Characterizing the proteomes of selected members of the Mycobacterium tuberculosis complex

Botha, Louise (2014-04)

Thesis (MScMedSC)--Stellenbosch University, 2014.

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. This pathogen is a member of the Mycobacterium tuberculosis complex (MTBC) which constitutes eleven members 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. caprae form part of the animal-adapted MTBC members that cause disease in a variety of animal hosts (primarily bovidae) and goats, respectively. Extensive genetic analyses have been done to try and explain virulence, phenotype and host-preferences of these members with no success. Recent advances in mass spectrometry techniques enable us to analyse thousands of proteins simultaneously and explore the possible proteomic variation between these members that could contribute to the phenotypic, virulence and host-specificity characteristics of the MTBC members. In this study, we aimed to characterize the proteomes of M. bovis and M. caprae by analysing the high and or low abundance proteins, relative to M. tuberculosis H37Rv, which could possibly explain virulence mechanisms and host-specificity of these MTBC members. Whole cell lysate protein extracts were extracted from mid-log phase cultures of M. 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 digests were done. Peptides were identified using LC-MS/MS on the Orbitrap Velos mass spectrometer and their corresponding proteins were identified by searching peptide databases. Protein functional groups were assigned according to TubercuList. To provide an integrated overview of the overall network of protein expression (rather than just limit analysis to individual proteins), pathway analysis was done on the differentially expressed proteins of M. bovis and M. caprae using PATRIC (Pathosystems Resource Integration Center) and pathways were visualized using iTUBY (Interactive Pathway Explorer database). We detected 2199, 2367 and 2350 proteins for M. tuberculosis H37Rv, M. bovis and M. caprae which correlate to 60% of the proposed M. tuberculosis proteins being expressed during log-phase. Considering similarities between genomes, it was no surprise that the functional distribution of the detected proteins extracted was similar. Metabolic pathways affected by the proteins which were in higher abundance in M. bovis and M. caprae included amino acid and lipid metabolism, oxidative phosphorylation and xenobiotic degradation. The over-abundant proteins in M. bovis and M. caprae were also involved in ribosomal proteins and carbohydrate metabolism, respectively. Lower abundance proteins in these species were found in lipid and pyrimidine metabolism. These affected pathways can be associated with the ability of M. bovis and M. caprae to adapt to their environment more readily which helps them to survive inside the hosts and cause severe pathogenesis. In this study the proteomes of M. tuberculosis H37Rv, M. bovis and M. caprae were characterized and the variation between detected proteins and protein abundances explored in order to describe differences between these closely related strains. Future research on animaladapted Mycobacterial species will address knowledge gaps that are needed to prevent transmission and spread of the disease. Understanding the mechanisms of virulence and pathogenicity could lead to development of efficient vaccines and diagnostic tests for a variety of animal hosts.

AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis is 'n patogene organisme wat 'n derde van die wêreld se bevolking infekteer en veroorsaak ongeveer 2 miljoen sterftes per jaar. Hierdie patogeen is 'n lid van die Mycobacterium tuberculosis kompleks (MTBK) wat bestaan uit elf lede wat 99,9% ooreenkoms op nukleotiedvlak toon en amper identiese 16S rRNA deel. MTBK lede veroorsaak Tuberkulose in 'n verskeidenheid van gasheerspesies. M. bovis en M. caprae vorm deel van die MTBK en veroorsaak Tuberkulose in 'n verskeidenheid van diere-gashere (hoofsaaklik Bovidae) en bokke, onderskeidelik. Verskeie genetiese ontledings is al gedoen om virulensie, fenotipe en gasheer-voorkeure van hierdie lede te ondersoek, maar was onsuksesvol. Die onlangse vooruitgang in massa-spektrometriese tegnieke stel ons in staat om duisende proteïene gelyktydig te analiseer en die moontlike proteomiese variasie tussen hierdie lede te identifiseer. Proteomiese analises kan bydra tot die fenotipiese-, virulensie- en gasheer-spesifieke eienskappe van die hierdie lede. Die doel met hierdie studie was om die proteome van M. bovis en M. caprae te beskryf deur die proteïene te identifiseer wat differentieel uitgedruk was, in vergelyking met M. tuberculosis H37Rv, wat moontlik die virulensie meganismes en gasheer-spesifisiteit van hierdie MTBK lede kan verduidelik. Proteïen ekstraksies is geneem uit die middellogaritmiese groeifase van M. tuberculosis H37Rv (A600 = 0.7), M. bovis (A600 = 0,65) en M. caprae (A600 = 0,7) kulture. Proteïene is gefraksioneer deur SDS-PAGE en in-jel vermindering/alkilering en tripsien vertering is gedoen. Peptiede is geïdentifiseer met behulp van LC-MS/MS op die Orbitrap Velos massa-spektrometer en die ooreenstemmende proteïene is geïdentifiseer. Proteïen funksionele groepe is toegeken aan proteïene volgens TubercuList. Om ‘n geïntegreerde oorsig van die totale netwerk van die proteïen uitdrukking te gee (eerder as ontleding van slegs individuele proteïene), is metaboliese weë analises op die differensieel uitgedrukte proteïene van M. bovis en M. caprae gedoen, deur gebruik te maak van PATRIC (Pathosystems Resource Integration Center). Metaboliese weë is gevisualiseer deur iTUBY (Interactive Pathway Explorer databasis). ‘n Totaal van 2199, 2367 en 2350 proteïene is ontdek vir M. tuberculosis H37Rv, M. bovis en M. caprae onderskeidelik, wat ooreenstem met 60% van die voorgestelde M. tuberculosis proteïene. A.g.v. genoom ooreenkomste, was dit geen verrassing dat die funksionele verspreiding van die proteïene soortgelyk was nie. Metaboliese weë wat geraak word deur die proteïene wat in hoë-oorvloed in M. bovis en M. caprae ontdek is, sluit die aminosuur- en lipiedmetabolisme, oksidatiewe fosforilering en xenobiotiese afbreking in. Die hoë-oorvloed proteïene in M. bovis en M. caprae is ook betrokke by ribosomale funksies en koolhidraatmetabolisme, onderskeidelik. Proteiene wat in laer-oorvloed in hierdie twee spesies geidentifiseer is, speel ‘n rol in lipied- en pirimidienmetabolisme Geaffekteerde metaboliese weë kan geassosieer word met die vermoë van M. bovis en M. caprae om meer geredelik by hul omgewing aan te pas wat die organimses help om te oorleef in die gasheer en patogenese te ontwikkel. In hierdie studie is die proteome van M. tuberculosis H37Rv, M. bovis en M. caprae beskryf en die variasie tussen die ontdekte proteïene en proteïen verspreidings ontleed om die verskille tussen hierdie nou verwante spesies te beskryf. Toekomstige navorsing op diereaangepaste mikobakteriële spesies sal die kennisgapings oorbrug wat nodig is om die oordrag en verspreiding van Tuberkulose te voorkom. Begrip van die meganismes van virulensie en patogenisiteit kan lei tot die ontwikkeling van doeltreffende entstowwe en diagnostiese toetse vir 'n verskeidenheid van diere-gashere.

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