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

Tuberculosis (TB), primarily caused by Mycobacterium tuberculosis ( M. tb ) and Mycobacterium bovis ( M. bovis ), 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 M. bovis intracellular survival. This gene was termed NMRAL2_Bovine due to its inclusion of the NmrA domain which has a relation to nitric oxide (NO) production. We used CRISPR/Cas9 to knock out NMRAL2_Bovine in bovine lung epithelial cells and observed a significant decrease in M. bovis -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 M. bovis 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.