Genetic validation of Trypanosoma brucei glutathione ...€¦ · 04.03.2014 · v vs Introduction vt Trypanosoma brucei is the causative agent of huma n African trypanosomiasis (HAT),

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Genetic validation of Trypanosoma brucei glutathione synthetase as an 1 essential enzyme 2

3 Chelsea Pratt, Suong Nguyen and Margaret A. Phillips# 4

5 Department of Pharmacology, University of Texas Southwestern Medical Center at 6

Dallas, 6001 Forest Park Rd, Dallas, Texas 75390-9041, United States of America 7 8 9

#Author to whom all correspondence should be addressed. Email: 10 [email protected]; Tel: (214) 645-6164 11

12 Running title: T. brucei glutathione synthetase 13

14 15 16 17

EC Accepts, published online ahead of print on 7 March 2014Eukaryotic Cell doi:10.1128/EC.00015-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Abbreviations. HAT, human African trypanosomiasis; TbGS, Trypanosoma brucei glutathione 18 synthetase; γ-GCS, γ-glutamylcysteine synthetase; TryS, trypanothione synthetase; TR, 19 trypanothione reductase; ODC, ornithine decarboxylase; AdoMetDC, S-adenosylmethionine 20 decarboxylase; DHODH, dihydroorotate dehydrogenase; BSF, Blood stream form; cDKO, 21 conditional double knockout T. brucei cell line. 22 23


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Abstract 24 Human African trypanosomiasis (HAT) is a debilitating and fatal vector borne disease. 25 Polyamine biosynthesis is the target of one of the key drugs (eflornithine) used for the treatment 26 of late stage disease suggesting the pathway might be exploited for the identification of 27 additional drug targets. The polyamine spermidine is required in trypanosomatid parasites for 28 formation of a unique redox cofactor termed trypanothione, which is formed from the 29 conjugation of glutathione to spermidine. Herein we characterize recombinant T. brucei 30 glutathione synthetase (TbGS) and show that depletion of TbGS in blood form parasites using a 31 regulated knockout strategy leads to loss of trypanothione and to cell death as quantified by 32 FACS analysis. These data suggest that >97% depletion of TbGS is required before 33 trypanothione is depleted and cell growth arrest is observed. Exogenous glutathione was able to 34 partially compensate for the loss of TbGS suggesting that parasites are able to transport intact 35 glutathione. Finally, reduced expression of TbGS leads to increased levels of upstream 36 glutathione biosynthetic enzymes and decreased expression of polyamine biosynthetic enzymes 37 providing evidence that the cells cross regulate the two branches of the trypanothione 38 biosynthetic pathway to maintain spermidine and trypanothione homeostasis. 39 40


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Introduction 41 Trypanosoma brucei is the causative agent of human African trypanosomiasis (HAT), a 42

vector-borne illness endemic in 36 African countries (1-4). The World Health Organization 43 (WHO) estimates that 30,000 people are currently infected, while millions in the region remain 44 at risk of contracting the disease (5). HAT is nearly always fatal if untreated, however, current 45 drug therapy is limited by toxicity and difficult treatment regimens. Eflornithine, a key drug for 46 treatment of late stage HAT, is a suicide inhibitor of the polyamine biosynthetic enzyme 47 ornithine decarboxylase (ODC), thus establishing the polyamine biosynthetic pathway as an 48 essential and targetable pathway in these parasites (6). Polyamines are positively charged, 49 flexible hydrocarbons that are essential for growth and proliferation of eukaryotic cells, with 50 roles in transcription, translation, chromatin structure and ion channel function (7-10). 51 Trypanosomes have evolved to use the polyamine spermidine in an additional role through 52 conjugation with two molecules of glutathione to form the essential redox cofactor trypanothione 53 (N1, N8-bis(glutathionyl)-spermidine) (11). 54

Trypanothione synthesis occurs via four ATP dependent enzymatic steps beginning with 55 the conjugation of L-Cys to L-Glu by γ-glutamyl cysteine synthetase (γ-GCS)(12) (Fig. 1). The 56 product of this reaction (γ-GC) is then ligated to Gly by glutathione synthetase (GS) to form the 57 thiol peptide glutathione that is used to maintain redox homeostasis in most cells. The third and 58 fourth steps are catalyzed by trypanothione synthetase (TryS), which conjugates glutathione to 59 spermidine forming N1-glutathionylspermidine and subsequently adds a second molecule of 60 glutathione to N1-glutathionylspermidine to form trypanothione (13, 14). TyrS also contains an 61 amidase domain and can catalyze the breakdown of trypanothione to glutathione and spermidine. 62


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Trypanothione is required for growth and proliferation of trypanosomatids and is 63 necessary to maintain cellular redox balance, replacing the role of glutathione found in 64 mammalian cells (12, 15). The presence of two novel enzymes in the trypanosomatids required 65 for trypanothione synthesis (TryS) and reduction (trypanothione reductase (TR)) has led to 66 interest in identifying inhibitors of these enzymes for drug discovery efforts, though none have 67 yet advanced beyond hit identification or early lead optimization (16-21). With the exception of 68 GS, the biosynthetic enzymes for both the polyamine and trypanothione biosynthesis have been 69 characterized and shown to be essential in T. brucei using genetic methods (14, 22-25). A crystal 70 structure of T. brucei GS (TbGS) has been reported and shows conservation of the overall fold 71 when compared to the human GS protein, and a similar active site composition (26). However, 72 no kinetic analysis of the T. brucei enzyme has been reported and genetic studies on its role and 73 essentiality are lacking. 74

In mammalian cells, glutathione biosynthesis is regulated primarily by modulation of γ-75 GCS expression, the first step in the pathway (27). While T. brucei γ-GCS has been 76 characterized and shown to be essential for growth in T. brucei, gene knockdown did not lead to 77 compensatory changes in the levels of the polyamine biosynthetic enzymes and no evidence for 78 cross regulation between the polyamine and trypanothione arms of the pathway was found (22, 79 28, 29). Regulation has been shown to occur within the T. brucei polyamine biosynthetic 80 pathway where gene knockdown or inhibition of S-adenosylmethionine decarboxylase 81 (AdoMetDC), an essential enzyme in spermidine biosynthesis, led to increased protein 82 expression of both the AdoMetDC regulatory subunit prozyme and ODC (23, 24, 30, 31). The 83 AdoMetDC regulatory protein prozyme is itself also an activator of AdoMetDC as heterodimer 84 formation with prozyme (an inactive paralog of AdoMetDC) increases enzymatic activity by 85


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three orders of magnitude. T. brucei deoxyhypusine synthase, an enzyme that uses spermidine as 86 a substrate to modify an essential elongation factor eIF5A also requires activation by an inactive 87 paralog (32). Despite these advances in understanding polyamine pathway regulation in T. 88 brucei, key questions remain about how the glutathione branch of the pathway is regulated, and 89 how or if the polyamine and glutathione pathways show evidence for cross regulation. 90

