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

[摘要] ENGLISH ABSTRACT: The investigation was aimed to improve the understanding of the binding interactionsbetween DevS and DevR that are implicated in the regulation of the dormancy response inMycobacterium tuberculosis. These binding interactions could provide good drug targets forthe treatment of persistent tuberculosis, the mechanistic understanding of their bindinginteractions is important for the development of a validated inhibitor screen. A detailed insilico analysis of the amino acid residues that play a role in the binding of receptor DevR toboth kinase DevS and the target DNA was undertaken. A reasonable approximation of theDevS structure was produced using homologous protein structures. In silico docking of DevSto DevR merely produced a set of probable candidate structures, since more than oneconformation with similar docked energies was observed. The decision on which one is themore correct form can only be estimated by crystallization of this complex. Therefore, thefunctional expression and purification of the Dev TCS components were pursued. DenaturingHIS™-select nickel affinity gel purification in the form of matrix-assisted refolding led to theproduction of functional Dev TCS proteins. To understand the binding of DevR to DNAconsensus sequences, as well as the nature of these interactions, a model was built of the fulllength DevR dimer binding to DNA consensus sequences. Based on this model, singlemutations were made to DevR in vitro and their effects assessed in order to validate themodel built. During Electrophoretic Mobility Shift Assay (EMSA) analysis, it was found thatK179I 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 tohave the protocols to accurately measure their interaction, in addition to developing afundamental understanding of how their interactions occur. The binding affinity of DevR toboth DevS and the truncated soluble fragment of DevS (DevS201) were explored, using theBIAcore instrument, an SPR-based biosensor. For sufficiently strong binding between ahistidine kinase and a response regulator, the KD needs to be in the nM range. The KD wascalculated to be 255 nM for DevS201 and 184 nM for DevS. Therefore it can be concludedthat DevS201 binds DevR strongly enough to be used in future studies, and that the BIAcorecould be used to screen small-molecule inhibitors of DevR-DevS interactions.

[发布机构] Stellenbosch University