Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The causative agent of human tuberculosis, Mycobacterium tuberculosis, is an intracellular
pathogen that secretes virulence factors, namely ESAT-6 and CFP-10, as substrates of the
ESX-1 secretion system. It is hypothesised that these substrates interact with host proteins in a
targeted manner in order to elicit a required immune response, and they have been shown to
be involved in processes related to pro-inflammatory responses, necrosis, apoptosis,
membrane lysis and cytolysis. However, the biological function of ESX-1 substrates during
host-pathogen interactions remains poorly and incompletely understood. Therefore, the
present study was designed to gain insight into the role of the ESX-1 secretion system
substrates in host-pathogen interactions and to identify how M. tuberculosis mediates the
response of the human host.
In this study, a cDNA yeast two-hybrid library was constructed from human lung mRNA, to
identify mycobacterial-host protein-protein interactions that occur within the lung alveoli. The
ESX-1 secretion system substrates, ESAT-6 and CFP-10, were cloned in-frame into the
pGBKT7 vector, which was used in the yeast two-hybrid system to screen the lung cDNA
library in Saccharomyces cerevisiae. The ESAT-6 and CFP-10 screens identified 79 and 19
positive colonies, respectively. Of the total number of clones characterised, only two in-frame
inserts were identified with the ESAT-6 screen, corresponding to the human proteins filamin
A and complement component 1, q subcomponent, A chain (C1QA). In addition, the screen
with CFP-10 also identified C1QA as binding partner.
Subsequent in vitro and in vivo experiments were unable to confirm the putative interactions
of C1QA with ESAT-6 and CFP-10. However, the interaction between filamin A and ESAT-6
was demonstrated and confirmed by both in vivo co-localisation and co-immunoprecipitation.
Furthermore, the degradation of filamin A in the presence of ESAT-6 was shown to be
reflective of cytoskeleton remodelling and the induction of cell death. The work presented
here suggests that as ESAT-6 gains access to the cytosol, it initiates cell death by inducing
destabilisation of the cytoskeleton cell structure. This may possibly be driven by the
interaction of ESAT-6 and filamin A.
Finally, we also initiated an investigation of the identified putative binding partners (filamin A and C1QA) as possible genetic markers for genetic susceptibility studies to tuberculosis. A case-control analysis was performed involving 604 cases, of which 109 were Tuberculous
Meningitis (TBM), and 486 were controls from the South African Coloured (SAC) population
within the Ravensmead-Uitsig catchment area. The results of this analysis demonstrated a
novel association of a regulatory variant (rs587585) located upstream of the C1QA gene and
demonstrated an increasing trend towards increased values in tuberculosis patients with the
associated genotype.
This study has contributed significantly to our understanding of human-mycobacterial hostpathogen
protein-protein interactions and has opened the way for future studies further
exploring the consequences and function of the identified ESAT-6-filamin A interaction. It
has also led to the identification of a novel genetic association with tuberculosis. Finally, it
demonstrates the usefulness of the yeast two-hybrid system to identify potential proteinprotein
(host-pathogen) interactions that can lead to additional important and exciting research. / AFRIKAANSE OPSOMMING: Die organisme wat tuberkulose veroorsaak, Mycobacterium tuberculosis, is `n intrasellulȇre
patogeen wat virulensie faktore afskei, naamlik ESAT-6 en CFP-10, as substrate van die
ESX-1 sekresiesisteem. Daar word vermoed dat hierdie substrate met gasheerproteïene in „n
teiken wyse interaksie het om `n vereiste immuunreaksie voort te bring. Hierdie substrate is
betrokke by prosesse soos pro-inflammatoriese reaksies, nekrose, apoptose, membraanlise en
sitolise. Die biologiese funksie van die ESX-1 substrate tydens gasheer-patogeen interaksies
word egter tans swak en onvolledig verstaan. Daarom was die huidige studie ontwerp om
insig te bekom oor die rol hiervan in gasheer-patogeen interaksies en om te identifiseer hoe M.
tuberculosis die reaksie teenoor die gasheer bemiddel.
In hierdie studie was `n komplementȇre deoksiribonukleïensuur (kDNS) gis twee-hibried
biblioteek gemaak vanaf long boodskapper ribonukleïensuur (bRNS) om proteïen-proteïen
interaksies wat in die long plaasvind, te identifiseer. Die substrate van die ESX-1
sekresiesisteem, ESAT-6 en CFP-10, is in volgorde gekloneer in die pGBKT7 vektor en is
gebruik om die long kDNS biblioteek in Saccharomyces cerevisiae te ondersoek. In die soeke
na interaksies met ESAT-6 and CFP-10, was 79 en 19 positiewe kolonies onderskeidelik
geïdentifiseer. Van die aantal klone, was slegs twee volgordes in-leesraam geïdentifiseer met
ESAT-6. Hierdie proteïene het ooreengestem met filamin A en “complement component 1, q
subcomponent, A chain” (C1QA). Bykomend hiertoe, is C1QA ook geïdentifiseer as „n
bindende vennoot met CFP-10.
Daaropvolgende in vitro and in vivo eksperimente kon nie die vermeende interaksie van
C1QA met ESAT-6 en CFP-10 bevestig nie. Maar die interaksie tussen filamin A en ESAT-6
kon wel gedemonstreer word deur die gebruik van mede-lokalisering en medeimunopresipitasie.
Die afbreek van filamin A in die teenwoordigheid van ESAT-6 is ook
aangetoon en blyk „n weerspieëling te wees van sitoskelet hermodellering en die induksie van
seldood. Die werk wat hier aangebied word, dui daarop dat soos ESAT-6 toegang kry tot die
sitosol, inisieër dit seldood deur die destabilisaisie van die sitoskelet selstruktuur. Dit word
moontlik aangedryf deur die interaksie van ESAT-6 met filamin A. Laastens het ons `n ondersoek van die geïdentifiseerde bindingsvennote (filamin A and
C1QA) as moontlike genetiese merkers vir genetiese vatbaarheidsstudies vir tuberkulose
uitgevoer. `n Pasiënt-kontrole studie is gedoen waarby 604 individue ingesluit is, waarvan 109
gediagnoseer is met Tuberculosis Meningitis (TBM), en die ander 486 kontrole individue was
van die Suid Afrikaanse Kleurling (SAC) bevolking binne die Ravenmead-Uitsig
opvanggebied. Die resultate het „n nuwe assosiasie van „n regulerende variant (rs587585) wat
stroomop van die C1QA geen gelokaliseer is, getoon. Hierdie variant het `n verhoogde
neiging in tuberkulose pasiënte met die geassosieërde genotipe getoon.
Hierdie studie het `n beduidende bydrae gemaak tot ons begrip van menslike-mikobakteriese
gasheer-patogeen proteïen-proteïen interaksies. Hierdie resultate het die weg oopgemaak om
die gevolge en funksie van die geïdentifiseerde ESAT-6-filamin A interaksie verder te
ondersoek. Dit het ook aanleiding gegee tot die identifikasie van `n genetiese assosiasie met
tuberkulose. Om saam te vat, hierdie werk bewys die bruikbaarheid van die gis twee-hibriede
sisteem, om potensiële proteïen-proteïen interaksies te ontdek wat die moontlikheid het om
aanleiding te gee tot addisionele navorsingsvrae. / The National Research Foundation, / Harry Crossley Foundation / Medical Research Council of South Africa / Stellenbosch University Postgraduate bursary / Prof. Paul van Helden
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/71977 |
| Date | 12 1900 |
| Creators | Bruiners, Natalie |
| 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 | xxix, 210 p. : col ill., maps |
| Rights | Stellenbosch University |
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