Volume 24, Issue 3, Pages 306-315 (March 2017) Fumarase Deficiency Causes Protein and Metabolite Succination and Intoxicates Mycobacterium tuberculosis  Nadine Ruecker, Robert Jansen, Carolina Trujillo, Susan Puckett, Pradeepa Jayachandran, Gerardo G. Piroli, Norma Frizzell, Henrik Molina, Kyu Y. Rhee, Sabine Ehrt  Cell Chemical Biology  Volume 24, Issue 3, Pages 306-315 (March 2017) DOI: 10.1016/j.chembiol.2017.01.005 Copyright © 2017 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2017 24, 306-315DOI: (10. 1016/j. chembiol. 2017 Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Fum Depletion Kills Mtb (A) Schematic depicting the TCA cycle, the urea cycle, and reactions leading to fumarate production. ArgH, argininosuccinate lyase; PurB, adenylosuccinate lyase. (B and C) Growth (B) and survival (C) of WT and Fum-DUC in 7H9 complete medium with (+) and without (−) atc. Colony-forming units (CFU) were determined by culturing serial dilutions on agar plates at different time points post inoculation. See also Figure S2. (D) Fum amounts in Fum-DUC were determined by immunoblot. The proteasome subunit B (PrcB) serves as loading control. (E) Quantitative Fum immunoblotting in Fum-DUC extracts from cultures on day 7 after atc addition. Serial dilutions of WT extract were used for relative quantification. PrcB served as loading control. (F) Impact of Fum depletion on starved Mtb. WT and Fum-DUC were cultured in PBS with 0.05% tyloxapol for 10 days, before atc was added (arrow). CFU were determined by culturing serial dilutions on agar plates at different time points post inoculation. (G) Fum depletion in Fum-DUC during PBS starvation was analyzed by immunoblot. PrcB serves as loading control. All data are representative of at least two independent experiments. See also Figures S1 and S2. Cell Chemical Biology 2017 24, 306-315DOI: (10.1016/j.chembiol.2017.01.005) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Mtb Requires Fum to Establish and Maintain Infection in Mice (A and B) Bacterial burden in lungs (A) and spleens (B) from mice infected with WT Mtb and the Fum-DUC strain. Mice were fed doxy-containing chow as indicated. Lung and spleen homogenates were cultured on agar plates to determine bacterial burden at the indicated time points post infection. Data are means ± SD of four mice per time point. Data are representative of two independent infection experiments. See also Figure S3. Cell Chemical Biology 2017 24, 306-315DOI: (10.1016/j.chembiol.2017.01.005) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Metabolic Consequences of Fum Deficiency Intrabacterial pool sizes of selected metabolites in the indicated Mtb strains after 24 hr cultivation on filters on top of 7H9 medium with 0.2% glucose with or without atc. Pool sizes are expressed as area under the curve normalized to protein content. Data are mean values ± SD of three biological replicates and are representative of two independent experiments. Statistically significant differences between atc-treated and untreated cultures that were observed in two biologically independent experiments are reported. *p ≤ 0.05, **p ≤ 0.005 by Student’s t test. The differences between pool sizes in atc-treated and untreated WT were not reproducibly statistically significant. Enzymatic reactions are depicted as arrows; the dashed arrow indicates that the respective enzyme has not been identified in Mtb; black dotted arrows indicate spontaneous dephosphorylation reactions. AICAR, 1-(5'-phosphoribosyl)-5-amino-4-imidazolecarboxamide; AMP, adenosine 5'-monophosphate; ArgH, argininosuccinate lyase; PurB, adenylosuccinate lyase; SAICAR, succinylaminoimidazole carboxamide ribose-5′-phosphate; SAICAr, succinylaminoimidazole carboxamide ribose. See also Figure S4. Cell Chemical Biology 2017 24, 306-315DOI: (10.1016/j.chembiol.2017.01.005) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Intracellular Fumarate Accumulation Results in Toxicity (A) Secretion of fumarate and succinate. Pool sizes of secreted metabolites are expressed as area under the curve (AUC) and normalized to the protein content of the bacteria on the filter. Values are the averages ± SD of biological triplicates. *p ≤ 0.05, **p ≤ 0.005 by Student’s t test. Data are representative of two independent experiments. (B) Addition of fumarate to the extracellular medium accelerates death of Fum-depleted Mtb. Fum-DUC was cultivated in medium containing increasing amounts of fumarate with or without atc. CFU were monitored at the indicated time points. Data are representative of three independent experiments. (C) Effect of extracellular fumarate and dimethylfumarate (DMF) on WT Mtb. CFU values are averages ± SD of biological triplicates and are representative of two independent experiments. (D) Effect of extracellular dimethylfumarate (DMF) on Fum-DUC. Fum-DUC was treated with atc for 48 hr to deplete Fum or left untreated and then exposed DMF. CFU values are averages ± SD of biological triplicates. Data are representative of three independent experiments. Cell Chemical Biology 2017 24, 306-315DOI: (10.1016/j.chembiol.2017.01.005) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Fumarate Accumulation Leads to Metabolite Succination (A) The electrophile fumarate can react with cysteine thiol residues in a Michael addition generating S-(2-succino) compounds. (B) Intracellular pool sizes of a metabolite with a mass to charge ratio (m/z) of 603.158 in extracts from Mtb Fum-DUC treated with atc for 24 hr. *p ≤ 0.05, by Student’s t test. (C) Comparison of MS-MS fragmentation spectra of 603.158 with fragmentation spectra of MSH after incubation with fumarate. (D) Intracellular pool sizes of a metabolite with an m/z of 238.038. *p ≤ 0.05, by Student’s t test. (E) Comparison of MS/MS fragmentation spectra of 238.038 with fragmentation spectra of succinocysteine. Cell Chemical Biology 2017 24, 306-315DOI: (10.1016/j.chembiol.2017.01.005) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 Fumarate Accumulation Leads to Protein Succination (A) Detection of 2SC proteins in lysates of Fum-DUC grown with or without atc. For each condition 5 μg protein was loaded. Succinated GAPDH (Alderson et al., 2006) was loaded as a positive control for succination (labeled C). PrcB serves as loading control. (B) Tandem MS spectra of the triply charged tryptic cysteine-containing peptide: LQV TYE GCE PHT VAD AYR, from 1,4-alpha-glucan branching enzyme GlgB (Rv1326c). Fragment ion (y and b ions)-annotated spectra are shown (top to bottom) of the iodoacetamide-modified cysteine peptide (m/z of 703.6605) matched in the “WT + atc” sample, the succinated peptide (m/z of 723.3422) matched in the “Fum-DUC + atc,” sample and the dimethylsuccinated peptide (m/z of 732.6677) matched in the “WT + DMF” sample, respectively. All peptides were matched with mass accuracy better than 2 ppm. See also Figure S5. (C) Fum deficiency increases susceptibility to hydrogen peroxide (H2O2). Fum-DUC was exposed to 5 μM H2O2 in the presence or absence of atc for 30 hr. CFU values are averages ± SD of biological triplicates. Data are representative of three independent experiments. Cell Chemical Biology 2017 24, 306-315DOI: (10.1016/j.chembiol.2017.01.005) Copyright © 2017 Elsevier Ltd Terms and Conditions