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Investigating the impacts of resistance mutations in Mycobacterium tuberculosis in order to improve the selection of new drug targets



Change log


Munir, Asma 


Drug-resistant tuberculosis (TB), one of the leading causes of death worldwide, arises mainly from spontaneous mutations in the genome of Mycobacterium tuberculosis (M. tb). There is an urgent need to understand the mechanism by which the mutations confer resistance in order to design new drugs to reduce its emergence in the future. The present study involves analyses of drug-resistant mutations in M. tb using both computational and experimental approaches.

In Chapter 2 in-silico analyses of mutations linked to isoniazid (INH) and rifampicin (RIF) resistance were performed in seven proteins. These proteins included the catalase-peroxidase (KatG), Enoyl-Acyl Carrier Protein reductase (InhA), β-ketoacyl ACP synthase (KasA), alkyl hydroxide reductase (AhpC) and NADH dehydrogenase (Ndh) for INH resistance and RpoB and RpoC for RIF resistance. The impacts of mutations on the structure and function of these proteins were predicted using software developed in the Blundell group, including impacts on stability using SDM, a statistical approach, and mCSM, a machine learning approach, while the effects of mutations on protein-protein interactions and protein-ligand affinity were predicted using mCSM-PPI and mCSM-lig respectively. The models of mutants were built using Modeller, and the atomic interactions of wild-type and mutant residues were analysed by Intermezzo and Arpeggio. Around 80% of the mutations that were analysed were predicted to be destabilizing for the respective protein. Mutations were found predominantly to target the active sites and interfaces of the proteins, and 100 % of the mutations present at the interface of the studied proteins were predicted to be destabilizing for protein-protein interactions by mCSM-PPI. Moreover, all the mutations present in the active sites of the proteins were predicted to decrease the affinity of the ligand for the protein.

KatG (a heme-dependent catalase peroxidase) and two KatG mutants W107R and T275P were selected for experimental analyses. KatG activates the prodrug INH and mutations in KatG are likely to be the first step in conferring resistance against the drug. The structures of wild-type KatG protein (with and without the addition of INH) were obtained using X-ray crystallography and cryo-EM and potential INH binding sites in KatG were identified using cryo-EM and illustrated in Chapter 4. The structure of KatGINH revealed the dynamic binding ability of INH and the power of cryo-EM as a technique to visualise small drug molecules

Two KatG mutants, W107R and T275P were created in Chapter 5 and the structures were obtained using cryo-EM. In combination with cryo-EM, the wild-type and mutant proteins were also characterised using UV-visible spectroscopy, circular dichroism and biochemical methods. The analyses revealed that the variants cause disorder in the heme environment preventing heme uptake and retention by the KatG protein likely to be the cause of INH resistance. This study helped in providing new insights into INH resistance caused by resistance mutations in KatG.





Blundell, Tom


Tuberculosis, Mutations, Cryo-EM


Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge
Cambridge Commonwealth, European and International Trust