Herein we investigate the function and regulation of TbGS, including steady-state kinetic 91 analysis of the recombinant enzyme and characterization of a TbGS knockout in T. brucei blood 92 stream form (BSF) cells. We show that TbGS has similar catalytic activity to other characterized 93 GS homologs, and we demonstrate through the use of a TbGS conditional knockout (cDKO) cell 94 line that TbGS is essential for trypanothione biosynthesis and for parasite growth. Furthermore, 95 we show that loss of TbGS protein leads to an increase in γ-GCS protein and a decrease in the 96 levels of AdoMetDC prozyme. Thus, similarly to the other polyamine and trypanothione 97 biosynthetic enzymes, TbGS is essential to T. brucei growth and it appears that TbGS protein 98 levels are linked to the regulatory mechanisms that control expression levels of both polyamine 99 and glutathione/trypanothione biosynthetic enzymes. 100

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Materials and Methods 103 Gene ID numbers 104 The TriTrypDB gene ID numbers are as follows: GS (Tb927.7.4000), γ-GCS 105 (Tb927.10.12370), TryS (Tb927.2.4370), TR (Tb927.10.10390), ODC (Tb927.11.13730), 106 AdoMetDC (Tb927.6.4410 and Tb927.6.4460), AdoMetDC prozyme (Tb927.6.4470), SpdSyn 107 (Tb927.9.7770) and DHODH (Tb427.05.3830). 108 T. brucei cell culture 109

Blood stream form (BSF) T. brucei strain 90-13 was cultured in HMI-9 media with 10% 110 FCS at 37°C and 5% CO2 with the appropriate antibiotics (G418 (2.5 μg/ml), hygromycin (5 111 μg/ml) or phleomycin (2.5μg/ml) as required) (33). When required, Tet (1 μg/ml) was added to 112 cultures fresh daily. Cells were split every 1 - 3 days to maintain log phase growth (103-106 113 cells/ml) and cell densities were calculated with a hemocytometer (Fisher). Growth curves 114 represent total cell number, which is a product of cell density and total dilution. 115 Genomic DNA Purification 116

Genomic DNA was isolated from T. brucei 427 BSF cells (1.0 x 108) harvested by 117 centrifugation and washed 3x with phosphate buffered saline (PBS) (137 mM NaCl, 2.7 mM 118 KCl, 10 mM Na2HPO4, 2.0 mM KH2PO4, pH 7.4). Pellet was resuspended in 500 μl DNA lysis 119 buffer (100 mM Tris-HCl (pH 8.0), 5 mM EDTA, 200 mM NaCl, 0.2% SDS, 60 μg/ml RNase 120 A), incubated for 30 min at 37°C, proteinase K (5 μl of 20 mg/ml) was added and the mixture 121 incubated at 55°C overnight. DNA was extracted by phenol/chloroform/ethanol precipitation. 122 Cloning of the TbGS construct for recombinant expression in E. coli 123

The TriTryp database contains a single gene annotated as TbGS (Tb927.7.4000). The 124 TbGS gene was synthesized and cloned into pUC57 by Genscript. The gene was PCR amplified 125


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from the plasmid to obtain a product flanked with BsaI and XbaI restriction sites. The PCR 126 product was digested with BsaI/XbaI and subcloned into BsaI-linearized pE-SUMO(Kan) (Life 127 Technologies), generating the pE-SUMO-TbGS construct for expression of an N-terminally 128 tagged His6-SUMO-TbGS. 129 Protein expression and purification of SUMO-TbGS from E. coli 130

The pE-SUMO-TbGS construct was transformed into T1 phage resistant E. coli 131 BL21(DE3) competent cells for protein expression. Protein expression was induced at A600 of 0.5 132 with isopropyl β-D-1-thiogalactopyranoside (200 uM, Sigma) for 18 h at 14°C. Cells were 133 harvested by centrifugation (1000 x g for 0.5 h). Pellet was resuspended in Buffer A (25 mM 134 Hepes pH 7.5, 300 mM NaCl, 1 mM MgCl2, 5% glycerol) and supplemented with protease 135 inhibitor mix (1 μg/ml Leupeptin, 2 μg/ml Antipain, 10 μg/ml Benzamidine, 1 μg/ml Pepstatin, 1 136 μg/ml Chymostatin, 200 μM PMSF). Cells were lysed by high-pressure disruption (EmulsiFlex-137 C5, Avestin), the lysate was clarified by centrifugation (15,000 x g for 0.5 h), and protein was 138 purified by Ni+2- affinity chromatography (HiTrap Chelating HP column, GE Life Sciences) 139 using an AKTA FPLC (GE Life Sciences). The column was washed with Buffer A plus 10 mM 140 imidazole before protein elution with Buffer A plus 250 mM imidazole. Fractions were analyzed 141 by SDS-PAGE and those containing His6-SUMO-TbGS were pooled and dialyzed against Buffer 142 A (16h, 4°C). To remove the His6-SUMO tag, Ulp1 (20 ug/ml) was added and the mixture was 143 incubated at 30° for 2 h. Protein was concentrated (Centricon-30, Millipore) and TbGS was 144 separated from the tag with a second Ni+2-affinity purification. TbGS protein fractions (flow-145 through) were pooled and dialyzed against storage buffer (25 mM Hepes pH 8.0, 150 mM NaCl, 146 1 mM MgCl2, 1mM DTT, 5% glycerol). Protein concentration was determined by absorbance at 147 280 nm using an extinction coefficient of 60.0 mM-1cm-1 (computed using ProtParam, ExPASy, 148


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Swiss Institute of Bioinformatics). The final yield was 0.8 mg of TbGS per liter of bacterial 149 culture. Ulp1 was expressed and purified as previously described (31, 32). All purification steps 150 were performed at 4°C. 151 Steady-state kinetic analysis of TbGS activity 152

TbGS activity was measured using a previously described ATP-coupled assay (34). All 153 assays were performed at 37°C on a Synergy H1 Hybrid Reader (BioTek) using UVStar 96-well 154 half-area plates (Greiner). Total reaction volume was 100 μl, containing 100 mM Tris-HCl (pH 155 8.2), 50 mM KCl, 20 mM MgCl2, 250 μM NADH, 5 mM phosphoenolpyruvate, 10 U lactate 156 dehydrogenase, 10 U pyruvate kinase and substrates. The assay was shown to be linearly 157 dependent on the TbGS concentration (range 10 - 50 nM). TbGS concentrations for substrate 158 titration experiments were 15 and 25 nM. Unless otherwise stated, substrate concentrations were 159 fixed at 3 mM γ-GC (BAC Chemicals), 30 mM Gly, or 10 mM ATP. For substrate titrations, the 160 varied substrate concentrations were as follows γ-GC (1 µM – 1 mM), Gly (100 µM – 100 mM), 161 and ATP (10 µM – 10 mM). Reactions were monitored by absorbance at 340 nM and rates were 162 fitted to the Michaelis-Menten equation using GraphPad Prism to determine Km and kcat values. 163 Cloning of the TbGS RNAi construct 164

