Investigation of the ESX-4 secretion system interactome of Mycobacterium tuberculosis

[摘要] 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 genecluster regions, which are directly involved in pathogenicity and phagosomal escape, containgenes encoding exported T-cell antigens ESAT-6 and CFP-10. The mechanism of action ofthe ESX secretion system however, remains largely unknown. This study focused on ESXgene cluster region 4 (ESX-4), which has been shown to be the most ancestral region and isalso present in other species of Mycobacteria and even in other high G+C Gram-positivebacteria, such as Corynebacterium diptheriae and Streptomyces coelicolor.This project aimed to investigate the protein-protein interactions of ESX-4 of M. tuberculosisin the model organism Mycobacterium smegmatis by means of Mycobacterial ProteinFragment Complementation (M-PFC). M-PFC is a two-hybrid technique which employs twocloning vectors, pUAB300 (conferring resistance to hygromycin B) and pUAB400 (conferringresistance to kanamycin). Genes of interest are cloned into these vectors and co-transformedinto 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 antibiotictrimethoprim. Various interactions were identified throughout this region, including selfinteractionsas well as the expected interaction between the ESAT-6 and CFP-10 proteinfamily members esxT and esxU. Since this region is ancestral, ESX-4 provides the basicmodel of the mechanism of secretion of the type VII secretion system. Many similarities wereapparent when the interactions identified for ESX-4 were compared to the interactionspreviously identified in ESX-3.Interactions identified by means of M-PFC provide a basis for the further study of thestructure of this secretion system, and should be confirmed by means of other techniques, suchas co-immunoprecipitation. Despite the ability of M-PFC to identify protein-proteininteractions in a mycobacterial system, and thus overcoming some of the limitations of theclassical yeast two-hybrid model, it must still be regarded as a fishing experiment for potentialinteractions.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. Homologousrecombination proved to be an effective technique for the construction of the knock-out, alsoindicating that ESX-4 is not essential for in vitro growth of M. smegmatis. The knock-outstrain showed no morphological differences to the wild type strain of M. smegmatis. Theknock-out strain will in future be compared to the wild type strain in various functional studiesin order to determine the function of the ancestral ESX region.

[发布机构] Stellenbosch University