Thesis (PhD)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: This study exploits the Mycobacterium tuberculosis H37Rv genome sequence data in
the context of M. tuberculosis clinical isolates, to elucidate genetic variation, and examine the
phenotypic and molecular epidemiological implications thereof.
The study was initiated by investigation of the insertion sequence IS6110, the primary
DNA fingerprinting probe for the molecular epidemiology of tuberculosis. The transposable
element is present in variable copy number and chromosomal location in clinical isolates of
M. tuberculosis strains, giving rise to extensive genetic diversity. At the inception of this
study, little was known about this element in terms of the genetic identity of its surrounding
regions, its chromosomal distribution, and the mechanisms contributing to genetic diversity.
These shortcomings were therefore addressed by a number of approaches.
Firstly, to establish their genetic identity and chromosomal distribution, IS6110
insertion sites from clinical isolates of M. tuberculosis were cloned and sequenced. This data
was examined in conjunction with available genome sequence data. The results demonstrated
that the majority of insertions occurred within coding regions. Furthermore, the element was
shown to have a non-random chromosomal distribution, and a number of preferential
integration sites were identified. Secondly, the stability of chromosomal domains flanking
IS611 0 elements was investigated by utilizing the insertion site clones as hybridization probes
against clinical isolates. This allowed the identification of extensive genetic variation
associated with these chromosomal domains, arising from IS6110 transpositions, deletions
and point mutations. These events were expressed in terms of a phylogenetic tree which
demonstrated ongoing genome evolution associated with IS6110. Thirdly, to investigate the
hypothesis that IS6110-mediated deletions occur via homologous recombination between
adjacent elements, deletion junctions were mapped and sequenced in clinical isolates
representing predecessor and descendant strains. While these results support the involvement
of IS6110 as a mediator of genetic deletion, they suggest either alternative mechanisms or the
existence of unidentified intermediates.
The investigation of IS6110 flanking regions identified the disruption of a number of
members of the PPE gene family, leading to the second main area of investigation. The PPE
gene family was newly identified as a result of the M. tuberculosis genome sequencing
project, and its products are speculated to be of antigenic importance. However, at the
commencement of this study very little data was available regarding the biological role of
PPE proteins. Therefore, to explore the phenotypic implications of PPE gene disruption, various aspects of the gene family were investigated.
Firstly, phylogenetic relationships between members of the PPE family were
elucidated, which suggested an evolutionary progression, and highlighted the possibility that
there may be functional subdivisions within the gene family. Secondly, the extent and
mechanisms of PPE gene variation were analyzed by a combination of hybridization, peR
and sequence analysis. This approach revealed extensive variation associated the gene family,
although different members of the family exhibit different levels of variation. Of special
interest was the discovery that long tandem repeat regions (~69 bp) found within 3 members
of the gene family demonstrate variation in the numbers of these tandem repeats. A third
avenue of investigation focused on in vitro and in vivo PPE gene expression profiles. RT-
, peR was utilized to demonstrate in vitro expression of PPE genes, while RNA:RNA in situ
hybridization demonstrated the expression of PPE genes in human tissue samples.
Intriguingly, in situ hybridization suggests that there is variable PPE gene expression within
the human granuloma. The final approach reported here focused on the subcellular
localization of one member of the PPE family, Rv1917c. A combination of cell fractionation
and whole-cell antibody binding experiments suggest that the Rv 1917c protein is a cell wallassociated,
surface exposed molecule.
In summary, the results obtained have potential implications for the interpretation of
molecular epidemiological data, support the role of IS6110 as an agent of genome evolution,
and emphasize the potential for IS6110 to impact on strain phenotype. Investigation of the
PPE family demonstrated that this gene family contributes to genetic variation, is expressed in
vitro and in vivo and that at least one protein encoded by the gene family is cell wall
associated. Together, the results obtained support the hypothesis that selected members of the
PPE gene family may encode products involved in antigenic variation. / AFRIKAANSE OPSOMMING: Dié studie maak gebruik van die Mycobacterium tuberculosis H37Rv genoom
volgorde data in die konteks van M. tuberculosis kliniese isolate, om genetiese variasie toe te
lig en die fenotipiese en molekulêre epidemiologiese implikasies daarvan te ondersoek.
Die studie het 'n aanvang geneem deur die ondersoek van die inset-volgorde /S6110,
wat die primêre DNS vingerafdruk pylfragment vir die molekulêre epidemiologie van
tuberkulose is. Hierdie transponerende element is in wisselende kopiegetal en chromosomale
posisies teenwoordig in kliniese isolate van M. tuberculosis stamme, en gee so oorsprong aan
omvangryke genetiese afwisseling. Met die aanvang van hierdie studie was min bekend
omtrent hierdie element betreffende die genetiese identiteit van die areas wat die insetsels
omring, die chromosomale distribusie van insetsels, asook die meganismes wat bydra tot
genetiese afwisseling. Hierdie gebreke is dus deur 'n aantal benaderings aangespreek.