The TbGS RNAi stem loop construct was generated according to previous methods from 165 the pLEW100 and pJM326 vectors (35). Using primers containing the appropriate restriction 166 enzyme sites (Table A1) a TbGS gene fragment (nucleotides 1-440) was PCR-amplified from T. 167 brucei 427 genomic DNA and subcloned into pLEW100 in the forward direction (using XbaI 168 and MluI) and into pJM326 in the reverse direction (using HindIII and NheI). The TbGS gene 169 along with the stuffer region was excised from pJM326 using HindIII and XbaI and ligated into 170 pLEW100 containing the first TbGS RNAi section. The construct generated contained the two 171


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fragments of the TbGS gene separated by the stuffer region in opposing directions under the 172 transcriptional control of a Tet-regulated promoter. These elements were flanked by a ribosomal 173 DNA sequence, allowing for homologous recombination into the multi-copy ribosomal locus of 174 T. brucei. Sequencing confirmed that the correct construct was obtained. 175 Cloning of GS allelic replacement constructs 176

The TbGS knockout cell lines were generated using plasmids and protocols that have 177 been previously described for gene disruption in T. brucei (23, 36, 37). Briefly, two 178 approximately 300 bp segments of the TbGS 5’ and 3’ UTR (corresponding to nucleotide -387 to 179 -61 and 1745 to 2135, respectively where base 1 represents the ATG start codon and base 1668 180 represents the TAA stop codon) were PCR amplified using primer sets containing the 181 appropriate restriction enzymes (Table A1) and the resultant PCR fragment was cloned into 182 TOPO-Blunt (Life Technologies). The UTRs were sequentially subcloned into the pLEW13 183 vector first by introducing the 5’ UTR region using NotI/MluI followed by the 3’ UTR region 184 using XbaI/StuI. The resulting pLEW13-TbGS knockout construct A (KO-A) contained the T7 185 polymerase and neomycin resistance cassette sandwiched between the TbGS UTRs. To 186 generate the pLEW90-TbGS knockout construct B (KO-B), the TbGS KO-A vector was digested 187 with XhoI/SwaI to remove to T7 polymerase and neomycin resistance cassette, and the pLEW90 188 vector was digested with XhoI/StuI to liberate the Tet repressor and Hygromycin resistance 189 cassette (SwaI/StuI are blunt end endonucleases). The TetR and Hygromycin resistance cassette 190 were ligated into the pLEW13-TbGS KO-A forming the pLEW90-TbGS KO-B vector. 191 Cloning TbGS Tet-regulatable T. brucei expression construct 192

To generate the C-terminal FLAG-tag TbGS T. brucei expression construct, the gene was 193 PCR amplified using primers with appropriate restriction enzymes (Table A1), digested with 194


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HindIII/KpnI, and ligated into HindIII/KpnI linearized pT7-FLAG-MAT to generate the pT7-195 GS-FLAG-MAT bacterial expression vector. The GS-FLAG insert was obtained by PCR 196 amplification from this vector, digested with BamHI/HindIII and ligated to pLEW100v5 vector, 197 creating the C-terminal FLAG-tag ectopic expression vector. 198 Generation of T. brucei transgenic cell lines 199

The pLEW100-TbGS stemloop RNAi construct was transfected as previously described 200 (23). Briefly, the pLEW100-TbGS stem loop RNAi vector was linearized by EcoRV (80 μg) and 201 transfected into log phase T. brucei 90-13 bloodstream form (BSF) cells using the Amaxa 202 Nucleofector system. Phleomycin resistant cells that integrated the construct into rRNA locus 203 were selected and clonal lines were obtained through limited dilution. To induce RNAi 204 knockdown of the TbGS gene, 1 μg/ml of Tet was added every 24 h during experiments. 205

To generate the TbGS double knockout (DKO) cell line, log phase bloodstream 427 cell 206 lines were transfected with the NotI linearized pLEW13-TbGS KO-A vector. After establishing 207 a TbGS single knockout (SKO) cell line with neomycin/G418, glutathione (80 μM) was added to 208 the medium and parasites were transfected with the NotI linearized pLEW90-TbGS KO-B 209 vector. The resulting transfectants were established in medium containing glutathione, 210 neomycin, and hygromycin. Analysis demonstrated that these clones retained a copy of the wild-211 type allele of the gene. 212

To generate the TbGS conditional double knockout (cDKO) cell line, TbGS SKO cells 213 were transfected with Tet-regulated TbGS ectopic expression vector and selected in the presence 214 of phleomycin. The resulting cell lines, single knockout with inducible TbGS (SKO+iGS), were 215 investigated for expression of a FLAG-tagged TbGS protein. After expression of the ectopic 216 copy of TbGS had been established, parasites were transfected with the pLEW90-TbGS KO-B 217


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vector in the presence of Tet to generate the TbGS cDKO cell line. Limited dilution was used to 218 obtain clonal lines and parasites were maintained in medium that contained Tet, hygromycin, 219 neomycin, and phleomycin. 220 Collection of TbGS cDKO growth curves 221 TbGS cDKO growth was evaluated by first washing cells twice with Tet-free media 222 followed by plating into fresh media at a density of 1-5 x 104 cells/ml with or without Tet. Cells 223 were counted on the days indicated and split into fresh media every 24 - 48 h, dependent on 224 growth. 225 Western blot analysis 226 Parasites (~108) were harvested by centrifugation (1900 x g for 10 min) and washed 3x 227 with cold (4°C) PBS pH 7.4. Pellets were resuspended in protein lysis buffer (50 mM HEPES, 228 pH 8.0, 100 mM NaCl, 5 mM β-mercaptoethanol, and protease inhibitor mix (described above). 229 Cells were lysed by three freeze/thaw cycles and clarified by high-speed centrifugation using a 230 bench top centrifuge. Protein concentration was determined by a colorimetric protein assay 231 (BioRad). 232 Protein obtained from samples were separated by a 12% SDS-PAGE gel and transferred 233 from the gel to a polyvinylidene difloride (PVDF) membrane (Hybond-P, Amersham) using a 234 wet transfer at 4°C for one hour using 100 V. Membranes were blocked in 5% non-fat dry milk 235 in Tris-buffered saline (20 mM Tris-HCl, 137 mM NaCl, pH 7.6) overnight at 4°C. Primary 236 rabbit TbGS polyclonal antibodies were generated under contract by Proteintech Group Inc 237 (Chicago, IL) from recombinant His6-TbGS protein isolated as above. Antibodies against 238 polyamine and trypanothione biosynthetic enzymes were used as previously described (22, 23, 239 25, 38, 39). Dilutions for primary antibodies in 5% Milk in TBS-T were: α-TbGS – 1:2,500, α-240


