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A metabolomics approach investigating the functionality of the ESX-1 gene cluster in mycobacteria / Conrad Cilliers SwanepoelSwanepoel, Conrad Cilliers January 2015 (has links)
Tuberculosis (TB) claims the lives of millions of individuals each year, and is
consequently the world’s second-most deadly infectious disease after acquired
immune deficiency syndrome (AIDS), responsible for 1.4 million deaths in 2010
alone. Developing countries carry the heaviest burden, with the occurrence of
multidrug-resistant (MDR) TB becoming more frequent, making more efficient
vaccination and treatment strategies a necessity to combat this epidemic. The ESX-1
gene cluster (encoding the virulence-associated proteins ESAT-6 and CFP-10) and
the Type Vll secretion system are thought to be responsible for the transport of
extracellular proteins across the hydrophobic, and highly impermeable, cell wall of
Mycobacterium, and consequently are thought to play a role in the virulence of this
organism. To date, our understanding of tuberculosis pathophysiology and virulence
has been described primarily using proteomic and genomic approaches.
Subsequently, using the relatively new research approach called metabolomics, and
interpreting the data using systems biology, we aimed to identify new metabolite
markers that better characterise virulence and the proteins involved, more
specifically related to the ESX-1 gene cluster. Using a GCxGC-TOFMS
metabolomics research approach, we compared the varying metabolomes of M.
smegmatis ESX-1 knock-out (ESX-1ms) to that of the wild-type parent strain and
subsequently identified those metabolite markers differing between these strains.
Multivariate and univariate statistical analyses of the analysed metabolome were
used to identify those metabolites contributing most to the differences seen between
the two sample groups. A general increase in various carbohydrates, amino acids
and lipids, associated with cell wall structure and function, were detected in the
ESX-1ms strain relative to the wild-type parent strain. Additionally, metabolites
associated with the antioxidant system, virulence protein formation and energy
production in these mycobacteria, were also seen to differ between the two groups.
This metabolomics investigation is the first to identify the metabolite markers
confirming the role of the ESX-1 gene cluster with virulence and the underlying
metabolic pathways, as well as its associated role with increased metabolic activity,
growth/replication rates, increased cell wall synthesis and an altered antioxidant
mechanism, all of which are believed to contribute to this organism’s increased
pathogenicity and survival ability. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015
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A metabolomics approach investigating the functionality of the ESX-1 gene cluster in mycobacteria / Conrad Cilliers SwanepoelSwanepoel, Conrad Cilliers January 2015 (has links)
Tuberculosis (TB) claims the lives of millions of individuals each year, and is
consequently the world’s second-most deadly infectious disease after acquired
immune deficiency syndrome (AIDS), responsible for 1.4 million deaths in 2010
alone. Developing countries carry the heaviest burden, with the occurrence of
multidrug-resistant (MDR) TB becoming more frequent, making more efficient
vaccination and treatment strategies a necessity to combat this epidemic. The ESX-1
gene cluster (encoding the virulence-associated proteins ESAT-6 and CFP-10) and
the Type Vll secretion system are thought to be responsible for the transport of
extracellular proteins across the hydrophobic, and highly impermeable, cell wall of
Mycobacterium, and consequently are thought to play a role in the virulence of this
organism. To date, our understanding of tuberculosis pathophysiology and virulence
has been described primarily using proteomic and genomic approaches.
Subsequently, using the relatively new research approach called metabolomics, and
interpreting the data using systems biology, we aimed to identify new metabolite
markers that better characterise virulence and the proteins involved, more
specifically related to the ESX-1 gene cluster. Using a GCxGC-TOFMS
metabolomics research approach, we compared the varying metabolomes of M.
smegmatis ESX-1 knock-out (ESX-1ms) to that of the wild-type parent strain and
subsequently identified those metabolite markers differing between these strains.
Multivariate and univariate statistical analyses of the analysed metabolome were
used to identify those metabolites contributing most to the differences seen between
the two sample groups. A general increase in various carbohydrates, amino acids
and lipids, associated with cell wall structure and function, were detected in the
ESX-1ms strain relative to the wild-type parent strain. Additionally, metabolites
associated with the antioxidant system, virulence protein formation and energy
production in these mycobacteria, were also seen to differ between the two groups.
