A versatile plasmid platform for auxotrophic complementation in attenuated Mycobacterium bovis BCG

Mycobacterium bovis BCG is widely used as a vaccine vector due to its safety, genetic stability, and immunomodulatory properties. However, current expression systems for BCG transformation rely on antibiotic resistance genes, raising biosafety concerns. Therefore, alternative strategies—such as the use of auxotrophic strains—are highly desirable. Our group previously employed BioBrick™ technology to construct the pUP500 plasmid series containing various mycobacterial promoters. We engineered these plasmids for the auxotrophic complementation of BCG ΔLeuD, a leucine-auxotrophic strain, using an antibiotic-free and BioBrick™-compatible approach. The auxotrophic complementation cassette pAN-leuD was cloned into pUP500 plasmids. Subsequently, the egfp gene was inserted downstream of the promoters, the kanamycin resistance gene was removed, and fluorescence was assessed by flow cytometry, qPCR and confocal microscopy in BCG grown in 7H9 medium and inside macrophages. Auxotrophic complementation was confirmed by growth of transformants in non-selective medium and analysis of leuD expression, and plasmid stability was evaluated along six BCG generations. Strains harboring plasmids containing the pAN and HspX promoters exhibited stronger fluorescence levels both in vitro and within macrophages. In contrast, the 18 kDa promoter demonstrated marked activation specifically in the intracellular environment, driving robust gene expression inside macrophages. These expression patterns were significantly higher compared to strains carrying the Ag85B or Hsp60 promoters, as well as to the negative control. Except for the Hsp60 construct, all plasmids exhibited robust in vitro stability across six consecutive passages, supporting their potential as reliable expression systems for heterologous antigens in therapeutic and vaccine applications. We developed five BioBrick™-compatible plasmids with distinct promoters for auxotrophic complementation and antigen expression in BCG ΔLeuD, eliminating the need for antibiotic-based selection. This standardized plasmid series reduces vector-related variability in comparative studies and provides a diverse promoter repertoire that may enhance the stability and efficacy of recombinant BCG ΔLeuD strains, representing a valuable advancement for prophylactic and therapeutic vaccine development.