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TbDHODH – 1:10,000, α-FLAG – 1:1,000 (Sigma), α-TbAdoMetDC – 1:2,500, α-Tbprozyme – 241 1:5,000, α-TbODC – 1:10,000, α-LdSpdSyn – 1:1,000, α-TbTryS – 1:1,000, and α-Tbγ-GCS – 242 1:10,000. The TbTryS antibody was a gift from Dr. Alan Fairlamb (University of Dundee) and 243 the LdSpdSyn antibody was a gift from Dr. Buddy Ullman (Oregon Health and Science 244 University). Secondary antibodies (anti-rabbit, anti-rat (TryS), and anti-mouse (FLAG)) were 245 used at 1:10,000 dilution in 5% nonfat milk TBS-T. For detection of protein by multiple 246 antibodies, parallel gels were loaded with 20-40 μg total protein per well, transferred, and each 247 membrane was sectioned and probed with a different antibody. Each membrane was used to 248 assess protein levels of two biosynthetic pathway proteins along with the DHODH loading 249 control. Protein levels were visualized by chemiluminescence detected when incubating the 250 membrane with ECL HRP substrate (ThermoFisher) and exposing to film (Fisher). 251 Bands on western blots were quanitified using ImageJ software. High resolution scans of 252 film were loaded into ImageJ and analyzed correcting for background using an area of the 253 corresponding film that was signal-free. Each band was quantified as a ratio to the DHODH 254 loading control, and the value was compared to the protein levels expressed on day 0. 255 Intracellular polyamine determination 256

Analysis of polyamines was done as previously described (23). For analysis of 257 intracellular polyamines, 1 x 107 cells were harvested by centrifugation (3000 rpm. for 10 min), 258 washed twice with 1 ml of PBS pH 7.4 and then resuspended in 25 μl polyamine lysis buffer 259 (100 mM MOPS, pH=8.0, 50 mM NaCl, 20 mM MgCl2). Cells were lysed by 3 freeze/thaw 260 cycles followed by acid precipitation of proteins by addition of 7.5 μl of 40% trichloroacetic acid 261 (TCA) and incubation on ice for 10–15 min. Cell debris was removed by centrifugation (20000 262 x g). Supernatant was fluorescently labeled using AccQ-Fluor reagent (6-aminoquinolyl-n-263


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hydroxysuccinimidyl, Waters) by incubating 5 µl of sample supernatant with 20 µl labeling 264 reagent and 75 µl borate buffer at 55°C for 10 min. Polyamines were separated by HPLC using a 265 Waters AccQtag (3.9x150mm) column run on a Beckman System Gold HPLC with a Rainin 266 Dynamax Fluorescent detector. Peaks were separated using a previously described linear 267 gradient with eluent A containing 450 mM sodium acetate pH 4.95, 17 mM triethylamine (TEA), 268 0.01% sodium azide, and eluent B containing 60% acetonitrile and 0.01% acetone (23, 24, 40). 269 The gradient was as follows: 0% B for 5 min, 20% B 45 min, 50% B 5 min, 100% B 3 min, 0% 270 B over 1 min, 0% B for 10 min. Polyamines were identified by retention time and quantified by 271 peak area in comparison to standards run under the same conditions. 272 Intracellular thiol determination 273

Reduced intracellular thiols were determined as described with slight modification (41, 274 42). Briefly, cells (1 x 108) were harvested by centrifugation (3000 rpm, 10 min at RT) and 275 washed twice with pre-warmed HMI-9 medium lacking fetal bovine serum (FBS), β-276 mercaptoethanol, and L-Cys. A final wash was done with cold (4°C) PBS pH 7.4 and cells were 277 pelleted by centrifugation (3000 rpm for 5 min). Cell pellets were immediately resuspended in 278 150 µl ice cold 5% TCA (trichloroacetic acid) in 10 mM HCl and incubated on ice for 5-10 min. 279 Denatured proteins and cell debris was removed by centrifugation at 4°C. The supernatant was 280 extracted four times with 450 μl ice-cold diethyl ether to remove excess TCA and 345 μl of 40 281 mM HEPPS (3-[4-(2-Hydroxyethyl)-1-piperazinyl]propanesulfonic acid) pH 8.0 containing 4 282 mM DTPA (diethylene triamine pentaacetic acid) was added to the extractant. 283

To derivatize thiols in the extracted supernatant, 5 µl of 200 mM monobromobimane was 284 added for a final concentration of 2 mM and vortexed. Samples were incubated at RT for 10-15 285


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min in the dark. To stop the labeling reaction and prevent oxidation of samples, 2.5 µl of 5 M 286 MSA (methanesulfonic acid) was added. Samples were stored at -80°C until analysis by HPLC. 287

HPLC analysis used a Phenomenex Kinetex 2.6 μ C18 column with a constant flow rate 288 of 1.25 ml/min at room temperature using gradient mixtures of eluent A (0.25% 289 camphorsulphonic acid (CSA) pH 2.64) and eluent B (0.25 % CSA pH 2.64 and 25% 1-propanol 290 both) as follows: 0% eluent B for 1 min, 0% to 5% eluent B over 0.5 min, 5% eluent B for 4.5 291 min, 5% to 12.5% eluent B over 29 min, 12.5% to 35% eluent B over 12 min, 35% to 60% eluent 292 B over 2 min, 60% eluent B for 4 min, then 60% eluent B to 0% eluent B over 1 min. The 293 column was re-equilibrated for 20 min between samples using the same flow rate and 0% eluent 294 B. Thiols were identified by retention time and quantified by reduced standard’s peak area. 295 Evaluation of the anti-trypanosomal activity of BSO 296

Parasites were grown with or without Tet for 2 d, quantified, and diluted to 1 x 103 297 cells/ml. Parasites were plated into 96 well plates (Greiner) with 200 μl/well. BSO 298 concentrations were varied from 0 to 3 mM using 10 different concentrations and incubated with 299 parasites for 72 h. Cell viability was measured using CellTiter-Glo (Promega), an 300 ATP/luminescence assay. BioTek Synergy H1 Hybrid reader measured cell viability in the form 301 of luminescence. Data were fitted by nonlinear regression to the log (agonist) v. response (four 302 parameter fit) in GraphPad Prism to determine EC50 for 3 replicates at each concentration. Final 303 data represent the average of three independent experiments and the standard error of the mean is 304 reported. The P value was calculated using a two-tailed unpaired t test with equal SD using 305 GraphPad Prism. 306 FACS analysis/Live/Dead cell assay 307


