Proximity‐dependent biotin identification links cholesterol catabolism with branched‐chain amino acid degradation in Mycobacterium smegmatis

Abstract

AbstractCholesterol is a crucial component in Mycobacterium tuberculosis virulence as it is required for phagocytosis of mycobacteria by macrophages. In addition, the tubercle bacilli can grow using cholesterol as the sole carbon source. Thus, cholesterol catabolism represents a valuable target for the development of new antitubercular drugs. However, the molecular partners of cholesterol catabolism remain elusive in mycobacteria. Here, we focused on HsaC and HsaD, enzymes involved in two consecutive steps of cholesterol ring degradation and identified putative partners, using a BirA‐based proximity‐dependent biotin identification (BioID) approach in Mycobacterium smegmatis. In rich medium, the fusion protein BirA‐HsaD was able to fish the endogenous cognate HsaC, thus validating this approach to study protein–protein interactions and to infer metabolic channeling of cholesterol ring degradation. In chemically defined medium, both HsaC and HsaD interacted with four proteins, BkdA, BkdB, BkdC, and MSMEG_1634. BkdA, BkdB, and BkdC are enzymes that participate in the degradation of branched‐chain amino acids. As cholesterol and branched‐chain amino acid catabolism both generate propionyl‐CoA, which is a toxic metabolite for mycobacteria, this interconnection suggests a compartmentalization to avoid dissemination of propionyl‐CoA into the mycobacterial cytosol. Moreover, the BioID approach allowed us to decipher the interactome of MSMEG_1634 and MSMEG_6518, two proteins of unknown function, which are proximal to the enzymes involved in cholesterol and branched‐chain amino acid catabolism. In conclusion, BioID is a powerful tool to characterize protein–protein interactions and to decipher the interconnections between different metabolic pathways, thereby facilitating the identification of new mycobacterial targets.