The Tuberculosis Necrotizing Toxin kills macrophages by hydrolyzing NAD

Mycobacterium tuberculosis (Mtb) induces necrosis of infected cells to evade immune responses. Recently, we found that Mtb utilizes the protein CpnT to kill human macrophages by secreting its C-terminal domain, named tuberculosis necrotizing toxin (TNT) that induces necrosis by an unknown mechanism. Here we show that TNT gains access to the cytosol of Mtb-infected macrophages, where it hydrolyzes the essential co-enzyme nicotinamide adenine dinucleotide (NAD(+)). Expression or injection of a non-catalytic TNT mutant showed no cytotoxicity in macrophages or zebrafish zygotes, respectively, demonstrating that the NAD(+)-glycohydrolase activity is required for TNT-induced cell death. To prevent self-poisoning, Mtb produces an immunity factor for TNT (IFT) that binds TNT and inhibits its activity. The crystal structure of the TNT-IFT complex revealed a novel NAD(+)-glycohydrolase fold of TNT, which constitutes the founding member of a toxin family wide-spread in pathogenic microorganisms.