Regulatory mechanism of cholesterol transport via LtmA in Mycobacterium smegmatis

Mycobacterium tuberculosis, the causative agent of tuberculosis, is responsible for over a million deaths annually. This pathogen survives within macrophage phagosomes, exploiting cholesterol as a primary carbon and nutrient source. Cholesterol is a crucial metabolite for M. tuberculosis, contributing to energy production, cell wall integrity, immune modulation, and drug resistance. The Mammalian Cell Entry (MCE) protein family, in which the mce4 operon is particularly pivotal, facilitates cholesterol transport. This study investigates the regulatory effects of the transcription factor LtmA on the mce4 operon in Mycobacterium smegmatis, a model organism closely related to M. tuberculosis. LtmA, which possesses a unique TetR-type HTH domain, targets genes associated with lipid transport and metabolism. Using bioinformatics and Electrophoretic Mobility Shift Assay (EMSA), we identified and validated LtmA binding sequences within the mce4 regulatory region. Functional studies using ltmA knockdown and overexpression mutants revealed that LtmA acts as a repressor of the mce4 operon, thereby reducing cholesterol transport and impairing the intracellular survival of M. smegmatis within macrophages under cholesterol-dependent growth conditions. This work advances our knowledge of M. tuberculosis survival strategies, contributing significantly to global tuberculosis control efforts.