Biosynthesis and recycling of nicotinamide cofactors in mycobacterium tuberculosis. An essential role for NAD in nonreplicating bacilli

Abstract

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.

FIGURE 1.

Genes encoding NAD biosynthetic enzymes. A, pathways for de novo and Preiss-Handler de novo synthesis of NAD in M. tuberculosis. PncB activity is encoded by both Rv1330c and Rv0573c. B, analysis of gene expression levels by quantitative RT-PCR of genes involved in the (a) de novo, (b) salvage, and (c) universal pathways of NAD(P) synthesis. Gene expression levels were normalized to the levels of sigA and expressed as ratios compared with aerobic growth. M. tuberculosis during the following are shown: 1, NRP-1 hypoxic adaptation; 2, NRP-2 survival; 3, growth in J774 macrophages; 4, growth in murine bone marrow macrophages; 5, growth in activated bone marrow macrophages; 6, survival in chronically infected mouse lung.

FIGURE 2.

Incorporation of nicotinamide into NAD through salvage synthesis. A, TLC analysis of nucleotides. In vitro grown M. tuberculosis (10⁹ cells), M. tuberculosis isolated from infected mouse lung (10⁶ cells), or M. tuberculosis released from infected J774 murine macrophages (5 × 10⁷ cells) were labeled with [¹⁴C]nicotinamide for 2 days (for in vitro grown M. tuberculosis) or 1 day (for in vivo grown M. tuberculosis) before analysis of pyridine nucleotides by TLC. Lane 1, WT H37Rv; lane 2, pncB1::aph; lane 3, pncB1::aph/pncB2::hyg; lane 4, nad::hyg; lane 5, pncB1::aph/pncB2::hyg/attB::pGhsp60pncB2; lane 6, WT from infected mouse lung; lane 7, WT from infected J774 macrophages. B, densitometric analysis of relative abundance of radiolabeled NADH (black bars) and NAD⁺ from TLC analysis. Numbering of lanes as in A. C, TLC analysis of [¹⁴C]nicotinamide incorporation into NADH (NAD⁺ not shown) by WT (lane 1) and pncB1::aph (lane 2) isolated from 4-week-old infected mouse lungs and labeled as above. D, TLC analysis of radiolabeled [¹⁴C]nicotinamide incorporation into NAD⁺ by 4 × 10⁹ aerobically growing M. tuberculosis (lane 1), 5 × 10⁸ 8-day-old microaerophilically adapted cells (lane 2), 5 × 10⁸ 3-week anaerobically adapted cells (lane 3), and 1 × 10⁸ 8-week-old anaerobically adapted M. tuberculosis (lane 4).

FIGURE 3.

Phosphoribosyltransferase assays of affinity-purified histidine-tagged proteins. Recombinant proteins were assayed for phosphoribosyltransferase activity with nicotinamide (NAM, lanes 1–3) or nicotinic acid (NA, lanes 4 and 5) as substrate for the formation of nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN), respectively. Lanes 1 and 4, PncB1-His; lane 2, PncB2-His, lanes 3 and 5, pET30(b)+ control.

FIGURE 4.

Survival of the de novo knock-out mutant in the absence of exogenous pyridine nucleotide precursors. Cells from 20 μg/ml nicotinamide-containing cultures were washed five times in an equal volume of medium before transfer to nicotinamide-free growth medium and survival (▪) over time measured by plating of serial dilutions onto nicotinamide-containing medium. Total NAD (▴) and NADH (Δ) levels in the nad::hyg mutant after transfer from nicotinamide-replete to nicotinamide-free medium were determined concurrently.

FIGURE 5.

Changes in redox status of NAD⁺/NADH and isoprenoid quinone-quinol pairs. A, effect of NAD biosynthetic capacity on NAD⁺/NADH ratio and levels of the oxidized and reduced pyridine nucleotides (inset) in WT as compared with NAD biosynthetic mutant strains. For x axis: 1, WT H37Rv; 2, nad::hyg in nicotinamide-replete medium; 3, nad::hyg starved for nicotinamide for 4 days; 4, pncB1::aph/pncB2::hyg; 5, WT H37Rv starved 3 days in PBST; 6, pncB1::aph. B, level of oxidized menaquinones. The x axis shows menaquinones with 8 (bar 1), 9(bar 2), 10 (bar 3) isoprene groups and total oxidized menaquinones (bar 4). Results shown as percentage of quinone for each pair.

FIGURE 6.

Inhibition of NAD synthetase results in depletion of NAD(H) pools. Treatment of aerobically growing M. tuberculosis with NAD synthetase inhibitor 2 (triangles) or rifampicin (squares) under aerobic (A) and anaerobic conditions (B) is shown. Total NAD(H) pools (solid symbols) and % survival (open symbols) were measured at various time intervals. NAD synthetase inhibitor 2 was used at 19.5 μm (dashed lines) and 39 μm (solid lines). An independent culture was treated with rifampicin at 0.6 μm (dashed lines) or 2.5 μm (solid lines).

FIGURE 7.

Virulence of NAD biosynthetic mutants in mice as determined by bacterial loads in lungs (A) and spleens (B) of aerosol-infected C57Bl/6 mice. Results shown are one of two similar experiments four mice plated per group for each time point.

FIGURE 8.

De novo NAD biosynthesis is not required during nonreplicating persistence. A, analysis of survival during starvation of the nad::hyg mutant in PBST in the presence (▪) and absence (224) of nicotinamide (NAM). B, survival of NAD biosynthetic mutants during adaptation and survival in hypoxic conditions. ▪, nad::hyg in the presence of nicotinamide; 224, nad::hyg in the absence of nicotinamide; •, WT H37Rv; ▴, pncB1::aph/pncB2::hyg.