Designing a robust whole-cell biosensor platform for detection of toxic metals using intein splicing inhibition of Mycobacterium tuberculosis SufB protein

Heavy metal contamination has emerged as a persistent environmental and public health challenge. Although existing analytical methods provide high sensitivity, they often require complex instrumentation with limited on-site applications, and generate hazardous waste, raising environmental concerns. At present, whole-cell biosensors provide an empirical alternative, utilizing cellular machinery for sensitive detection of metals. In a relevant context, intein-mediated protein splicing, a spontaneous post-translational process, can be adapted for biosensing by coupling metal-dependent splicing inhibition with whole-cell sensing units. Current research elucidates how toxic metal ions, including cadmium (Cd2+), mercury (Hg2+), chromium (Cr3+), and lead (Pb2+), influence the splicing and N-terminal cleavage reactions of Mycobacterium tuberculosis (Mtb) SufB precursor protein. While Cd2+; and Hg2+; significantly attenuated the splicing activity of Mtb SufB precursor protein over a concentration range of 25 uM to 2 mM, Pb2+; and Cr3+; failed to do so. Next, an innovative biosensor platform was designed to detect these metal ions by utilizing splicing inhibition in metal-sensitive Mtb SufB precursor protein within attenuated Mtb (H37Ra) cells. Metal-induced splicing inhibition led to loss of viability in H37Ra indicator cells, while specificity of this detection system was confirmed by adding respective metal chelators. Multiplexing and quantitative efficacy of the designed biosensor was evaluated by including known splicing inhibitors like Cu2+, Zn2+, and Pt4+ over various concentration range alongside Cd2+; and Hg2+;. The developed biosensor offers a cost-effective, user-friendly, specific, and sensitive assay that does not require advanced instrumentation facility. It can be employed in standard biological laboratories utilizing whole-cell microorganisms harbouring precursor proteins with metal-sensing intein sequences. This approach holds potential for multiplexing metal screening in environmental and industrial effluents, significantly enhancing toxic metal monitoring capabilities.