Understanding the evolution and function of the mycobacterial Type VII ESX secretion systems (T7SSs) and their substrates

[摘要] ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative agent of tuberculosis disease, contains five copies of the ESAT-6gene cluster, each encoding a dedicated ESX protein secretion system which has been defined as a novelType-VII secretion system. The ESX have been implicated in virulence and survival of M. tuberculosis, andas such present a promising target for novel treatment interventions. This study has investigated theevolution, regulation, functions and substrates of the ESX secretion systems.The evolutionary history of the ESX secretion systems was established using in silico and phylogeneticanalyses of the sequenced mycobacteria, closely related actinomycetes and WXG-FtsK clusters from otherbacteria. The ESX-4 gene cluster appears to have evolved with the start of the evolution of themycomembrane, followed by the duplication of ESX-3, which marks the evolution of the genusMycobacterium. The ESX-1 duplication occurred next, followed by ESX-2 and ESX-5 which occur only in theslow growing mycobacteria. Five additional ESX gene clusters were newly identified and named ESX-P1 to -P5. These additional ESX clusters occur, or are predicted to occur, on plasmid DNA, and appear to beprogenitors of the genomic ESX-1 to -5 gene clusters, possibly indicating a plasmid-mediated mechanism ofESX duplication and evolution.The promoters expressing the M. tuberculosis ESX-1 to ESX-5 secretion systems were investigated using apromoter probe assay, and characterised using in silico analyses. Promoters were identified for ESX-1, -2, -3and -5.The functions of the mycobacterial ESX secretion systems were investigated using whole proteomic,secretomic and metabolomic analyses of the fast growing, non-pathogenic M. smegmatis, which containsthree of the ESX secretion systems, ESX-1, 3, and 4. ESX knockout strains of M. smegmatis were generatedand used in comparative analyses with wild-type M. smegmatis. ESX-1 was highly expressed in wild-type M.smegmatis, however no specific pathways showed considerable variation when ESX-1 was deleted. Deletionof ESX-3 resulted in substantial variation to multiple cellular pathways, including amino acid, carbohydrateand fatty acid metabolism and oxidative stress. These and other differences indicate possible perturbedpolyamine metabolism in the absence of ESX-3. Although no ESX-4 protein components were detected inwild type M. smegmatis, the ESX-4 knockout displayed substantial proteomic variation. Reduced levels ofESX-3 component proteins in the ESX-4 knockout suggest that ESX-4 influences ESX-3 expression. Othervariation linked ESX-4 to cell division and molybdenum metabolism.Secretomic analyses of wild-type and ESX knockout M. smegmatis strains were used to search for novelsubstrates of the M. smegmatis ESX secretion systems. No prototype ESX substrates were identified in theculture filtrates, however 10 possible substrates of the ESX-1, -3 and -4 secretion systems, containing thegeneral ESX secretion signal, YxxxD/E, were identified. The functions of some of these proteins correlatewith the ESX functions identified in the proteomic and metabolomic analyses.This study sets the groundwork for future work in understanding the functional roles and expression patternsof these ESX secretion systems and in using global proteomic and metabolomic analyses to understandcellular changes in response to specific signals or genomic changes.

[发布机构] Stellenbosch University