Functional analysis of the rv1371/2/3 operon in mycobacteria

Mycobacterium tuberculosis is known for its lipid-rich cell wall. Additionally, lipids are also involved in various mycobacterial pathways, such as signaling and energy storage. In this study, using BCG as a model, we aimed to characterize the rv1371/2/3 operon, which is predicted to be involved in lipid production. Western blotting reveals that single nucleotide polymorphisms in BCG Moreau’s rv1371 and rv1373 do not hinder the expression but alter the primary structure of BCG’s Rv1371. Mass spectrometry analysis with the extracted lipids shows that the complemented strains’ (with Mtb or BCG alleles) global profiles are quite similar, but slightly differ from those of the knockout (Δ rv1371/2/ 3) in the negative mode. Functionally, Δ rv1371/2/ 3:: Mtb shows a disadvantage during the early stages of infection in the J774 macrophage model, whereas a significantly higher bacterial load is recovered from cells infected with this strain in later infection stages. Additionally, this strain also shows a higher survival when exposed to SDS compared to the others. These data suggest that this operon may be partially functional in BCG Moreau due to mutations present in the genes, but further experiments are needed to identify the molecule produced from the Mtb allele, which leads to the differential phenotype observed in macrophage infection and SDS resistance assays. IMPORTANCE This study establishes a functional link between a genetic locus and phenotypic modifications in Mycobacterium tuberculosis that may impact the bacillus’s life cycle. We demonstrate that the rv1371/2/3 operon modulates bacterial lipid composition, which in turn affects macrophage uptake and intracellular persistence. Our findings highlight a functional distinction between the pathogenic M. tuberculosis and the attenuated BCG vaccine, providing a model to study how lipid-driven manipulation of host cells alters infection outcomes. Consequently, this operon and its products emerge as potential targets for therapeutic development aimed at disrupting the bacterial mechanisms associated with critical events that culminate in successful infection.