Despite the presence of genes that apparently encode
NAD salvage-specific
enzymes in its genome, it has been previously thought that Mycobacterium tuberculosis can only synthesize
NAD de novo. Transcriptional analysis of the de novo synthesis and putative salvage pathway genes revealed an up-regulation of the salvage pathway genes in vivo and in vitro under conditions of
hypoxia. [14C]
Nicotinamide incorporation assays in M.
tuberculosis isolated directly from the lungs of infected mice or from infected macrophages revealed that incorporation of exogenous
nicotinamide was very efficient in in vivo-adapted cells, in contrast to cells grown aerobically in vitro. Two putative
nicotinic acid phosphoribosyltransferases, PncB1 (Rv1330c) and PncB2 (Rv0573c), were examined by a combination of in vitro enzymatic activity assays and allelic exchange studies. These studies revealed that both play a role in cofactor salvage. Mutants in the de novo pathway died upon removal of exogenous
nicotinamide during active replication in vitro. Cell death is induced by both cofactor
starvation and disruption of cellular redox homeostasis as electron transport is impaired by limiting
NAD. Inhibitors of
NAD synthetase, an essential
enzyme common to both recycling and de novo synthesis pathways, displayed the same bactericidal effect as sudden
NAD starvation of the de novo pathway mutant in both actively growing and nonreplicating M.
tuberculosis. These studies demonstrate the plasticity of the organism in maintaining
NAD levels and establish that the two
enzymes of the universal pathway are attractive chemotherapeutic targets for active as well as
latent tuberculosis.