Regulatory mechanisms of the cholesterol catabolism repressor KstR in Mycobacterium tuberculosis

Abstract

There is currently extensive data establishing Mycobacterium tuberculosis (Mtb) uses cholesterol as a carbon and energy source. The ability for Mtb to utilize cholesterol as a carbon source is maintained through transcriptional regulation, yet the mechanisms are not fully understood. The Rv3574 gene encodes for the self‐regulated TetR‐like transcriptional repressor KstR that controls the expression of a large cluster of cholesterol catabolic genes in Mtb. This study aims to provide insight on the regulatory mechanisms of side‐chain cholesterol catabolism and the downstream effect on lipid metabolites, such as phthiocerol pthiocerol dimycocerocate (PDIM), which plays a key role during infection. To study the interaction between KstR, and its physiological ligand(s) in‐vitro, the KstR protein has been expressed in wild‐type and mutant forms and electrophoresis mobility shift assays (EMSA) conducted in the presence or absence of other potential ligands. Surface plasmon resonance (SPR) and X‐ray crystallographic studies of KstR with its corresponding kstR DNA operator sequence as well as with the substrate 3‐oxo‐cholest‐4‐en‐26‐oyl‐CoA ester (25,26‐CoA) have been performed to better understand the conformational changes KstR undergoes in physiological conditions. CDC1551, H37Rv, and Erdman Mtb knockout mutant strains lacking kstR (ΔkstR) and complements have been generated and cultured in the presence of cholesterol. Lipid extractions with these strains and growth conditions have been harvested and analyzed using mass spectrometry. EMSA studies revealed the KstR regulator protein is able to bind to the substrate 25,26‐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 25,26‐CoA. Mutant variants of KstR have shown to interact with the DNA operator in different manners. Crystallization of KstR in its ligand bound condition with the 25,26‐CoA, as well as bound to the DNA operator, were accomplished showing different orientations of the repressor. Lipid analysis shows the production of PDIM to be varied dependent on the individual Mtb strains. The characterization of KstR interaction with its corresponding binding DNA motifs in the presence of produced substrates will provide significant and fundamental knowledge of the regulatory mechanisms of cholesterol metabolic processes for Mtb. The information garnered within this and future research will assess KstR as a potential therapeutic drug target for the treatment of tuberculosis disease.Support or Funding InformationThis research was funded by the University of Texas at El Paso Border Biomedical Research Center Grant Support: NCRR (5G12RR008124) and NIMHHD (8G12MD007592) from NIH and by NIH/NIAID/NIGMS SC1 AI116567‐01A1 to HO. CA was supported by the Louis Stokes Alliance for Minority Participation under Grant No. HRD‐1301858This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.