Regulators of dormancy/viability of Mycobacterium tuberculosis inside the human macrophages

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dc.contributor.advisor Wiid, I. J. F. en_ZA
dc.contributor.advisor Kenyon, C. P. en_ZA
dc.contributor.author Botha, Maria Magdalena en_ZA
dc.contributor.other Stellenbosch University. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. en_ZA
dc.date.accessioned 2012-01-23T09:09:19Z en_ZA
dc.date.accessioned 2012-03-30T10:42:25Z
dc.date.available 2012-01-23T09:09:19Z en_ZA
dc.date.available 2012-03-30T10:42:25Z
dc.date.issued 2012-03 en_ZA
dc.identifier.uri http://hdl.handle.net/10019.1/20099
dc.description Thesis (PhD)--Stellenbosch University, 2012. en_ZA
dc.description.abstract ENGLISH ABSTRACT: The investigation was aimed to improve the understanding of the binding interactions between DevS and DevR that are implicated in the regulation of the dormancy response in Mycobacterium tuberculosis. These binding interactions could provide good drug targets for the treatment of persistent tuberculosis, the mechanistic understanding of their binding interactions is important for the development of a validated inhibitor screen. A detailed in silico analysis of the amino acid residues that play a role in the binding of receptor DevR to both kinase DevS and the target DNA was undertaken. A reasonable approximation of the DevS structure was produced using homologous protein structures. In silico docking of DevS to DevR merely produced a set of probable candidate structures, since more than one conformation with similar docked energies was observed. The decision on which one is the more correct form can only be estimated by crystallization of this complex. Therefore, the functional expression and purification of the Dev TCS components were pursued. Denaturing HIS™-select nickel affinity gel purification in the form of matrix-assisted refolding led to the production of functional Dev TCS proteins. To understand the binding of DevR to DNA consensus sequences, as well as the nature of these interactions, a model was built of the full length DevR dimer binding to DNA consensus sequences. Based on this model, single mutations were made to DevR in vitro and their effects assessed in order to validate the model built. During Electrophoretic Mobility Shift Assay (EMSA) analysis, it was found that K179I and N183L mutants prevented the binding of DevR to the DNA consensus sequences. If DevR and DevS binding are to be used in a drug development program, it is essential to have the protocols to accurately measure their interaction, in addition to developing a fundamental understanding of how their interactions occur. The binding affinity of DevR to both DevS and the truncated soluble fragment of DevS (DevS201) were explored, using the BIAcore instrument, an SPR-based biosensor. For sufficiently strong binding between a histidine kinase and a response regulator, the KD needs to be in the nM range. The KD was calculated to be 255 nM for DevS201 and 184 nM for DevS. Therefore it can be concluded that DevS201 binds DevR strongly enough to be used in future studies, and that the BIAcore could be used to screen small-molecule inhibitors of DevR-DevS interactions. en_ZA
dc.description.abstract AFRIKAANSE OPSOMMING: Die Dev twee komponent sisteem (TKS) bestaan uit ‘n histidine kinase naamlik (DevS) en ‘n reaksie reguleerder DevR. DevS en DevR is betrokke by die regulering van die dormante stadium van Mycobacterium tuberculosis. Hierdie meganisme kan ‘n deurbraak dwelm teiken vir die behandeling van sluimerende tuberkulose wees. Die meganisme van hierdie bindings interaksies is van kritieke belang, tesame met die ontwikkeling van 'n erkende inhibeerder toets. ‘n Gedetaileerde in silico analise van die aminosuur volgordes wat 'n rol speel in die binding van die reseptor DevR aan beide DevS sowel as die teiken DNS is voltooi. ‘n Model van die DevS struktuur is saamgstel met behulp van homoloë proteïen strukture. In silico mering van DevS aan DevR het `n stel van die waarskynlike kandidaat strukture verskaf, aangesien meer as een konformasie met soortgelyke merings energieë waargeneem is. Die mees waarskynlike vorm kan alleenlik geïndentifiseer word na kristallisasie van hierdie kompleks. Die funksionele uitdrukking en suiwering van die Dev TKS proteine is gevolglik uitgevoer. Funksionele Dev TKS proteïene is verkry deur denaturerende HIS-select nikkel affiniteit jel suiwering, in die vorm van matriks-geassisteerde hervouing te gebruik. Ten einde die binding te verstaan tussen DevR en DNS konsensus volgordes, sowel as die aard van hierdie interaksies, is 'n model gebou van die volle lengte DevR dimeer binding aan DNS konsensus volgordes. Hierdie model is gevalideer deur punt mutasies in DevR te skep en die gevolge daarvan te beoordeel met elektroforetiese mobiliteits verskuiwing reaksie analises. Dit is bevind dat K179I en N183L mutante, verhoed die binding van DevR aan die DNS konsensus volgordes. Die gebruik van DevR en DevS bindings in ‘n dwelm ontwikkelingsprogram, benodig die fundamentele begrip van hoe die interaksies plaasvind, sowel as akkurate protokolle om die interaksies te meet. Die BIAcore instrument, ’n SPR-gebaseerde biosensor, is ingespan om die bindings affiniteit van DevR aan beide DevS en die fragment van DevS (DevS201) te ondersoek. Om voldoende sterk binding tussen DevS en die DevR te verseker, moet die KD in die nM omgewing wees. Die KD is bepaal as 255 nM en 184 nM vir DevS201 en DevS, onderskeidelik. Die afleiding kan dus gemaak word dat DevS201 sterk genoeg aan DevR bind om in verdere studies gebruik te kan word, en dat die BIAcore gebruik kan word om klein-molekule inhibeerders van DevR-DevS interaksies te toets. af_ZA
dc.format.extent 136 p. : ill. en_ZA
dc.language.iso en_ZA en_ZA
dc.publisher Stellenbosch : Stellenbosch University en_ZA
dc.subject TB en_ZA
dc.subject Mycobacterium tuberculosis en_ZA
dc.subject Tuberculosis en_ZA
dc.subject Interaction between DevS and DevR en_ZA
dc.subject Amino acid residues en_ZA
dc.subject Dissertations -- Medical biochemistry en_ZA
dc.subject Theses -- Medical biochemistry en_ZA
dc.title Regulators of dormancy/viability of Mycobacterium tuberculosis inside the human macrophages en_ZA
dc.type Thesis en_ZA
dc.rights.holder Stellenbosch University en_ZA


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