NAD+ Depletion Triggers Macrophage Necroptosis, a Cell Death Pathway Exploited by Mycobacterium tuberculosis

Abstract

SUMMARY Mycobacterium tuberculosis (Mtb) kills infected macrophages by inhibiting apoptosis and promoting necrosis. The tuberculosis necrotizing toxin (TNT) is a secreted nicotinamide adenine dinucleotide (NAD+) glycohydrolase that induces necrosis in infected macrophages. Here, we show that NAD+ depletion by TNT activates RIPK3 and MLKL, key mediators of necroptosis. Notably, Mtb bypasses the canonical necroptosis pathway since neither TNF-α nor RIPK1 are required for macrophage death. Macrophage necroptosis is associated with depolarized mitochondria and impaired ATP synthesis, known hallmarks of Mtb-induced cell death. These results identify TNT as the main trigger of necroptosis in Mtb-infected macrophages. Surprisingly, NAD+ depletion itself was sufficient to trigger necroptosis in a RIPK3- and MLKL-dependent manner by inhibiting the NAD+ salvage pathway in THP-1 cells or by TNT expression in Jurkat T cells. These findings suggest avenues for host-directed therapies to treat tuberculosis and other infectious and age-related diseases in which NAD+ deficiency is a pathological factor.

Plasma membrane damage causes NLRP3 activation and pyroptosis during Mycobacterium tuberculosis infection

“…Tuberculosis necrotizing toxin (TNT, the C-terminal end of the protein CpnT) is secreted from Mtb and released into the host cell cytosol in an ESX-1 dependent manner, leading to macrophage necroptosis 34, 53, 54 . We therefore wanted to investigate if TNT is contributing to the ESX-1 dependent inflammasome activation.…”

“…Finally, we assessed the possible involvement of mitochondrial membrane perturbation prior to inflammasome activation and pyroptosis, an event that could also be linked to the activity of ESX-1 and related proteins. Mitochondrial damage has been strongly implicated in inflammasome activation in general, and in Mtb-induced necrosis 25, 29, 33, 34, 55 . In particular, Mtb H37Rv is reported to cause a loss in mitochondrial membrane potential (ΔΨ m ), which could be inhibited by cyclosporine A (CsA), a cyclophilin D inhibitor that prevents formation of the mitochondrial permeability transition pore 34, 56 .…”

“…by the small-molecule compound Imiquimod 29 . Mitochondrial dysfunction seems closely tied to Mtb-induced necrosis as well 33, 34 , but whether this is related to inflammasome activation is not clear. Inflammasome activation and pyroptosis by Mtb is dependent on the type VII secretion system 6-kDa early secretory antigenic target (ESAT-6) secretion system 1, ESX-1.…”

The TIR-domain containing effectors BtpA and BtpB fromBrucella abortusblock energy metabolism

“…A switch to Warburg metabolism has also been observed upon immune activation of many cell types, for example macrophages, following pattern recognition receptor activation[34]. In addition, low NAD + levels are a trigger for cell death via necroptosis in macrophages [35].…”

“…M. tuberculosis infection reduces intracellular NAD + and down-regulates SIRT1, which can be reversed by the addition of SIRT1-activating compounds that represent a potential therapeutic option [43]. The Tuberculosis Necrotizing Toxin (TNT) of M. tuberculosis bears NAD + glycohydrolase activity, which was shown to be important for mycobacterial replication in macrophages, and is involved in triggering necroptosis as a consequence of NAD + depletion [35, 44]. In addition, it has been proposed that reduced NAD + and ATP levels in Salmonella -infected macrophages, a process dependent on the Salmonella Pathogenicity Island 2 (SPI-2) type 3 secretion system [45], trigger the modulation of TORC1 to protect intracellular bacteria from xenophagy via lysosomal degradation of its upstream SIRT1/LKB1/AMPK regulators [45].…”