This metabolomics investigation is the first to identify the metabolite markers
confirming the role of the ESX-1 gene cluster with virulence and the underlying
metabolic pathways, as well as its associated role with increased metabolic activity,
growth/replication rates, increased cell wall synthesis and an altered antioxidant
mechanism, all of which are believed to contribute to this organism’s increased
pathogenicity and survival ability. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015
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Molecular Motors of ESX-Type Secretion SystemsRamsdell, Talia Lynn 17 December 2012 (has links)
Tuberculosis is an enormous global health problem. Despite decades of research, the mechanism(s) by which Mycobacterium tuberculosis (Mtb) mediates virulence remains incompletely understood. The ESX-1 secretion system is critical for Mtb to survive and cause disease in vivo, but its primary function and mechanism of action are unclear. The many inherent challenges of working with this slow-growing pathogen often limit the experimental approaches that can be used to address these questions. Thus, we have developed a model system in the nonpathogenic bacterium Bacillus subtilis to study ESX-type secretion systems. Here, we demonstrate that the B. subtilis yuk operon encodes an ESX-type secretion system responsible for the secretion of YukE. Additionally, we demonstrate that the yuk system is active in B. subtilis during conditions of nutrient deprivation and is required for normal biofilm formation. Interestingly, this is similar to our findings that the Mtb ESX-1 system plays dual roles in protein secretion and modulating cell wall integrity. One defining feature of all ESX loci is the presence of an FtsK/SpoIIIE family ATPase. Interestingly, these ATPases have a domain structure unique to ESX-associated ATPases, where each protein contains multiple (2-3) enzymatic domains. We used our B. subtilis system to dissect the mechanism of action of this unique class of motor proteins. We find that the yuk-encoded ATPase YukBA dimerizes to form a hexamer of enzymatic subunits that are differentially required for secretion. Strikingly, we find a unique requirement for rotational symmetry in the nucleotide binding activity of the subunits. Finally, we compared the energy requirements of the Mtb ESX-1 system and the B. subtilis yuk system. We find that these systems have some overlapping ATPase requirements for protein secretion and cell wall integrity/biofilm formation, suggesting that there is a conservation of function among ESX-type systems. We also find that some ATPase domains are differentially required for function between these two systems, which we postulate is due to the split protein architecture of the ESX-1-encoded ATPases. Together, these findings highlight the power of using a B. subtilis model system to understand the function and mechanism of action of ESX-type secretion systems.
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Interplay of human macrophages and Mycobacterium tuberculosis phenotypesRaffetseder, Johanna January 2016 (has links)
Mycobacterium tuberculosis (Mtb) is the pathogen causing tuberculosis (TB), a disease most often affecting the lung. 1.5 million people die annually due to TB, mainly in low-income countries. Usually considered a disease of the poor, also developed nations recently put TB back on their agenda, fueled by the HIV epidemic and the global emergence of drug-resistant Mtb strains. HIV-coinfection is a predisposing factor for TB, and infection with multi-drug resistant and extremely drug resistant strains significantly impedes and lengthens antibiotic treatment, and increases fatality. Mtb is transmitted from a sick individual via coughing, and resident macrophages are the first cells to encounter the bacterium upon inhalation. These cells phagocytose intruders and subject them to a range of destructive mechanisms, aiming at killing pathogens and protecting the host. Mtb, however, has evolved to cope with host pressures, and has developed mechanisms to submerge macrophage defenses. Among these, inhibition of phagosomal maturation and adaptation to the intracellular environment are important features. Mtb profoundly alters its phenotype inside host cells, characterized by altered metabolism and slower growth. These adaptations contribute to the ability of Mtb to remain dormant inside a host during latent TB infection, a state that can last for decades. According to recent estimates, one third of the world’s population is latently infected with Mtb, which represents a huge reservoir for active TB disease. Mtb is also intrinsically tolerant to many antibiotics, and adaptation to host pressures enhances tolerance to first-line TB drugs. Therefore, TB antibiotic therapy takes 6 to 9 months, and current treatment regimens involve a combination of several antibiotics. Patient noncompliance due to therapeutic side effects as well as insufficient penetration of drugs into TB lesions are reasons for treatment failure and can lead to the rise of drug-resistant populations. In view of the global spread of drug-resistant strains, new antibiotics and treatment strategies are urgently needed. In this thesis, we studied the interplay of the primary host cell of Mtb, human macrophages, and different Mtb phenotypes. A low-burden infection resulted in restriction of Mtb replication via phagolysosomal effectors and the maintenance of an inactive Mtb phenotype reminiscent of dormant bacteria. Macrophages remained viable for up to 14 days, and profiling of secreted cytokines mirrored a silent infection. On the contrary, higher bacterial numbers inside macrophages could not be controlled by phagolysosomal functions, and intracellular Mtb shifted their phenotype towards active replication. Although slowed mycobacterial replication is believed to render Mtb tolerant to antibiotics, we did not observe such an effect. Mtb-induced macrophage cell death is dependent on ESAT6, a small mycobacterial virulence factor involved in host cell necrosis and the spread of the pathogen. Although well-studied, the fate of ESAT6 inside infected macrophages has been enigmatic. Cultivation of Mtb is commonly carried out in broth containing detergent to avoid aggregation of bacilli due to their waxy cell wall. Altering cultivation conditions revealed the presence of a mycobacterial capsule, and ESAT6 situated on the mycobacterial surface. Infection of macrophages with this encapsulated Mtb phenotype resulted in rapid ESAT6-dependent host cell death, and ESAT6 staining was lost as bacilli were ingested by macrophages. These observations could reflect the earlier reported integration of ESAT6 into membranes followed by membrane rupture and host cell death. In conclusion, the work presented in this thesis shows that the phenotype of Mtb has a significant impact on the struggle between the pathogen and human macrophages. Taking the bacterial phenotype into account can lead to the development of drugs active against altered bacterial populations that are not targeted by conventional antibiotics. Furthermore, deeper knowledge on Mtb virulence factors can inform the development of virulence blockers, a new class of antibiotics with great therapeutic potential.
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Investigating the Human-M. tuberculosis interactome to identify the host targets of ESAT-6 and other mycobacterial antigensBruiners, Natalie 12 1900 (has links)
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
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