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This protocol was based on previously published methods (43) and optimized for TbGS 308 cDKO cell lines. Cells (~106) were harvested by centrifugation (3000 rpm for 10 m) and washed 309 using pre-warmed (37°C) PBSG (PBS plus 10 mM glucose, pH 7.4). Cells were then 310 resuspended in 500 μl PBSG and calcein and ethidium homodimer were added to final 311 concentrations of 50 nM (Life Technologies). Cells were incubated for 15-20 min at 37°C and 312 then analyzed immediately by FACS. Samples were analyzed on a Beckman Coulter FACS 313 Calibur flow cytometer set to measure forward and side scatter to determine cell size and 314 morphology as well as fluorescence using filters 530/40 (calcein) and 692/40 (ethidium). Data 315 obtained were analyzed using FlowJo software with single-stained cells used for compensation 316 of spectral overlap within the software. Eflornithine treated cells were used to determined FACS 317 gating for unhealthy populations while gating for healthy cells was determined by unstained 318 proliferating cells. 319 320 on M

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Results 321 322 Recombinant expression and steady-state kinetic analysis of TbGS 323

T. brucei GS was expressed as a His6-SUMO-tagged fusion protein in E. coli, purified by 324 Ni+2-agarose affinity column chromatography and the N-terminal fusion was then removed by 325 the protease Ulp1. The purified recombinant protein was tested for GS activity using an 326 ATPase-coupled assay. The reaction was well modeled by simple Michaelis-Menten kinetics. 327 TbGS catalyzed the synthesis of glutathione with a kcat between 7.0 – 7.3 s-1 (Table 1), which is 328 comparable to previously published eukaryotic GS enzymes from humans, plants and T. cruzi 329 (44-46). The apparent Km (Km.app) values were measured for each of the three substrates at fixed 330 concentrations of the other substrates (Table 1). Km.app for γ−GC,glycine, and ATP were similar 331 to reports for other GS homologs. 332 Evaluation of the essentiality of TbGS by RNA interference (RNAi) 333

In order to decipher the role of TbGS in the trypanothione biosynthesis pathway, a Tet-334 inducible TbGS RNAi stem loop BSF cell line was generated and the effects of gene knockdown 335 were studied. Upon addition of Tet (1 μg/ml), the TbGS RNAi stem-loop was expressed and 336 TbGS protein levels were reduced by 80-90% based on western blot analysis (Fig. 2). Because 337 trypanothione biosynthesis enzymes were previously shown to be essential in T. brucei (22, 25), 338 we anticipated cells would display a growth phenotype upon reduction of TbGS protein levels. 339 However, TbGS RNAi cell lines continued to grow at the same rate as wild-type cells (Fig. 2) 340 and the reduced intracellular thiol levels as measured by HPLC remained unchanged (Fig. A1). 341 Three independent clones were evaluated and all showed the same phenotype. Our previous 342 studies showed that the addition of glutathione to the cultures was sufficient to rescue the RNAi 343 knockdown of γ-GCS (22) so to rule out the possibility that glutathione in the medium was 344


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leading to rescue of cell growth we analyzed glutathione levels using HPLC and verified that 345 medium did not contain glutathione (data not shown). These results suggested that even though 346 the TbGS protein levels were reduced, TbGS was not sufficiently depleted by the RNAi 347 mechanism to yield a phenotype. 348 Evaluation of the essentiality of TbGS by gene knockout 349

To generate a more robust gene knockdown that would allow investigation of the role of 350 TbGS in trypanothione biosynthesis, we generated TbGS knockout cell lines. TbGS is a single 351 copy gene, but T. brucei is a diploid organism, requiring that any knockout approach target both 352 alleles. Initially we attempted to knock out both alleles of the TbGS gene in the absence of a 353 regulatable copy of the gene, but in the presence of exogenous glutathione (80 μM). A cell line 354 was selected that exhibited resistance to both selectable markers used to target the TbGS gene 355 (neomycin and hygromycin). However, Southern blot showed that the cells retained the TbGS 356 gene along with the 2 resistance genes, and thus it was not possible to select cells with a 357 knockout of TbGC under these conditions (Fig. A2). This phenomenon has been previously 358 described in Leishmania and is suggestive of an essential gene (47). Therefore, in order to 359 investigate the role of TbGS a conditional double knockout (cDKO) was generated by 360 replacement of the first allele with the neomycin resistance gene, followed by insertion of a Tet-361 regulatable ectopic copy of TbGS into the ribosomal repeat region of the genome and then 362 knockout of the second TbGS allele with the hygromycin resistance gene. Cultures were grown 363 in the presence of Tet to maintain TbGS expression during cloning and selection of the cDKO 364 cell line. 365

The effects of TbGS depletion on growth of the cDKO cell line were studied. Removal of 366 Tet from the media of TbGS cDKO cells led to reproducible growth arrest that began on day 4 367


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and lasted through day 6, after which growth resumed at rates similar to the +Tet controls (Fig. 368 3A). TbGS protein levels were decreased to less than 3-4% of control levels by day 2 and were 369 undetectable (97% of TbGS must be depleted to maintain cell growth arrest. 374

We next tested the effects of adding either exogenous glutathione or glutathione ethyl 375 ester to determine if the cell growth effects caused by loss of TbGS expression could be reversed. 376 Glutathione ethyl ester was shown to be more readily transported into some mammalian cells 377 than glutathione (46). Exogenous glutathione at 80 uM did not rescue the growth defect that 378 occurred upon TbGS depletion consistent with the finding that we could not generate the DKO 379 cell line in the absence of the Tet regulated copy of TbGS. We found that concentrations of 380 glutathione and glutathione ethyl ester above 1 mM were toxic to the cells so additional rescue 381 experiments were carried out using the maximum tolerated concentration of 1 mM. Glutathione 382 at 1 mM, but not glutathione ethyl ester, was able to partially rescue the cell growth phenotype 383 that occurred upon Tet removal and loss of TbGS expression (Fig. 4). Cells grown with 384 glutathione ethyl ester (1 mM) did show slightly cell numbers compared to control cells but 385 growth rates were comparable suggesting that toxicity of the ester did not contribute to the lack 386 of rescue. 387 TbGS knockout leads to trypanothione but not polyamine depletion 388

Intracellular thiol and polyamine levels were measured at different time points after Tet 389 withdrawal from the TbGS cDKO cell lines (Fig. 3C and 3D). While polyamine levels remained 390


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relatively unchanged, trypanothione levels were depleted by 50% on day 3 and by 100% on day 391 4, corresponding to the time point where cells stopped growing. Trypanothione levels returned to 392 normal by days 8 - 10 corresponding to the point where TbGS protein was again detected and 393 cell growth resumed. Glutathionylspermidine levels were 3-4% of trypanothione levels, and were 394 depleted with a similar time course to trypanothione after Tet removal (data not shown). Thus, 395 the observed growth arrest correlates with both depletion of the TbGS protein and the complete 396 loss of the reduced trypanothione and glutathionylspermidine pools, supporting the conclusion 397 that TbGS is an essential protein required for trypanothione production. 398 Effects of TbGS knockout on expression of polyamine and trypanothione biosynthetic enzymes 399

