Characterization of the NarS/NarL and KdpD/KdpE Two-Component Signal Transduction Systems in Mycobacterium tuberculosis: Recombinant Protein Expression, Polyclonal Antibody Development, and Construction of Gene Knockout Vectors

Abstract

Mycobacterium tuberculosis survives hostile host environments through two-component regulatory systems (TCSs), including the NarS/NarL and KdpD/KdpE pathways, which play important roles in environmental sensing and bacterial adaptation. This study aimed to characterize these signaling systems by expressing recombinant NarS, NarL, KdpD, and KdpE proteins in Escherichia coli. The proteins were purified using Ni-NTA affinity chromatography, and polyclonal antibodies against NarL and KdpE were generated in rabbits. Antibody specificity and titre were evaluated using ELISA and Western blotting. A suicide delivery vector was also constructed by cloning the downstream homologous region of the NarS/NarL genes into the p2NIL vector for targeted gene knockout in Mycobacterium bovis BCG. The recombinant proteins and antibodies demonstrated high specificity and are suitable for functional studies. The constructed knockout vector provides a valuable tool for investigating gene function through homologous recombination. Overall, this study establishes important molecular resources for understanding bacterial stress adaptation, signal transduction, and virulence regulation in M. tuberculosis, thereby supporting future tuberculosis research and therapeutic target identification.