Eerstens is IS6110 insettingsetels van kliniese M. tuberculosis isolate gekloneer en hul
nukleotiedvolgorde bepaal om sodoende hul genetiese identiteit en chromosomale
verspreiding vas te stel. Hierdie data is in oorleg met beskikbare genomiese volgorde data
geanaliseer. Die resultate het gewys dat die meerderheid van insetsels binne koderende
gebiede plaasgevind het. Verder is gewys dat hierdie element nie na willekeur deur die
chromosoom versprei is nie, en 'n aantal gebiede waar insetting by voorkeur plaasvind, is
geïdentifiseer. Tweedens is die stabiliteit van die chromosomale gebiede wat IS6110
elemente flankeer ondersoek deur die insettingsetel klone as pylfragmente te gebruik in
hibridisasie van kliniese isolate. Dit het die identifisering van omvangryke genetiese
afwisseling binne hierdie chromosomale gebiede, wat ontstaan deur IS611 0 transposisies,
delesies en puntmutasies, tot gevolg gehad. Hierdie afwisselings is uitgedruk as 'n
filogenetiese boom waarin die voortdurende genomiese evolusie wat geassosieer word met
IS6110 gewys word. Derdens, om die teorie dat IS6110-gedrewe delesies deur middel van
homoloë rekombinasie tussen naasliggende elemente plaasvind te ondersoek, is die grense
van delesies gekarteer en die nukleotiedvolgorde daarvan bepaal in kliniese isolate wat
voorganger- en afstammelingstamme verteenwoordig. Alhoewel die resultate die
betrokkenheid van IS6110 as 'n bemiddelaar van genetiese delesie ondersteun, stel dit ook die
bestaan van of alternatiewe meganismes of van onbekende intermediêre vorme voor.
Ondersoek van die IS6110-flankerende gebiede het gelei tot die ontdekking van
ontwrigting van 'n aantal gene wat behoort tot die PPE geenfamilie, en het so gelei tot die
tweede hoof ondersoek tema. Die PPE geenfamilie is ontdek as gevolg van die M. tuberculosis genoomprojek, en dit word gespekuleer dat die produkte van hierdie gene van
antigeniese belang mag wees. Daar was egter met die aanvang van hierdie studie baie min
data beskikbaar omtrent die biologiese rol van die PPE proteïene. Om die fenotipiese
implikasies van ontwrigting van PPE gene te ondersoek is daar dus ondersoek ingestel na
verskeie aspekte van hierdie geenfamilie.
Eerstens is filogenetiese verwantskappe tussen lede van die PPE familie bepaal, wat
gedui het op 'n evolusionêre progressie en wat ook aangedui het dat daar moontlik
funksionele onderverdelings binne hierdie geenfamilie mag bestaan. Tweedens is die omvang
en meganismes van PPE geenvariasie geanaliseer deur 'n kombinasie van hibridisasie, PKR
en nukleotiedvolgorde analise. Hierdie benadering het omvangryke afwisseling binne hierdie
geenfamilie getoon, alhoewel verskillende lede van die familie verskillende vlakke van
afwisseling demonstreer. Wat veral interessant was, was die ontdekking dat lang tandem
herhalingsvolgordes (~69 bp) wat in 3 lede van hierdie geenfamilie voorkom, variasie toon in
die getalle van hierdie tandem herhalingsvolgordes. 'n Derde been van ondersoek het gefokus
op in vitro en in vivo PPE geen uitdrukkingsprofiele. RT-PKR is gebruik om te toon dat PPE
gene in vitro uitgedruk word, terwyl RNA:RNA in situ hibridisasie getoon het dat PPE gene
ook in menslike weefsel uitgedruk word. Interessant genoeg dui in situ hibridisasie daarop
dat daar wisselende PPE geen uitdrukking binne die menslike granuloom voorkom. Die
laaste benadering wat hier gerapporteer word fokus op die sub-sellulêre lokalisering van een
lid van die PPE familie, Rv1917c. 'n Kombinasie van selfraksionering en heel-sel
antiliggaam-bindingseksperimente dui daarop dat Rv1917c 'n selwand-geassosieerde
molekuul is wat aan die oppervlak blootgestel word.
Ter opsomming het die resultate wat bereik IS potensiële implikasies vir die
interpretasie van molekulêr-epidemiologiese data, dit ondersteun die rol van IS6110 as 'n
bemiddelaar van genoom evolusie en beklemtoon die potensiaal vir IS6110 om 'n invloed te
hê op die fenotipe van die stam. Ondersoek van die PPE familie het getoon dat hierdie
geenfamilie bydra tot genetiese afwisseling, dat dit uitgedruk word beide in vitro en in vivo en
dat ten minste een lid van hierdie geenfamilie geassossieer word met die selwand. Tesame
ondersteun hierdie resultate die teorie dat geselekteerde lede van die PPE geenfamilie wel
produkte enkodeer wat betrokke is by antigeniese variasie.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52948 |
| Date | 03 1900 |
| Creators | Sampson, Samantha Leigh |
| Contributors | Warren, R. M., Van Helden, P. D., Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 252 p. : ill. |
| Rights | Stellenbosch University |
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