Fig 1.
S. mansoni utilizes extracellular NAD to support intracellular NAD homeostasis and egg production.
(A) Genomic reconstruction of NAD metabolism in S. mansoni. Genes (red boxes with gene names listed below) encoding enzymes predicted to participate in the NAD-biosynthesis salvage pathways (left side of cartoon) and NAD catabolism (right side of cartoon) are shown. Metabolites (blue boxes) in the pathways and inhibitors (yellow boxes) of NAMPT and SmNACE are indicated. No genes encoding candidate orthologs of enzymes in the de novo NAD synthesis pathway from tryptophan or aspartate were identified. Enzymes include: PNP (Purine Nucleotide Phosphorylase), MTAP (Nucleoside hydrolase), NAMPT (Nicotinamide phosphoribosyltransferase), NAPRT (Nicotinic acid phosphoribosyltransferase), NMNAT (Nicotinamide mononucleotide adenylyltransferase), GAT (Glutamine amidotransferase), NADS (NAD Synthetase), NADK (NAD Kinase), SmNACE (S. mansoni NAD-catabolizing enzyme) and Poly-ADP-ribose polymerase (PARP). Metabolites include: NAD(P) (nicotinamide adenine dinucleotide (phosphate)), NAM (nicotinamide), NA (nicotinic acid), NR (nicotinamide riboside), NMN (nicotinamide mononucleotide), NaMN (nicotinic acid mononucleotide) and NAAD (nicotinic acid adenine dinucleotide). (B) S. mansoni genes encoding putative NAD salvage pathway biosynthetic enzymes are transcribed. PCR amplification of NADS, NAPRT, GAT, NAMPT, NMNAT, MTAP, NADK1 and NADK2 from cDNA prepared from RNA isolated from adult S. mansoni. Transcripts for PNP were not detected. (C-D) Recombinant SmNACE (rSmNACE, panel C) and live male or female S. mansoni (n = 1 parasites/well; panel D) catabolize extracellular NAD. NAD glycohydrolase activity measured by monitoring hydrolysis of the NAM-ribose bond in etheno-NAD (ε-NAD) resulting in release of NAM and ε-ADPR, which is detected using a fluorimeter. Data reported as Relative Fluorescence Units (RFU) measured over time. (E) SmNACE is expressed by male and female S. mansoni. A SmNACE standard curve, which was generated by measuring the NAD glycohydrolase activity of increasing concentrations of rSmNACE (see panel C), was used to determine the amount of enzymatically active native SmNACE expressed by live S. mansoni parasites. Data pooled from 2 experiments with n = 4 wells/group and 1 parasite/well. (F) Intracellular NAD levels in S. mansoni females cultured for 48 h in serum free media (SFM) ± 2mM extracellular NAD. Data represents n = 5 wells/group with 2 female parasites/well. (G) Native SmNACE activity is blocked by the SmNACE inhibitor CMP1. NAD glycohydrolase activity of adult schistosomes incubated with ε-NAD ± increasing concentrations of CMP1. The IC50 of CMP1 on live parasites is indicated. n = 2 wells/group with 4 parasites/well. (H) Intracellular NAD levels in S. mansoni females cultured for 48h with 2mM extracellular NAD ± CMP1 (200μM) or vehicle (2% DMSO). n = 5 wells/group with 2 female parasites/well. (I) Egg production in S. mansoni females cultured for 48h in SFM ± 2mM extracellular NAD. Data are pooled from 3 experiments with n = 15 wells/group and 2 female parasites/well. (J) Egg production in S. mansoni females cultured for 48h with 2mM extracellular NAD ± CMP1 (200μM) or vehicle (2% DMSO). n = 6 wells/group with 2 female parasites/well. (K) S. mansoni egg production is restored following removal of SmNACE inhibitor. Egg production by S. mansoni females cultured 48h with 2mM extracellular NAD + CMP1 (200μM) and then washed and recultured for an additional 48h with 2mM extracellular NAD ± CMP1 (200μM) or vehicle (2% DMSO). n = 5 wells/group with 2 female parasites/well). Data are representative of 2 (C-H, J-K) or 3 (I) independent experiments and are represented as the mean ± SD (bars) of individual samples (squares) (E-F, H-K). Statistical analyses were performed using two-tailed Student’s t test.
Fig 2.
NAD biosynthesis in S. mansoni requires the NAD salvage pathway.
(A) Structure-based sequence alignment of S. mansoni NAMPT with mammalian NAMPTs. Secondary structure elements are placed on top of the alignment according to the structure of R. norvegicus (rat) NAMPT in complex with FK866 (PDB 2G97 [42]). Residues interacting with FK866 [42] are indicated with blue arrows. (B-D) Best docking poses of FK866 in the superimposed active sites of human NAMPT and SmNAMPT dimers. In panels B and D, the chain-A and chain-B of each NAMPT dimer are represented in light (chain-A) and dark (chain-B) colors. Human NAMPT is represented by the white/grey ribbons and SmNAMPT is represented by the pink ribbons. The FK866 crystal structure is shown in green in panels B-D. The best optimized docking poses of FK866 into human NAMPT (panel B) and SmNAMPT (panel D) are shown in purple and magenta, respectively. Panel C is an overlay of the SmNAMPT (transparent) and hNAMPT (grid) active site cavities with the hydrophobic surface represented in green and hydrophilic surface in purple. Side chains of species-specific residues lining the active site are shown in white (human NAMPT) and pink (SmNAMPT). Two water molecules used during docking are also represented. Predicted affinities of FK866 for hNAMPT (ΔG = -10kcal/mol) and smNAMPT (ΔG = -11 kcal/mol). (E) Intracellular NAD levels in S. mansoni females cultured for 48h with vehicle (0.001% DMSO) or increasing concentrations of the NAMPT inhibitor FK866. Data are pooled from 2 experiments with n = 8 wells/group and 2 female parasites/well. (F) Intracellular NAD levels in S. mansoni females cultured for 48h with vehicle or 250nM FK866± 2mM NMN. Data are pooled from 3 experiments with n = 11 wells/group and 2 female parasites/well. (G) Intracellular NAD levels in S. mansoni females cultured for 48h with vehicle or 250nM FK866± 2mM NAM, NR, NA, Asp or Trp. n = 3 wells/group with 2 parasites/well. Data in E-G are representative of 2–3 independent experiments and are shown as the mean ± SD (bars) and individual samples (squares). Statistical analyses were performed using one-way ANOVA multiple comparison tests. *Two-way comparisons between vehicle and all other groups P≤ 0.002.
Fig 3.
The NAD salvage pathway controls S. mansoni metabolism, egg production, and survival in vitro.
(A-B) Intracellular NAD levels in female S. mansoni cultured between 0-48h (A) or for 48h (B) with vehicle (0.001% DMSO) or 250nM FK866 ± 2mM NMN or Trp. n = 5 wells/group with 2 parasites/well. (C) Lactic acid levels in female S. mansoni cultured for 6 days with vehicle or 250nM FK866 ± 2mM NMN or Trp. n = 5 wells/group with 2 female parasites/well. (D-F) Loss of membrane integrity in FK866-exposed male S. mansoni. Representative H&E stained cross sections (D) and SEM images (F) of male S. mansoni cultured for 7 days with vehicle or 250nM FK866. Severity of tissue damage reported as vacuolization scores (E) as determined by blinded assessment of the H&E cross sections (see S2 Fig for pathology scoring methodology. See S2 Fig and S3 Fig for additional H&E and SEM micrographs). n = 5–7 worms/group/analysis. (G) Egg production by S. mansoni females cultured for 96h with vehicle or increasing concentrations of FK866. n = 5 wells/group with 2 female S. mansoni per well. (H) Egg production by S. mansoni females cultured for 72 h with vehicle or 250nM FK866 ± 2mM NMN or Trp. n = 3 wells/group with 2 female parasites/well. (I) Parasite mobility by S. mansoni cultured between 0–12 days with vehicle or 250nM FK866 ± 2mM NMN or Trp. n = pooled averages from 3 individual experiments consisting of 3 wells/condition with each well containing 6 parasites (3 male and 3 female). Representative videos documenting parasite motility can be found in S1–S4 Videos. (J) Survival of male and female S. mansoni cultured for 12 days with vehicle or 250nM FK866 ± 2mM NMN or Trp. Data are shown as the percentage of parasites alive at each timepoint. n = 18 parasites (3 males + 3 females per well in triplicate wells). Data are representative of 1 (G), 2 (A, C-F, H) or 3 (B, I-J) independent experiments. Data shown as the mean ± SD (A-C, E, G-H, bars) or mean ± SEM (I) with individual samples (squares). Statistical analyses were performed using one-way ANOVA multiple comparison tests for experiments with more than two groups, two tailed Student’s t-test for experiments with two groups and Log-rank (Mantel-Cox) for survival.
Fig 4.
The NAD salvage pathway controls metabolism and survival of immature S. mansoni.
(A) Intracellular NAD levels in S. mansoni schistosomula cultured for 48h with vehicle or 250nM FK866 ± 2mM NMN or Trp. n = 6 wells/group with 2 schistosomula/well. (B) Representative light microscopy images of schistosomula 7 days post-treatment with vehicle or 250nM FK866 ± 2mM NMN or Trp. (C) Parasite mobility by S. mansoni schistosomula cultured for 10 days with vehicle or 250nM FK866 ± 2mM NMN or Trp. n = 3 wells/group with 45–60 schistosomula/well. (D) Survival of schistosomula cultured for 10 days with vehicle or 250nM FK866 ± 2mM NMN or Trp. Data are shown as the percentage of parasites alive at each timepoint. n = 45–60 parasites/group. Data are representative of 2 independent experiments. Data shown as the mean ± SD with individual samples in squares (A, C). Statistical tests were performed using one-way ANOVA multiple comparison tests or Log-rank (Mantel-Cox) test for survival.
Fig 5.
The NAD salvage pathway controls metabolism, and survival of adult and immature S. japonicum.
(A) Intracellular NAD levels in female S. japonicum cultured for 48h with vehicle or 250nM FK866 ± 2mM NMN or Trp. n = 5–6 wells/group with 1 female parasite/well. (B) Egg production by female S. japonicum cultured for 48h with vehicle or 250nM FK866 ± 2mM NMN or Trp. n = 4–5 wells/group with 1 female parasite/well. (C-E) Loss of membrane integrity in day 7 FK866-exposed male S. japonicum. Representative H&E stained cross sections (C) and SEM images (E) of male S. japonicum cultured for 7 days with vehicle or 250nM FK866. Severity of tissue damage reported as vacuolization scores (D) as determined by blinded assessment of the H&E cross sections (see S2 Fig for pathology scoring methodology and S3 Fig for additional SEM micrographs). n = 7–10 worms/group/analysis. (F) Parasite mobility by adult S. japonicum cultured for 0–12 days with vehicle or 250nM FK866 ± 2mM NMN or Trp. n = pooled averages from 3 individual experiments consisting of 3 wells/condition with each well containing 2 male and 2 female parasites. (G) Survival of S. japonicum cultured for 12 days with vehicle or 250nM FK866 ± 2mM NMN or Trp. n = 12 parasites (2 males + 2 females/well in triplicate wells). (H) Parasite mobility by S. japonicum schistosomula cultured for 0–10 days with vehicle or 250nM FK866 ± 2mM NMN or Trp. n = 3 wells/group with 6–9 schistosomula/well. (I) Survival of S. japonicum schistosomula cultured for 10 days with vehicle or 250nM FK866 ± 2mM NMN or Trp. Data are shown as the percentage of parasites alive at each timepoint. n = 6–9 parasites/group. Data are representative of 2 (A-E, H-I) or 3 (F-G) independent experiments. Data shown as the mean ± SD (A-B, D, H) or mean ± SEM (F) with individual samples shown in squares. Statistical tests were performed using one-way ANOVA multiple comparison tests for experiments with more than two groups, two-tailed Student’s t-test for experiments with two groups or a Log-rank (Mantel-Cox) test for the survival experiments.
