Bovine NMRAL2 Protein Blunts Nitric Oxide Production and Inflammatory Response in Mycobacterium bovis Infected Bovine Lung Epithelial Cells

Tuberculosis (TB), primarily caused by <i>Mycobacterium tuberculosis</i> (<i>M. tb</i>) and <i>Mycobacterium bovis</i> (<i>M. bovis</i>), remains the leading cause of death from a single infectious agent globally. Intracellular survival is crucial for their virulence; yet, the underlying mechanisms are not fully understood. This study aimed to demonstrate the significance of a previously unannotated bovine gene ENSBTAG00000011305 in <i>M. bovis</i> intracellular survival. This gene was termed <i>NMRAL2_Bovine</i> due to its inclusion of the NmrA domain which has a relation to nitric oxide (NO) production. We used CRISPR/Cas9 to knock out <i>NMRAL2_Bovine</i> in bovine lung epithelial cells and observed a significant decrease in <i>M. bovis</i>-induced cell death and the intracellular bacterial count, alongside increased NO levels. A transcriptome analysis revealed the upregulation of pathways linked to NO, IL-6, and TNF-α production, which was confirmed by the increased expression of iNOS, IL-6, and TNF-α. Correspondingly, Western blotting indicated that key signaling pathways, including NF-κB and MAPK, were activated. In conclusion, our findings determined that NMRAL2_Bovine functions as a negative regulator of the inflammatory response induced by <i>M. bovis</i> infection at the cellular level and, thereby, provide a novel insight into TB pathogenesis and a potential target for developing novel host-directed therapies against TB.