Thesis (MScMedSc (Biomedical Sciences. Medical Biochemistry))--University of Stellenbosch, 2010. / Bibliography / ENGLISH ABSTRACT: The genome of the pathogen Mycobacterium tuberculosis contains five copies of the ESAT-6 (ESX) gene cluster region, which encodes for a novel type VII secretion system. These gene cluster regions, which are directly involved in pathogenicity and phagosomal escape, contain genes encoding exported T-cell antigens ESAT-6 and CFP-10. The mechanism of action of the ESX secretion system however, remains largely unknown. This study focused on ESX gene cluster region 4 (ESX-4), which has been shown to be the most ancestral region and is also present in other species of Mycobacteria and even in other high G+C Gram-positive bacteria, such as Corynebacterium diptheriae and Streptomyces coelicolor. This project aimed to investigate the protein-protein interactions of ESX-4 of M. tuberculosis in the model organism Mycobacterium smegmatis by means of Mycobacterial Protein Fragment Complementation (M-PFC). M-PFC is a two-hybrid technique which employs two cloning vectors, pUAB300 (conferring resistance to hygromycin B) and pUAB400 (conferring resistance to kanamycin). Genes of interest are cloned into these vectors and co-transformed into the model organism M. smegmatis after which it is expressed as fusion proteins. Interaction of the proteins allows selective growth on a medium containing the antibiotic trimethoprim. Various interactions were identified throughout this region, including selfinteractions as well as the expected interaction between the ESAT-6 and CFP-10 protein family members esxT and esxU. Since this region is ancestral, ESX-4 provides the basic model of the mechanism of secretion of the type VII secretion system. Many similarities were apparent when the interactions identified for ESX-4 were compared to the interactions previously identified in ESX-3. Interactions identified by means of M-PFC provide a basis for the further study of the structure of this secretion system, and should be confirmed by means of other techniques, such as co-immunoprecipitation. Despite the ability of M-PFC to identify protein-protein interactions in a mycobacterial system, and thus overcoming some of the limitations of the classical yeast two-hybrid model, it must still be regarded as a fishing experiment for potential interactions. A further aim of the project was to construct a knock-out of ESX-4 in the model organism M. smegmatis, which contains three ESX regions, namely ESX-1, -3 and -4. Homologous recombination proved to be an effective technique for the construction of the knock-out, also indicating that ESX-4 is not essential for in vitro growth of M. smegmatis. The knock-out strain showed no morphological differences to the wild type strain of M. smegmatis. The knock-out strain will in future be compared to the wild type strain in various functional studies in order to determine the function of the ancestral ESX region. / AFRIKAANSE OPSOMMING: Die genoom van die patogeen Mycobacterium tuberculosis bavat vyf kopieë van die ESAT-6 geen groep gebiede wat kodeer vir ‘n unieke tipe VII sekresie sisteem. Die geen groep gebiede, wat direk betrokke is by patogenisiteit en fagosomale ontsnapping, bevat gene wat kodeer vir die gesekreteerde T-sel antigene ESAT-6 en CFP-10. Die meganisme van die ESX sekresie sisteem is egter steeds tot ‘n groot mate onbekend. Hierdie studie het gefokus op die ESX geen groep gebied 4 (ESX-4), wat voorheen bepaal is om die vroegste kopie van die gebied te wees en wat ook in ander species van Mikobakterieë en hoë G+C Gram-positiewe bakterieë, soos Corynebacterium diptheriae en Streptomyces coelicolor, voorkom. Hierdie projek was daarop gemik om die proteïen-proteïen interaksies van ESX-4 van M. tuberculosis in die model organisme Mycobacterium smegmatis te ondersoek deur middel van Mikobakteriële Proteïen Fragment Komplementasie (M-PFK). M-PFK is ‘n twee-hibried tegniek wat van twee kloningsvektore, naamlik pUAB300 (wat weerstand teen hygromycin B bied) en pUAB400 (wat weerstand teen kanamycin bied) gebruik maak. Gene van belang word in die vektore ingekloneer en in die model organisme, M. smegmatis geko-transformeer, waarna dit as fusieproteïene uitgedruk word. Indien ‘n interaksie tussen die proteïene plaasvind, sal selektiewe groei op ‘n medium wat die antibiotikum trimethoprim bevat, waargeneem word. Verskeie interaksies is in hierdie gebied geïdentifiseer, insluitende self-interaksies, sowel as die verwagte interaksie tussen die ESAT-6 en CFP-10 proteïen familielede esxT en esxU. Aangesien hierdie gebied die vroegste kopie is, bied ESX-4 die basiese model vir die meganisme van sekresie van die tipe VII sekresie sisteem. Wanneer interaksies wat vir ESX-4 geïdentifiseer is met die wat voorheen vir ESX-3 geïdentifiseer is vergelyk word is daar heelwat ooreenkomste. Interaksies wat deur middel van M-PFK geïdentifiseer is, verskaf ‘n basis vir die vêrdere studie van interaksies van hierdie gebied, en sal bevestig moet word deur gebruik te maak van aanvullende tegnieke, soos ko-immunopresipitasie. Ten spyte van die vermoë van M-PFK om proteïen-proteïen interaksies in ‘n mikobakteriële sisteem, wat dus sommige van die beperkings van die klassieke gis twee-hibriedmodel oorkom, te bestudeer, behoort dit steeds as ‘n voorlopige metode van identifikasie beskou te word. ‘n Vêrdere doel van die projek was om ‘n uitslaanmutant van ESX-4 in die model organisme M. smegmatis, wat drie van die ESX gebiede, naamlik ESX-1, -3 en -4 bevat, te skep. Homoloë rekombinasie is bewys om ‘n effektiewe tegniek te wees vir die skep van ‘n uitslaanmuntant en het daarop gedui dat ESX-4 nie essensieel is vir die in vitro groei van M. smegmatis nie. Die uitslaanstam het ook geen morfologiese verskille getoon teenoor die oorspronklike stam nie. Die uitslaanmutant sal in die toekoms gebruik word in ‘n verskeidenheid funksionele studies waar dit vergelyk sal word met die oorspronklike stam, ten einde die funksie van die vroegste ESX-gebied te bepaal. / Medical Research Council of South Africa / National Research Foundation of South Africa / Ernst and Ethel Eriksen Trust