Date of Award

2020-01-01

Degree Name

Doctor of Philosophy

Department

Biological Sciences

Advisor(s)

Hugues Ouellet

Abstract

Mycobacterium tuberculosis (Mtb) is a bacterium that has been able to establish itself throughout evolution and is currently still an ever present threat resulting in approximately 2 million deaths per year worldwide. The ability for Mtb to utilize cholesterol as a carbon source is maintained through transcriptional regulation mechanisms not fully understood. The Rv3574 gene codes for the self-regulated TetR-like transcriptional repressor KstR that controls the expression of large clusters of cholesterol catabolic genes in Mtb. This study aims to provide insights on the regulatory mechanisms of cholesterol catabolism, which could be used as therapeutic target to treat and control Mtb infection. To study the interaction between KstR, and its physiological ligand(s) in vitro, the KstR protein has been expressed and electrophoresis mobility shift assays (EMSA) conducted in the presence or absence of other potential ligands. X-ray crystallographic studies of KstR with its corresponding kstR DNA operator sequence as well as with its effector, 3-oxo-cholest-4-en-26-oyl-CoA ester (3OCh(25S)26-CoA), have been performed to better understand the conformational changes KstR undergoes in physiological conditions. Surface plasmon resonance (SPR) and EMSA studies revealed that the KstR regulator protein binds to the substrate 3OCh(25S)26-CoA with apparent higher affinity than that of its 3-oxo-4-cholestenoic acid precursor and SPR data has shown the ability for KstR to interact with both -S and -R stereoisomers of 3OCh26-CoA. Crystallization of KstR in its ligand bound condition with the 3OCh(25S)26-CoA, as well as bound to the DNA operator, were accomplished showing different orientations of the repressor. The possibility for KstR isoforms exists in Mtb strains that could provide additional signaling functionality. Therefore, an extended KstR protein and mutant derivatives were examined and tested through SPR studies and showed hypothetical phosphorylation at the N-terminus still allows for binding to KstR but at reduced affinity. The characterization of kstR gene deletion studies in CDC1551 strains of Mtb illustrate alterations in growth that demonstrate regulatory aspects under the control of KstR are strain dependent and do not behave in similar manners. Growth curves were performed and showed Mtb clinical isolate CDC1551 ∆kstR growth is much more deficient than that of the WT. Thin layer chromatography experiments displayed how both CDC1551 WT and ∆kstR strains differ in how they metabolize the virulence factor PDIM after kstR deletion CDC1551 ∆kstR produces less PDIM after kstR deletion and complementation was unable to recover the PDIM loss to that of the WT. Proteomic studies for CDC1551 wild-type and the ∆kstR deletion strain showed KstR plays a role in Mtb, apart from cholesterol degradation, with downstream changes in enzyme quantities in various pathways, such as the glyoxylate cycle and PDIM production. In CDC1551, both deletion and over-expression conditions of kstR have shown to cause attenuation at the onset and in persistence with RAW264.7 macrophage infection experiments. ACDC1551 KDKstR strain have been created and has shown similar results to that of kstR deletion in macrophage infection studies.. Western blots, thin layer chromatography, and RAW264.7 macrophage infection studies have shown that the CDC1551 KDKstR strain behaves in the same manner as the ∆kstR strain and a viable model for future mouse infection experimentation. Overall, the data presented provide significant and fundamental knowledge regarding the regulatory effects of KstR deletion and over-expression in Mtb, and distinguishes KstR as viable a drug therapeutic target.

Language

en

Provenance

Received from ProQuest

File Size

115 pages

File Format

application/pdf

Rights Holder

Chenoa D Arico

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Biology Commons

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