Characterizing the proteomes of selected members of the Mycobacterium tuberculosis complex

[摘要] ENGLISH ABSTRACT: Mycobacterium tuberculosis is a pathogenic organism that infects a third of theworld's population and causes approximately 2 million deaths per year. This pathogen is amember of the Mycobacterium tuberculosis complex (MTBC) which constitutes elevenmembers that share 99.9% similarity at nucleotide level and have near identical 16S rRNA.MTBC members cause Tuberculosis in a variety of host species. M. bovis and M. capraeform part of the animal-adapted MTBC members that cause disease in a variety of animalhosts (primarily bovidae) and goats, respectively. Extensive genetic analyses have been doneto try and explain virulence, phenotype and host-preferences of these members with nosuccess. Recent advances in mass spectrometry techniques enable us to analyse thousands ofproteins simultaneously and explore the possible proteomic variation between these membersthat could contribute to the phenotypic, virulence and host-specificity characteristics of theMTBC members.In this study, we aimed to characterize the proteomes of M. bovis and M. caprae byanalysing the high and or low abundance proteins, relative to M. tuberculosis H37Rv, whichcould possibly explain virulence mechanisms and host-specificity of these MTBC members.Whole cell lysate protein extracts were extracted from mid-log phase cultures ofM. tuberculosis H37Rv (A600 = 0.7), M. bovis (A600 = 0.65) and M. caprae (A600 = 0.7).Proteins were fractionated by SDS-PAGE and in gel reduction/alkylation and trypsin digestswere done. Peptides were identified using LC-MS/MS on the Orbitrap Velos massspectrometer and their corresponding proteins were identified by searching peptide databases.Protein functional groups were assigned according to TubercuList. To provide an integratedoverview of the overall network of protein expression (rather than just limit analysis toindividual proteins), pathway analysis was done on the differentially expressed proteins of M. bovis and M. caprae using PATRIC (Pathosystems Resource Integration Center) andpathways were visualized using iTUBY (Interactive Pathway Explorer database).We detected 2199, 2367 and 2350 proteins for M. tuberculosis H37Rv, M. bovis andM. caprae which correlate to 60% of the proposed M. tuberculosis proteins being expressedduring log-phase. Considering similarities between genomes, it was no surprise that thefunctional distribution of the detected proteins extracted was similar. Metabolic pathwaysaffected by the proteins which were in higher abundance in M. bovis and M. caprae includedamino acid and lipid metabolism, oxidative phosphorylation and xenobiotic degradation. Theover-abundant proteins in M. bovis and M. caprae were also involved in ribosomal proteinsand carbohydrate metabolism, respectively. Lower abundance proteins in these species werefound in lipid and pyrimidine metabolism. These affected pathways can be associated withthe ability of M. bovis and M. caprae to adapt to their environment more readily which helpsthem to survive inside the hosts and cause severe pathogenesis.In this study the proteomes of M. tuberculosis H37Rv, M. bovis and M. caprae werecharacterized and the variation between detected proteins and protein abundances explored inorder to describe differences between these closely related strains. Future research on animaladaptedMycobacterial species will address knowledge gaps that are needed to preventtransmission and spread of the disease. Understanding the mechanisms of virulence andpathogenicity could lead to development of efficient vaccines and diagnostic tests for avariety of animal hosts.

[发布机构] Stellenbosch University