The levels of other polyamine and trypanothione biosynthetic pathway proteins were 400 measured by western blot to determine if depletion of TbGS protein led to any potential 401 regulatory responses (Fig. 3B and Fig. A3). While no changes were observed in SpdSyn, TryS 402 or AdoMetDC protein levels, γ-GCS protein levels were increased (2-3-fold) and AdoMetDC 403 prozyme and ODC levels were reduced on days 4 and 5 (3-fold for prozyme and 1.5 fold for 404 ODC compared to day 0, and up to 6-fold for prozyme from the peak at day 3). The observed 405 changes in expression correlated with the time point where trypanothione levels were most 406 reduced. The observed trends were reproducible as analyzed for three independent biological 407 replicates, though the effects were not always observed on the same day after Tet withdrawl. 408 TbGS cDKO cells have increased sensitivity to a pathway inhibitor 409

To determine of knockdown of TbGS sensitized cells to inhibitors of glutathione 410 biosynthesis, the TbGS cDKO cells were treated with the γ-GCS inhibitor buthionine 411 sulfoximine (BSO). TbGS cDKO cells were grown without Tet for 2 days to deplete GS and 412 trypanothione before incubation with various levels of BSO. The EC50 for BSO was increased 3-413


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fold in the absence of Tet relative to the control TbGS cDKO cells that were grown in the 414 presence of Tet, which was shown to be a statistically significant effect (Fig. 4A). After 415 resumption of growth around day 8, cells displayed similar EC50 values to control cells. 416 Re-expression of TbGS in the cDKO TbGS cell line was not the result of genetic mutation 417

To assess if parasites had acquired a genetic mutation leading to loss of Tet-regulation, 418 TbGS cDKO parasites capable of growing 10 days after removal of Tet were used to generate 419 clonal lines using limited dilution. These cell lines were then grown in media containing Tet for 420 3 days prior to removal of Tet to monitor the effects on cell growth (Fig. A4). Re-cloned 421 parasites underwent a period of stalled growth after the removal of Tet similar to the original 422 clonal lines suggesting that escape from Tet-regulation did not result from an inheritable genetic 423 mutation. 424 FACS analysis of cDKO cells provides evidence for multiple populations 425

We investigated the possibility that clonal cDKO cell lines contained multiple 426 populations with variable responses to Tet withdrawal that would suggest that not all cells in the 427 population experienced the same level or kinetics of TbGS depletion. A previously described 428 live/dead cell based fluorescence activated cell sorting (FACS) assay (43) was used to evaluate 429 the TbGS cDKO cell line for mixed populations of live versus dead or dying cells. Ethidium and 430 calcein staining were used for the analysis. Ethidium enters cells with compromised membranes 431 and becomes fluorescent upon binding to nucleic acid indicating dead or dying cells. Calcein is 432 a cell permeable non-fluorescent compound that is cleaved by non-specific esterases upon 433 crossing the cell membrane to produce a fluorescent compound that is retained within the cell. 434 Cells that are positive for calcein have retained esterase activity, and calcein is considered to be a 435 live cell marker. 436


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Cells were analyzed using side-scatter (SSC) and forward-scatter (FSC) followed by 437 FACS analysis of calcein and ethidium fluorescence in the FL1 and FL3 channels, respectively 438 (Fig. 5). Control cells contained 94% calcein only positive cells, consistent with a healthy 439 growing cell population, 5% cells double labeled with ethidium/calcein, which we define as 440 unhealthy, and there was no evidence for completely dead cells (ethidium-positive only) (Fig. 5A 441 and Fig. A6). Cells treated with the anti-trypanosomal drug eflornithine (200 µM) for 24 h on the 442 other hand showed an increase in cells with higher side-scatter, and an increase in cells staining 443 positive with ethidium suggesting they were dead or in the process of dying (Fig. 5A). 444 Eflornithine treated cells were used to define the gating regions between the unhealthy and 445 healthy populations of cells. 446

To analyze and compare GS cDKO cells by FACS at different time points after Tet 447 withdrawal, three staggered growth curves were set up where Tet was removed at 24 h intervals 448 allowing up to three different time points to be analyzed together by FACS on the same day. 449 The growth curves from the study were identical with growth arrest occurring 4 d after Tet 450 withdrawal (Fig. A5). As the TbGS cDKO cells progressed into growth arrest (days 3-5 after Tet 451 withdrawal), a new population of cells was observed on the FSC v. SSC plot (quadrant marked 452 as unhealthy). This new population fell within the same region as control dead parasite cells that 453 had been treated with eflornithine and that stained positive for ethidium. When the two 454 populations were analyzed for ethidium and calcein staining (gated populations), the unhealthy 455 population showed a mixture of cells that were either ethidum positive alone (dead) or stained 456 positive for both ethidium and calcein, suggesting cells with damaged membranes that still 457 contained esterase activity. The percentage of ethidium or dual ethidium/calcein cells increased 458 on day 3 and peaked on days 5 after Tet withdrawal (Figs. 5B and 5C). On day 5 ~75% of the 459


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cells were in the dead or dying population, corresponding to the time point of most significant 460 growth arrest and to greatest trypanothione depletion. These data confirm that that TbGS is 461 essential for parasite survival. However, 25% of cells were not yet compromised enough by 462 trypanothione depletion to stain with ethidium in this analysis. These data suggest that Tet 463 withdrawal from the GS cDKO cells results in minimally two cell populations, one that is 464 dead/dying and the other that shows delayed response to GS knockdown, allowing these cells 465 time to upregulate GS expression and escape selective pressure. 466

467 468


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Discussion 469 Polyamine and trypanothione biosynthesis has been shown to be a key pathway for 470

proliferation and infectivity of the African trypanosome, T. brucei (6). Knockdown of pathway 471 proteins by gene disruption using RNAi leads to growth arrest, and several inhibitors of 472 polyamine biosynthesis have been shown to have anti-trypanosomal activity, the most important 473 to date being the ODC inhibitor eflornithine that is currently a front-line treatment for late stage 474 T. brucei gambiense infection. The polyamine and glutathione pathways are highly regulated in 475 eukaryotic cells, however, the regulatory mechanisms that have been observed in other 476 eukaryotes do not seem to be present in T. brucei. Instead, polyamine biosynthesis is uniquely 477 regulated by the presence of a novel regulatory subunit of AdoMetDC, which activates the 478 enzyme and also appears to be translationally regulated (23, 30, 31). Less is known about how 479 the glutathione branch of the trypanothione biosynthetic pathway is regulated. Herein we show 480 that TbGS encodes a functional GS enzyme that is essential for growth of mammalian blood 481 form parasites, and we demonstrate that knockdown of TbGS leads to compensatory effects on 482 the expression levels of other pathway enzymes. 483

Knockout of the TbGS gene in mammalian blood stage parasites led to >99% reduction 484 in TbGS protein levels, cell growth arrest and complete depletion of trypanothione pools. These 485 data show that like other enzymes in the pathway, TbGS is essential for parasite growth and 486 confirm that trypanothione is an essential cofactor. The observed increase in sensitivity to the γ-487 GCS inhibitor BSO is also consistent with increased oxidative stress in the cells after depletion 488 of TbGS. As was observed for knockout of TR (48), knockout of TbGS led to an initial stalling 489 of growth but eventually parasites were able to escape the selective pressure leading to re-490 expression of TbGS and growth restoration. Cells that escaped growth arrest were recloned and 491


