprofitiese organisme M. smegmatis nie. Hierdie studie was gemik om die sub-sellulêre lokalisering van ESX-3 te identifiseer in die niepatogeniese en vinnig-groeiende organisme, M. smegmatis. Die “esx conserved component” (ecc) gene van ESX-3 is uitgedruk vanaf die episomale uitdrukkingsvektor pDMNI as gekombineerde proteïene met die groen fluoreserende proteïen (GFP). MSMEG_0615 (eccA3), MSMEG_0616 (eccB3), MSMEG_0623 (eccD3) en MSMEG_0626 (eccE3) is suksesvol gekloneer en die uitdrukking van die gekombineerde proteïene is bevestig deur Western oordrag vir MSMEG_0615-GFP, MSMEG_0616-GFP en MSMEG_0626-GFP in M. smegmatis. In die M. smegmatis ESX-3 uitklopmutant (met MSMEG_0615 tot MSMEG_0626 uitgeslaan) is uitdrukking bevestig vir MSMEG_0615-GFP en MSMEG0626-GFP. Fluoresensie mikroskopie het bepaal dat MSMEG_0615-GFP gelokaliseer is by ‘n enkele mikobakteriese pool in beide stamme. MSMEG_0616-GFP en MSMEG_0626-GFP was membraan-geassosieerd in M. smegmatis, terwyl en MSMEG_0626-GFP geassosieer het met die membraan in die M. smegmatis uitklopmutant. MSMEG_0615 het gelokaliseer by ‘n enkele pool in M. smegmatis en dit dui aan dat die saamgestelde ESX-3 sekresie sisteem apparaat slegs by ‘n enkele pool voorkom in M. smegmatis. Ons hipotiseer dat MSMEG_0615 dalk mag optree as ‘n werwer proteïen wat betrokke is by die samestelling van die ESX-3 sekresie sisteem by die mikrobakteriese pool.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/71959
This item appears in the following collections: