Metabolic and Bactericidal Effects of Targeted Suppression of Nadd and Nade Enzymes In
Supporting Information
Metabolic and Bactericidal Effects of Targeted Suppression of NadD and NadE Enzymes in Mycobacteria
Irina A. Rodionovaa,1, Brian M. Schusterb,1, Kristine M. Guinnb, Leonardo Sorcia,c, David A. Scotta, Xiaoqing Lia, Indu Kheterpald, Carolyn Shoene, Michael Cynamone, Christopher Locherf, Eric J. Rubinb,2 and Andrei L. Ostermana,2
a Infectious and Inflammatory Disease Center, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037; b Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115; c Department of Clinical Sciences, Section of Biochemistry, Polytechnic University of Marche, Ancona 60131, Italy; d Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, LA 7080; e Department of Medicine, Veterans Affairs Medical Center, Syracuse, NY 13210; f Vertex Pharmaceuticals Incorporated, 130 Waverly Street, Cambridge, MA 02139-4242
1 these two authors made equal contribution to this work;
SUPPLEMENTARY METHODS
Chemicals and strains
Genomic DNA from Mycobacterium tuberculosis H37Rv was a kind gift of Dr. M. Pavelka Jr (University of Rochester Medical Center). A cleavable SUMO-tag expression system, vector pSMT3 and protease Ulp1 [1], were gifts from Dr. C. Lima (Sloan Kettering Institute). PCR reagents and enzymes for cloning were from New England Biolabs; plasmid purification kits and Ni-NTA agarose beads were from Qiagen. N-Ribosylnicotinamide (NmR) was prepared by dephosphorylation of 1 mM solutions of NMN (Sigma) with alkaline phosphatase (Fermentas) at 37°C for ~ 12h in 200 mM Tris-HCl (pH 8.0) and 10 mM MgCl2. The reaction was monitored by HPLC (see below). All other chemicals were from Sigma. Oligonucleotide primers were custom ordered from Invitrogen or Sigma Aldrich; DNA sequencing – from GENEWIZ). Reagents for Western blots were from ThermoScientific); anti–flag antibodies (used at 1:10,000 dilution) - from Sigma).
Cloning of M. tuberculosis nad-genes, heterologous expression, and purification.
Gene nadD encoding a predicted nicotinate mononucleotide (NaMN) adenylyltransferase from M. tuberculosis H37Rv (Rv2421c) was PCR-amplified from genomic DNA and cloned into BamHI-HindIII sites of the pSMT3 vector. Two sets of primers were used (Table S4) producing: (i) the full-size nadD gene within GenBank boundaries (2718173..2718808 bp, complement), and (ii) the extended form containing an additional 24 bp at the 5’-end, relative to the originally designated start codon. The latter form encodes 8 additional N-terminal amino acids (MHGRRLGV) that were predicted and confirmed to be essential for NadD functional activity. Both gene forms were expressed as fusion proteins with the N-terminal His6 - (SUMO)-tag and purified using a standard two-step procedure (e.g. as in [2]), which includes metallo-affinity and FPLC size-exclusion chromatography (Fig. S2). To remove the SUMO-tag, the protein was treated by Ulp1 protease (1:100 w/w) in 50 mM Tris, pH 8.0, 350 mM NaCl at 4°C overnight and recovered in the flow-through of a Ni-NTA column (which separates it from the cleaved SUMO peptide, Ulp1 protease and remaining uncleaved SUMO-NadD, all containing His6-tag).
Gene nadRh (Rv0212c), a homolog of a bi-functional enzyme, nicotinamide riboside kinase/nicotinamide mononucleotide adenylyltransferase (described in H. influenzae [3]) was amplified (Table S4, cloned (253669..254640 bp, complement) and expressed, using the same approach. Purification of a relatively small soluble fraction was performed using the same procedure. In addition, a predominantly insoluble form of overexpressed enzyme was solubilized in 7M urea-containing buffer, refolded by dialysis against 2M urea and purified on a Ni-NTA column. SUMO-tag removal was performed in 1M urea-containing buffer. The resulting protein was subject to final purification by FPLC size-exclusion chromatography on a HiLoad Superdex 200 16/60 column yielding a fraction with retention volume consistent with a dimer of 70 KDa (Fig. S1). Gene nadRh from M. tuberculosis was also cloned in vector pMV261 and introduced into M. smegmatis (which does not contain nadRh in the genome) to probe for its possible contribution to NAD synthesis under conditions of nadD or nadE induced degradation (see below).
Enzyme activity assays and steady-state kinetic analysis.
Nicotinate mononucleotide adenylyltransferase (NaMNAT) activity of purified recombinant NadD of M. tuberculosis was measured as previously described for this enzyme from other species [4] using coupled enzymatic assays based on conversion of the reaction product, NaAD, to NAD by excess NAD synthetase and reduction of NAD to NADH by alcohol dehydrogenase with monitoring at 340 nm. The reaction mixture contained from 0.25 to 4 mM NaMN and 2 mM ATP in 50 mM Hepes pH 7.4 with 5 mM ammonium chloride, 20 mM semicarbazide, 58 mM ethanol, 10 mM MgCl2 and coupling enzymes - 0.6 U/ml B. anthracis NAD synthetase and 6 U/ml alcohol dehydrogenase (from Sigma). The same assay with variable concentrations of NaMN or NMN was used to assess steady-state kinetic parameters. To determine the NaMNAT activity in crude lysates of M. smegmatis (with and without NadD knockdown) the reaction mixture containing only 2 mM NaMN and 2 mM ATP, 10 mM MgCl2 in PBS buffer pH 7.4 was incubated for 4 or 8 hrs and formation of NaAD was detected by HPLC as previously described [4]. NAD synthetase (NADS) activity in crude lysates was determined using the same HPLC - based method detecting NAD formation in the reaction mixture containing 1 mM NaAD, 5 mM ATP, 50 mM KCl, 10 mM MgCl2, in PBS buffer pH 7.4 with 10 mM glutamine. Nicotinamide riboside kinase (NRK) activity of the recombinant purified NadR of M. tuberculosis was assessed using NmR substrate prepared as described above in a coupled assay (modified from [4,5]) using recombinant NadM from A. baylyi [6] and alcohol dehydrogenase to couple the conversion of NMN to NAD and NADH. Nicotinamide mononucleotide adenylyltransferase (NMNAT) activity, the second enzymatic activity of typical NadR enzymes [4,5] was assessed by a coupled assay described above. Both activities were analyzed in crude lysates by direct HPLC-based assay monitoring the conversion of NmR to NMN and/or NMN to NAD. Recombinant purified NadR enzymes of E. coli or H. influenzae [4,5] were used for positive controls. All enzymatic activities were normalized to total protein in lysates measured by Lowry [7] in triplicates against BSA calibration curve.
Degradation of NadD and NadE enzymes in Msm model.
The ClpP proteolytic complex recognizes specific amino acid sequences, and we specifically utilize the SsrA tag that must be exposed on the C-terminus of a protein to initiate degradation. To generate a knockdown strain with induced degradation (ID), the SsrA tag is introduced downstream of the target gene, and then masked with a downstream protecting peptide. An HIV-2 protease cleavage site is engineered between the SsrA tag and the protecting peptide, so that induction of HIV-2 protease (introduced on a plasmid under transcriptional control of a tetracycline-inducible system) cleaves the peptide and exposes the C terminus of the SsrA-tagged protein, which is then targeted for Clp-mediated proteolysis. A flag-tag is also engineered in the C-terminus so that degradation can be confirmed as experiments proceed. Phage-mediated recombineering of ID-tagged NadD and NadE was performed using methods described in (13). Briefly, synthetic constructs were designed to have ≥ 120 bp chromosomal homology to flanking regions of genes, with BamHI and XhoI sites, separating the flanks. Respective ID-taged genes containing myc- and flag- tags (for visualization) and hyg cassette were subcloned into the BamHI and XhoI site from pUC57::alr-ID-hyg. Primer pairs (NadDF, NadDR, NadEF, NadER) were used to create recombinant substrate PCR products from pUC57::NadD-ID-hyg and pUC57::NadE-ID respectively (See Table S4). Cells were prepared from a stationary culture of M. smegmatis with pNit::ET and diluted 1:100. Isovaleronitrile was added to a final concentration of 10 μM for induction of the recombinase. The culture was grown to OD600 = 0.8–1.0, and competent cells were prepared using standard procedures. Electroporation was performed and positive clones (by Western blot) were plated on LB agar containing 10% sucrose to counterselect against the recombinase plasmid. Isolates were then scored for growth on kanamycin-containing medium to confirm the loss of pNit::ET.
Effects of NadD and NadE knockdown on viability.
Media and cultivation. M. smegmatis strains were grown in Middlebrook 7H9 medium (Difco) supplemented with 0.2% glycerol, 0.05% Tween80, 0.5% BSA, 0.2% dextrose, and 0.085% sodium chloride. Strains were grown at 37 °C to log phase (OD600 ∼ 0.4). Cultures were diluted to OD600 of 0.01 into fresh medium with or without ATc. The OD600 was measured every 2 hrs unless otherwise noted, cells were plated on 7H10 (Difco) or LB (Difco) to determine CFU/mL, and a subset of cells was pelleted, washed, and snap frozen for analysis by Western blot and metabolomics. For the GC-MS experiment 7H9 defined medium was prepared without glycerol. Mycobacterial transformations were performed as described [8], and transformants were grown on either 7H10 (Difco) or LB agar (Difco). The final concentrations of antibiotics used for Mycobacterium species were 50 μg/mL for hygromycin and 20 μg/mL for kanamycin. Where indicated, the inducer anhydrotetracycline (ATc) was added to cultures at a final concentration of 100 ng/mL. Strains were grown at 37 °C to log phase (OD600 ∼ 0.4). Cultures were diluted to OD600 of 0.01 into fresh medium with or without ATc. The OD600 was measured every 2 hrs unless otherwise noted, cells were plated on 7H10 (Difco) or LB (Difco) to determine CFU/mL, and a subset of cells were pelleted, washed, and snap frozen for downstream analysis. Total protein lysates were prepared and standardized by OD600 values, then lysed by bead beating.
Expression of nadRh gene from Mtb in Msm did not suppress the effect of nadD knockdown even when NmR was added to the media. Mtb nadRh gene was subcloned into a tetracycline inducible vector, pMV261 and was introduced to NadD KD M. smegmatis strain using selection on kanamycin. Growth experiments were performed as described for NadD KD strain (Fig. 2C) showing the same viability effect in the absence or presence of 100 uM NmR in the medium.
Induced degradation of NadD or NadE in M. smegmatis showed bactericidal effects under condition of carbon starvation. NadD and NadE KD M. smegmatis strains were grown to OD600 ∼ 1, washed 3 times with PBS, and incubated in PBS supplemented by tyloxapol (0,05%) for 1 day. Then NadD and NadE KDs were induced by 100 ng/ml of Atc followed by CFU analysis. Measurements after 3 days of incubation showed a substantial decrease in CFU titer (100-1,000 fold) upon induction of NadD or NadE in contrast to nearly no decrease for noninduced NadD and NadE KD controls (Fig. S4).
Effects of NadD and NadE knockdown on levels of NAD-related metabolites by LC-MS analysis (Table S2).
The extraction and LC-MS based analysis were performed using a modified protocol published in [9]. Briefly, cell lysates in 0.5 N perchloric acid were defrosted on ice, centrifuged and supernatant containing NAD and related metabolites in perchloric acid was neutralized with an equal volume of 1 M ammonium formate (Sigma-Aldrich). Reverse phase LC separations were conducted using a flow rate of 0.15 mL/min with 5 mM ammonium formate as mobile phase A and methanol as mobile phase B. Samples were injected on an Atlantis dC18 column maintained at 30 °C (3 µm, 2.1 X 150 mm, Waters Corp.) and eluted with a linear gradient of 0-70% B over 10 min. The injection volume was 2 µL for all samples, and all samples were analyzed in triplicate. Samples eluted from the chromatographic column were ionized by electrospray ionization (ESI) and analyzed by a triple quadrupole mass spectrometer (XEVO TQMS, Waters Corp.). The mass spectrometer was operated in positive ionization mode with an electrospray voltage 3.5 kV, extraction cone voltage 3 V, source temperature 150 °C and desolvation temperature 600 °C. The MRM transitions (precursor to fragment ion transition) monitored for each compound and their respective cone voltage (CV) and collision energy (CE) were as follows: NAD: 664(precursor ion)/136 (fragment ion), 22 (CV), 48 (CE); NaAD: 665(precursor ion)/136 (fragment ion), 22 (CV), 48 (CE) and NaMN: 336(precursor ion)/124 (fragment ion), 14 (CV), 13 (CE). Calibration standards containing NAD, NaAD and NaMN purchased from Sigma-Aldrich Corp. were prepared in 0.5 N perchloric acid and neutralized with an equal amount of 1 M ammonium formate. The calibration standards containing final concentrations of 0.25 nM – 2.5 µM NAD, 0.15 nM – 1.5 µM NaAD and 0.5 nM – 5 µM NaMN were prepared daily from 0.1-100 mM stock solutions in water. Standard calibration curves were generated using 1/X-weighted linear regressions and used to determine the concentration of unknown samples. TargetLynx (Waters Corp.) was used for data processing. In addition, ATP levels were assessed using the BacTiter-Glo (Promega) reagent per the manufacturer’s specifications. Luminescence was determined in a 96-well format Fluoroskan Ascent FL plate reader (Thermo).
13C biosynthetic labeling and GC-MS-based metabolic profiling.
Cell Culture, Labeling, and Sample Collection. The labeling experiments were performed with the engineered M. smegmatis strains NadD KD and NadE KD. In a typical experiment, bacterial cultures grown to mid-log phase were diluted in 7H9 supplemented with glucose (2g/L) to OD600 = 0.02 and then grown overnight for 16 hours. Labeling was started when cell cultures reached OD600 0.4-0.5. At that point (t) 0 h, 1 g/liter [U-13C6] glucose (Sigma-Aldrich) and anhydrous tetracycline were added. The final glucose concentration at (t) 0 h was 2.1 g/liter with 47% [U-13C] glucose (confirmed by measurement of glucose in medium samples using a YSI 7100 analyzer). A sample of the labeling media was taken at t (0) and stored as a reference for analysis. Additional samples of the labeling media were collected at 2, 4, 6, 8, 10 hours. Following 6, 8, or 10-h labeling periods, cells were collected, rinsed with phosphate-buffered saline, and frozen in liquid nitrogen. Cell pellets were stored at -80 °C for subsequent analysis.
Analysis of polar metabolites by GC-MS: Cell pellets (1.5 x 109 cells) were resuspended in 0.4 ml cold (-20 °C) 40:40:20 methanol: acetonitrile: water (v/v/v) containing 100 uM L-norvaline (as internal standard). Cells were lysed by bead beating 4 times at 4 °C for 1 min before centrifugation. The liquid phase was removed to a separate tube, and dried by centrifugal evaporation. Derivatization of metabolites (using ethylhydroxylamine and N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide), separation and determination of mass spectral (13C-labeling) patterns by GC-MS, and correction of mass spectra for natural heavy isotope occurrence were done as previously [10]. Metabolite labeling is expressed as metabolite percentage derived from glucose, based on the average per-carbon 13C labeling of metabolites (or metabolite fragments for tyrosine and phenylalanine), divided by the percentage 13C labeling of glucose in the medium (47%).