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retained sensitivity to Tet withdraw and GS depletion suggesting that escape did not result from 492 a genetic mutation in the Tet promotor. Instead, epigenetic differences between different 493 subpopulations of the TbGS cDKO cells may lead to cell survival. Similarly, in the TR knockout 494 line gene deletions or nucleotide mutations were not identified, and growth restoration was 495 postulated to be due to epigenetic effects on TR expression (48). In another example, in the 496 conditional knockout of UDP-Glc-4'-epimerase, deletion of the TetR gene was responsible for 497 the growth restoration phenotype (49). 498

FACS analysis using live cell and dead cell markers was used to provide insight into the 499 mechanism of escape and regrowth that occurred after TbGS knockdown. These data suggest that 500 at least two cell populations arise after TbGS expression is shut off by Tet withdrawal. At days 4 501 and 5 after loss of GS expression the cells are in a transition. While most of the TbGS-depleted 502 cells (75%) stain with ethidium homodimer by day 4, suggesting that they have compromised 503 membranes and are committed to cell death, a significant proportion of cells (25%) do not. Thus, 504 at least by this marker of cell death, 25% of the cells appear not to be severely compromised by 505 depletion of TbGS and trypanothione pools. This result could be explained by differences in the 506 kinetics of TbGS depletion in the two cell populations, the result of a stochastic process or 507 epigenetic differences. If trypanothione depletion in this subset of cells is delayed, they may not 508 have experienced trypanothione depletion for long enough to lead to cell damage. While FACS 509 analysis provides data about individual cell populations, the growth curve analysis and 510 trypanothione measurements can show only average results. Trypanothione levels on day 3 are 511 reduced by about 50%. That could be interpreted to mean that all cells had lost half their 512 trypanothione content, or it could reflect an average value between a cell population that was 513 fully depleted of trypanothione and a second population that lags behind and still retains a 514


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substantial amount. It may take at least 24 h of trypanothione depletion before cells become 515 ethidium-positive, so only cells that reached complete depletion faster (by day 3) have been 516 irreversibly damaged before TbGS expression is upregulated, trypanothione pools are restored 517 and growth reversal occurs. For the fraction of cells where onset of trypanothione depletion is 518 slower they will have more time for an epigenetic event to cause upregulation of GS expression 519 before irreversible damage has been done. Our data suggest that upregulation of GS by only a 520 few percent would be enough to restore trypanothione biosynthesis to the parasites leading to 521 normal growth. These data highlight the importance of considering individual cell populations 522 and not just an average response in order to fully understand the mechanism and function of 523 analyzed genes. 524

In previous studies, we demonstrated that the first enzyme in the glutathione biosynthetic 525 pathway γ-GCS was essential for parasite growth (22). In these studies we also showed that 526 exogenously added glutathione was able to rescue cell growth arrest that occurred after γ-GCS 527 knockdown by RNAi. The ability of glutathione to similarly rescue the TbGS double knockout 528 suggests that like in yeast (27, 50) glutathione may be transported into the parasite intact, though 529 there is no obvious homolog of the yeast-like glutathione transporter in the T. brucei genome. 530 These data are in contrast to the glutathione transport mechanism that has been described in 531 mammalian cells, which use a transpeptidase transporter mechanism that couples glutathione 532 cleavage to cysteinyl-glycine and γ-glutamyl with conjugation of the γ-glutamyl moiety to an 533 amino acid, and subsequent uptake of the respective products of both reactions (27, 50). Notably 534 the glutathione levels (1 mM) that were required to rescue growth after TbGS knockdown are 535 higher than is present in human plasma ( range 4 – 40 uM) (www.hmdb.ca;(15, 20)) so parasites 536 would be unable to overcome inhibition of GS through uptake of exogenous glutathione in the 537


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blood. However, quantitation of protein expression levels in the TbGS cDKO and RNAi lines 538 suggests that >97% depletion of TbGS is required before trypanothione is depleted and cells 539 begin to die, showing that a small molecule inhibitor of TbGS would need to nearly fully inhibit 540 the enzyme. This is in contrast to ODC and AdoMetDC where ~80% inhibition is sufficient (23, 541 30). 542

Regulatory control of the polyamine biosynthetic pathway in T. brucei appears to center 543 around the AdoMetDC regulatory subunit prozyme, which responds to decarboxylated AdoMet 544 depletion by upregulation of prozyme expression and to a lesser extent ODC (23, 30). 545 Interestingly, knockdown of TbGS led to a decrease in prozyme and ODC protein and to an 546 increase in γ-GCS protein. These data suggest that the cells respond to trypanothione depletion 547 by increasing levels of the first enzyme required for glutathione synthesis in a classical feedback 548 type mechanism, though whether the metabolic trigger is glutathione or trypanothione levels 549 remains unknown. The concomitant decrease in levels of two key polyamine biosynthetic 550 enzymes appears to be a cell response to limit buildup of polyamine intermediates in the 551 situation where trypanothione is no longer a sink for spermidine flux. Spermidine levels remain 552 unchanged after depletion of TbGS, consistent with this result. In our previous study, we showed 553 that decarboxylated AdoMet levels were inversely proportional to AdoMetDC prozyme levels, 554 suggesting that elevated decarboxylated AdoMet leads to reduced prozyme expression (30). 555 SpdSyn from human and Plasmodium have been shown to be feedback inhibited by 556 methylthioadenosine, a reaction product of SpdSyn chemistry (51, 52) and both spermidine and 557 methylthioadenosine have been demonstrated to bind SpdSyn by crystallographic studies (53). 558 Thus, a working model that explains the available data suggests when T. brucei cells no longer 559 require spermidine for trypanothione biosynthesis, spermidine or methylthioadenosine might 560


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feedback inhibit SpdSyn, leading to build up of dcAdoMet and to down regulation of prozyme 561 expression. Down regulation of ODC would then also be needed to maintain constant putrescine 562 levels, since in this model putrescine flux into spermidine would be decreased. Direct studies 563 with T. brucei SpdSyn would be required to confirm this hypothesis. 564

In conclusion TbGS is an essential protein in T. brucei and our studies suggest that >97% 565 inhibition of the protein will be required to lead to cell growth arrest. We find that glutathione is 566 unable to rescue the TbGS cDKO cell line at physiological concentrations, providing a path 567 forward for exploring TbGS as a potential drug target in the parasite. We also find that 568 knockdown of TbGS leads to an increase in γ-GCS, suggesting that γ-GCS may be the key 569 regulatory control point for glutathione biosynthesis. Additionally, prozyme and ODC levels 570 decrease, providing additional evidence that the AdoMetDC prozyme is the key regulatory 571 protein in the polyamine biosynthetic pathway in T. brucei and providing evidence for cross talk 572 between the polyamine and glutathione branches of the pathway. 573 574 on M

