Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative agent of tuberculosis disease, contains five copies of the ESAT-6
gene cluster, each encoding a dedicated ESX protein secretion system which has been defined as a novel
Type-VII secretion system. The ESX have been implicated in virulence and survival of M. tuberculosis, and
as such present a promising target for novel treatment interventions. This study has investigated the
evolution, regulation, functions and substrates of the ESX secretion systems.
The evolutionary history of the ESX secretion systems was established using in silico and phylogenetic
analyses of the sequenced mycobacteria, closely related actinomycetes and WXG-FtsK clusters from other
bacteria. The ESX-4 gene cluster appears to have evolved with the start of the evolution of the
mycomembrane, followed by the duplication of ESX-3, which marks the evolution of the genus
Mycobacterium. The ESX-1 duplication occurred next, followed by ESX-2 and ESX-5 which occur only in the
slow 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 be
progenitors of the genomic ESX-1 to -5 gene clusters, possibly indicating a plasmid-mediated mechanism of
ESX duplication and evolution.
The promoters expressing the M. tuberculosis ESX-1 to ESX-5 secretion systems were investigated using a
promoter probe assay, and characterised using in silico analyses. Promoters were identified for ESX-1, -2, -3
and -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 contains
three of the ESX secretion systems, ESX-1, 3, and 4. ESX knockout strains of M. smegmatis were generated
and 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. Deletion
of ESX-3 resulted in substantial variation to multiple cellular pathways, including amino acid, carbohydrate
and fatty acid metabolism and oxidative stress. These and other differences indicate possible perturbed
polyamine metabolism in the absence of ESX-3. Although no ESX-4 protein components were detected in
wild type M. smegmatis, the ESX-4 knockout displayed substantial proteomic variation. Reduced levels of
ESX-3 component proteins in the ESX-4 knockout suggest that ESX-4 influences ESX-3 expression. Other
variation 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 novel
substrates of the M. smegmatis ESX secretion systems. No prototype ESX substrates were identified in the
culture filtrates, however 10 possible substrates of the ESX-1, -3 and -4 secretion systems, containing the
general ESX secretion signal, YxxxD/E, were identified. The functions of some of these proteins correlate
with 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 patterns
of these ESX secretion systems and in using global proteomic and metabolomic analyses to understand
cellular changes in response to specific signals or genomic changes. / AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis, die veroorsakende agent van tuberkulose, bevat vyf kopieë van die ESAT-6
geengroep, wat elk 'n toegewyde ESX proteïen sekresiesisteem, omskryf as 'n nuwe Tipe-VII
sekresiestelsel, kodeer. Die ESX sekresiesisteme is betrokke by virulensie en oorlewing van M.
tuberculosis, en is dus belowende teikens vir nuwe behandelings. Hierdie studie het die evolusie, regulasie,
funksies en substrate van die ESX sekresiesisteme ondersoek.
Die evolusionêre geskiedenis van die ESX sekresiesisteme is bepaal met behulp van in silico en
filogenetiese analises van die volgordebepaalde mikobakterieë, nouverwante actinomisete en WXG-FtsK
groepe van ander bakterieë. Die ESX-4 geengroep het saam met die evolusie van die mikomembraan
ontwikkel, gevolg deur die duplisering van ESX-3, wat die evolusie van die genus Mycobacterium merk. Die
ESX-1 duplisering het volgende plaasgevind, gevolg deur ESX-2 en ESX-5, wat slegs in die stadiggroeiende
mikobakterieë voorkom. Vyf addisionele ESX geengroepe is nuut geïdentifiseer in hierdie studie
en is ESX-P1 tot -P5 genoem. Hierdie addisionale ESX groepe is op, of word voorspel om op, plasmied DNS
voor te kom, en mag voorlopers van die genomiese ESX-1 tot -5 geengroepe wees, wat moontlik dui op 'n
plasmied-gemedieërde meganisme van ESX duplisering en evolusie.
Die promoters wat verantwoordelik is vir die uitdrukking van die M. tuberculosis ESX-1 tot ESX-5
sekresiesisteme is ondersoek deur middel van 'n promoter aktiwiteitstoets, en gekarakteriseer deur in silico
analises. Promoters is geidentifiseer vir ESX-1, -2, -3 en -5.
Die funksies van die mikobakteriële ESX sekresiesisteme is ondersoek deur proteomiese, sekretomiese en
metabolomiese analises van die vinnig-groeiende, nie-patogeniese mikobakterium M. smegmatis, wat ESX-
1, -3 en -4 sekresiesisteme besit. ESX uitslaanmutante van M. smegmatis is gegenereer en gebruik in die
vergelykende analises met die wilde-tipe M. smegmatis. ESX-1 is hoogs uitgedruk in wilde-tipe M.
smegmatis, maar geen spesifieke metabolise weë het aansienlike variasie getoon wanneer ESX-1 verwyder
is. Delesie van ESX-3 het gelei tot aansienlike variasie in verskeie sellulêre weë, insluitend aminosuur-,
koolhidraat- en vetsuur-metabolisme en oksidatiewe stres. Hierdie en ander verskille dui op moontlike
versteurde poli-amien metabolisme in die afwesigheid van ESX-3. Hoewel geen ESX-4 proteïenkomponente
opgespoor is in wilde-tipe M. smegmatis nie, vertoon die ESX-4 uitslaanmutant aansienlike proteomiese
variasie. Laer vlakke van ESX-3 proteïne dui daarop dat ESX-4 die uitdrukking van ESX-3 beinvloed. Baie
van die proteomiese variasie kan geassosieer word met verlaagde ESX-3 uitdrukking, maar ander variasie
mag ESX-4 koppel met seldeling en molibdeen metabolisme.
Sekretomiese analises van wilde-tipe en ESX uitslaanmutant M. smegmatis stamme is gebruik om nuwe
substrate van die M. smegmatis ESX sekresiesisteme te identifiseer. Geen prototipe ESX substrate is
geïdentifiseer in die kultuurfiltraat, maar 10 moontlike substrate van die ESX-1, -3 en -4 sekresiesisteme, met
die algemene ESX sekresiesein, YxxxD/E, is geïdentifiseer. Die funksies van sommige van hierdie proteïene
korreleer met die funksies geïdentifiseer in die proteomiese en metabolomiese analises.
Hierdie studie stel die grondslag vir toekomstige werk in die begrip van die funksionele rol en
uitdrukkingspatrone van die ESX sekresiesisteme en in die gebruik van globale proteomiese en
metabolomiese analises om sellulêre veranderinge in reaksie op spesifieke seine of genomiese veranderinge te verstaan. / The National Research Foundation / German Academic Exchange Service (DAAD), / The Harry Crossley Foundation / The Ernst and Ethel Erikson Trust / Stellenbosch University
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/79805 |
| Date | 03 1900 |
| Creators | Newton-Foot, Mae |
| Contributors | Gey van Pittius, Nicolaas Claudius, Warren, Robin Mark, Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Molecular Biology and Human Genetics. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xviii, 176 p. : col. ill. |
| Rights | Stellenbosch University |
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