Fig 6.
FK866 treatment reduces egg burden and liver pathology in S. mansoni-infected mice.
(A) Schematic showing treatment schedule for mice infected on day 0 with ~200 S. mansoni cercariae. Mice were injected (i.p.) with vehicle (45% propylene glycol, 5% tween-80 and 50% ddH2O) or 20mg/Kg FK866 2x/day for 4 consecutive days beginning on day 35. Following a 3-day rest period, animals were injected for an additional 4 days with FK866 or vehicle and then analyzed on day 49. (B) Egg burden in liver of vehicle and FK866-treated groups. Data reported as the total number of eggs per infected liver. n = 5–6 mice/group. (C-D) Hepatomegaly in vehicle and FK866-treated groups. Representative images of livers from infected mice (C) and liver weights (D) are shown. n = 5–6 mice/group. (E-F) Quantitation of granulomas from livers of vehicle and FK866-treated groups. Representative H&E stained cross sections (E) of livers from infected mice and numbers of granulomas per 40x field (F) as determined by blinded assessment of H&E cross sections. n = 6 mice/group. (G) Intracellular NAD levels in parasites flushed from the portal vein of vehicle or FK866-treated groups. n = 7 parasite pools/group with 2 female parasites/pool. (H) Recovered live parasites flushed from portal vein of vehicle and FK866-treated groups. n = 6 mice/group. Data are representative of 2 (E-F) or 4 (B-D, G-H) independent experiments and shown as the mean ± SD (B, D, F-H) of the groups with individual animals/samples shown in squares. Statistical analyses were performed using two-tailed Student’s t test.
Fig 7.
FK866 + NA treatment reduce egg and worm burden in S. mansoni-infected mice.
(A)Schematic showing treatment schedule for mice infected on day 0 with ~200 S. mansoni cercariae. On day 42 post-infection, mice were injected (i.p.) 2x/day for 4 consecutive days with vehicle or 50mg/kg FK866. Both groups were given an additional injection (i.p.) 1x/day of 50mg/kg NA for 4 consecutive days. Animals were analyzed on day 49 post infection. (B-D) Hepatomegaly and splenomegaly in uninfected controls and in vehicle+NA or FK866+NA treated infected mice. Representative images of isolated spleens and livers (B) and weights of spleens (C) and livers (D) from uninfected and infected mice are shown. n = 3–6 mice/group. (E) Egg burden in livers of vehicle+NA or FK866+NA treated infected mice. Data reported as the total number of eggs per infected liver. n = 5–6 mice/group. (F-G) Quantitation of granulomas from livers of vehicle+NA or FK866+NA-treated infected mice. Representative H&E stained cross sections (F) of livers from infected mice and numbers of granulomas per 40x field (G) as determined by blinded assessment of H&E cross sections. n = 5–6 mice/group. (H) Recovered live parasites flushed from portal vein of vehicle+NA and FK866+NA treated infected mice. n = 5–6 mice/group. (I) Intracellular NAD levels in parasites flushed from the portal vein of vehicle+NA or FK866+NA treated infected mice. n = 7 parasite pools/group with 2 female parasites/pool. Data are representative of 2 independent experiments. Data shown as the mean ± SD of the groups (C-E, G-I) with individual animals/samples shown in squares. Statistical tests were performed using one-way ANOVA multiple comparison tests for experiments with more than two groups or two-tailed Student’s t-test for experiments with two groups.