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Acknowledgements: This work was supported by National Institutes of Health grants 575 (R37AI034432) (to MAP) and the Welch Foundation grant I-1257 (to MAP). MAP holds the 576 Beatrice and Miguel Elias Distinguished Chair in Biomedical Science and the Carolyn R. Bacon 577 Professorship in Medical Science and Education. The authors thank Dr. Alan Fairlamb 578 (University of Dundee) for supplying the TbTryS antibody and Dr. Buddy Ullman (OHSU) for 579 supplying the LdSpdSyn antibody. 580 581


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50. Bourbouloux A, Shahi P, Chakladar A, Delrot S, Bachhawat AK. 2000. Hgt1p, a 721 high affinity glutathione transporter from the yeast Saccharomyces cerevisiae. J Biol 722 Chem 275:13259-13265. 723

51. Haider N, Eschbach ML, Dias Sde S, Gilberger TW, Walter RD, Luersen K. 2005. 724 The spermidine synthase of the malaria parasite Plasmodium falciparum: molecular and 725 biochemical characterisation of the polyamine synthesis enzyme. Mol Biochem Parasitol 726 142:224-236. 727

52. Wu H, Min J, Ikeguchi Y, Zeng H, Dong A, Loppnau P, Pegg AE, Plotnikov AN. 728 2007. Structure and mechanism of spermidine synthases. Biochemistry 46:8331-8339. 729

53. Ikeguchi Y, Bewley MC, Pegg AE. 2006. Aminopropyltransferases: function, structure 730 and genetics. J Biochem 139:1-9. 731

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Table 1. Steady-state kinetic parameters of recombinant TbGS 735 736

γ-GC Glycine ATP

kcat (s-1) Kmapp (mM) kcat/Km,app Kmapp (mM) kcat/Km,app Kmapp (mM) kcat/Km,app

T. brucei 7.1 ± 0.1 0.040 ± 0.004 1.8x105 1.5 ± 0.1 4.7x103 0.075 ± 0.006 9.5x104

T. cruzia 4.25 0.04 1.1x105 1.2 3.5x103 0.03 1.4x105

H. sapiensb 6.5 0.66 9.9x103 1.75 3.7x103 0.07 9.3x104

A. thalianac 12.3 0.039 3.2x105 1.51 8.2x103 0.057 2.2x105

Error represents the standard error for n=6. Units for kcat/Km,app are M-1 s-1. Published values 737 from a(11); b(45); c(44). 738 739


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Figure Legends 740 741 Figure 1. Trypanothione biosynthetic pathway in T. brucei. GS, glutathione synthetase; γ-742 GCS, γ-glutamylcysteine synthetase; TryS, trypanothione synthetase; TR, trypanothione 743 reductase; ODC, ornithine decarboxylase; AdoMetDC, S-adenosylmethionine decarboxylase 744 Figure 2 TbGS RNAi growth curve. RNAi was induced using a Tet-inducible stem loop 745 vector. Tet was added every 24 h to maintain knockdown. Protein levels were evaluated by 746 western with DHODH as a loading control. Data are shown for three replicates and error bars 747 represent the standard error of the mean, though because of the log plot they are typically not 748 visible. 749 Figure 3. Analysis of TbGS cDKO cell line. A. Growth curve of the TbGS cDKO cell line in 750 the presence and absence of Tet. For the +Tet group, Tet was added daily to maintain expression 751 of the ectopic copy of TbGS protein. Cell number is represented as the product of cell density 752 and total dilution. Three clonal lines were evaluated with triplicate data collected for each. A 753 representative data set for one line is displayed. B. Representative western blots of selected 754 polyamine and glutathione biosynthetic enzymes. For the prozyme antibody * marks the specific 755 prozyme band, whereas the band above arises from non-specific labeling. DHODH was used as a 756 loading control. C. Intracellular polyamine levels measured by HPLC. Data for 3 biological 757 replicates, each in quadriplicate, (n = 12 in total) is displayed. D. Intracellular reduced 758 trypanothione levels measured by HPLC. Data for 3 biological replicates, each in triplicate, (n = 759 9 in total) is displayed. Error bars represent the standard error of the mean. 760 Figure 4. Effects of glutathione biosynthetic inibitors and glutaione on the GS cDKO line. 761 A. Knockdown of GS increases sensitivity to BSO. The EC50 of BSO was measured on cells 762


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grown in the presence (EC50 = 270 ± 14 uM) and absence of Tet (EC50 = 88 ± 25 uM). Cells 763 were grown without Tet for 2 days to deplete GS and trypanothione before incubation with 764 various levels of BSO. EC50 values were measured independently three times and were 765 calculated in each case from triplicate data points. The plot represents the mean of the three 766 experiments and error bars are the standard error of the mean. The P values were calculated 767 using the student test. B. Metabolic rescue of growth in the GS cDKO cell line. Glutathione (1 768 mM) or glutathione ethyl ester (1 mM) were added to parasite cultures simultaneously to the 769 removal of Tet and the effects on cell growth were monitored. The +Tet control is included as a 770 comparator. Data were collected in triplicate and errors represent the standard error of the mean. 771 Figure 5. FACS analysis of TbGS cell line. A. Analysis of control TbGS cDKO cells (+Tet) in 772 the absence and presence of eflornithine (200 µM) using forward scatter v. side scatter to 773 observe cell shape and morphology and FL1 v. FL3 channels to observe calcein or ethidium 774 positive cells, respectively. B. Bar Graph summarizing the effects of TbGS knockdown (-Tet) 775 on the percentage of cells that are either calcein positive (white bar), ethidium positive (grey bar) 776 or calcein and ethidium double positive (hatched bar). Numbers were obtained from ungated 777 fluorescent populations where unstained cells set quadrants. Data represent the average and 778 standard deviation of the mean for n=3 independent experiments C. Representative samples of 779 FACS data obtained from TbGS cDKO cells on days 3, 5 and 7 minus Tet. Ungated populations 780 forward v. side scatter plots are shown on the left while the ungated fluorescent populations are 781 shown in left center plots. Within the fluorescent population plots, the upper left quadrant 782 represents cells staining with ethidium only, the lower right quadrant represents cells staining 783 with calcein only, and the upper right quadrant represents cells that stain with both ethidium and 784 calcein. The populations that were gated by their forward versus side scatter plot (gates shown in 785


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first plot) are shown in the two right hand columns. The health/unhealthy gates were determined 786 by healthy proliferating cells and cells that had been treated with drug (eflornithine) for 24 h 787 where the majority of cells were dead (stained with ethidium only). 788 789


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Genetic validation of Trypanosoma brucei glutathione ...€¦ · 04.03.2014 · v vs Introduction vt Trypanosoma brucei is the causative agent of huma n African trypanosomiasis (HAT),