1 Auranofin resistance in Toxoplasma gondii decreases the ......2020/04/23 · 1 Auranofin...

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Auranofin resistance in Toxoplasma gondii decreases the accumulation of 1

reactive oxygen species but does not target parasite thioredoxin reductase 2

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Christopher I. Maa, James A. Tirtorahardjob, Sharon Janb, Sakura S. Schweizera, Shawn 5

A. C. Rosario1, Yanmiao Du1; Jerry J. Zhanga, Naomi S. Morrissettec and Rosa M. 6

Andradea,b# 7

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a Department of Medicine, Division of Infectious Diseases, University of California Irvine 9

b Department of Microbiology and Molecular Genetics, University of California Irvine 10

c Department of Molecular Biology and Biochemistry, University of California Irvine, 11

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Running title: Auranofin resistance in T. gondii 13

Keywords: gold, repurposing, anti-parasitic, resistance, redox, Toxoplasma, auranofin, 14

superoxide 15

#Address correspondence to Rosa Andrade, [email protected] 16

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Shawn Rosario, Yanmiao Du; Jerry Zhang contributed equally to this work. Author order 18

was determined on the basis of seniority. 19

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was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 25, 2020. ; https://doi.org/10.1101/2020.04.23.058842doi: bioRxiv preprint

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ABSTRACT: 23

Auranofin, a reprofiled FDA-approved drug originally designed to treat rheumatoid 24

arthritis, has emerged as a promising anti-parasitic drug. It induces the accumulation of 25

reactive oxygen species (ROS) in parasites, including Toxoplasma gondii. We generated 26

auranofin resistant T. gondii lines through chemical mutagenesis in order to identify the 27

molecular target of this drug. Resistant clones were confirmed with a competition assay 28

using wild-type T. gondii expressing yellow fluorescence protein (YFP) as a reference 29

strain. The predicted auranofin target, thioredoxin reductase, was not mutated in any of 30

our resistant lines. Subsequent whole genomic sequencing analysis (WGS) did not reveal 31

a consensus resistance locus, although many have point mutations in genes encoding 32

redox-relevant proteins such as superoxide dismutase (TgSOD2) and ribonucleotide 33

reductase. We investigated the SOD2 L201P mutation and found that it was not sufficient 34

to confer resistance when introduced into wild-type parasites. Resistant clones 35

accumulated less ROS than their wild type counterparts. Our results demonstrate that 36

resistance to auranofin in T. gondii enhances its ability to abate oxidative stress through 37

diverse mechanisms. This evidence supports a hypothesized mechanism of auranofin 38

anti-parasitic activity as disruption of redox homeostasis. 39

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INTRODUCTION 43

For more than 50 years, the mainstay of treatment for acute toxoplasmosis has 44

been a combination of a dihydrofolate reductase inhibitor and a sulfa antimicrobial. 45

While this is currently a critical therapeutic strategy, currently approved drugs cannot 46

eliminate latent parasites in cysts that are found in chronically infected individuals. 47

Moreover, these drugs have significant bone marrow toxicity, are suspected teratogens 48

and the emergence of resistance remains a potential threat to treatment. Repurposing 49

FDA-approved drugs will accelerate drug discovery for neglected parasitic diseases. 50

Most recently, auranofin, a reprofiled drug that is FDA-approved for treatment of 51

rheumatoid arthritis, has emerged as a promising anti-parasitic agent. It has 52

antiproliferative activity against Plasmodium falciparum (1) , Schistosoma mansoni (2), 53

Leishmania infantum (3) and Entamoeba histolytica (4) as well as other parasites with 54

public health significance (5-11). Despite this promising broad spectrum of anti-parasitic 55

activity, the molecular target of auranofin has only been indirectly implicated as 56

thioredoxin reductase. 57

Thioredoxin reductase enzymes are found in archaea, bacteria and diverse 58

eukaryotes and play a key role in reduction of disulfide bonds that is essential for cell 59

replication and survival. Inhibition of human thioredoxin reductase 1 induces expression 60

of heme oxygenase-1 to repress inflammation(12). We previously demonstrated that 61

auranofin reduces T. gondii infection of host cells in vitro and a single dose of auranofin 62

allows survival of chicken embryos infected with this parasite (13). It is hypothesized 63

that the anti-parasitic activity of auranofin comes from inhibition of the thioredoxin 64

reductase enzyme of these parasites, akin to its action on human cells (4, 8, 14). This 65


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postulated mechanism of action explains why parasites treated with auranofin 66

accumulate ROS (4). Intriguingly, auranofin may improve infection outcome by 67

decreasing pathogenic host inflammatory damage in addition to its direct inhibition of 68

parasite replication. 69

In this paper we describe our work to investigate resistance to auranofin in T. 70

gondii parasites. This approach illuminates mechanisms of resistance that might arise 71

during its use as an anti-parasitic. In some circumstances, identification of resistance 72

mechanisms validates proposed drug targets (i.e.(15)). In this study, we found that 73

resistance was not associated with changes to the Toxoplasma thioredoxin reductase 74

gene and no other single locus emerged as a consistent site underlying resistance. 75

However, we observed that resistant clones accumulated less ROS than their wild type 76

counterparts, demonstrating that auranofin resistance enhances oxidative stress 77

responses. 78

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MATERIAL AND METHODS 80

1. Host cell and parasite cultures: 81

Parasite clones and human foreskin fibroblasts (HFF) were grown and 82

maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine 83

serum (Hyclone), penicillin and streptomycin (50 μg/ml each) and 200 μM L-glutamine. 84

We will refer to this complete medium as D10. T. gondii were maintained by serial 85

passage in HFF monolayers at 37°C in a humid 5% CO2 atmosphere, including RH 86

parasites (National Institutes of Health AIDS Reference and reagent Repository, 87

Bethesda, MD), RH tachyzoites expressing cytoplasmic yellow fluorescent protein 88


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(YFP) (kindly provided by M.J. Gubbels, Boston College, Boston, Massachusetts) (16) 89

and Ku80 parasites knock-out parasites (ΔKu80, kindly provided by V. Carruthers, 90

University of Michigan Medical School Dexter, Michigan) (17, 18). 91

92

2. Generation of T. gondii clones resistant to auranofin: 93

We mutagenized T. gondii with ENU as previously described (19, 20). Briefly, 94

approximately 1.5x107 intracellular tachyzoites of wild-type RH strain T. gondii were 95

mutagenized with ENU (N-Ethyl-N-nitrosourea, N3385-1G, Sigma Aldrich; 100-200 96

µg/ml) in DMEM without FBS for 2 hours at 37°C. These parasites were washed and 97

transferred to a new confluent HFF monolayer for selection with auranofin (2.5 μM-3 98

μM) until normal pace of replication was observed (~2 weeks). Auranofin resistant 99

clones were single cell cloned by limiting dilution in 2 μM auranofin. Isolated clones 100

were propagated in D10 media with 1 μM auranofin to maintain selection pressure 101

without cytotoxic effects on host cells (data not shown). 102

We selected ten representative T. gondii clones from independent ENU-103

generated pools to investigate resistance. To assess auranofin resistance, we modified 104

a tachyzoite growth competition assay (21). Using wild-type YFP-expressing T. gondii 105

parasites as a control, confluent monolayers of HFF cells were inoculated with 106

approximately equal numbers (2x106; 1:1 ratio) of ENU-mutagenized parasites and 107

YFP- expressing wild-type RH parasites. Upon lysis, a new T25 flask with confluent 108

HFF was inoculated with 0.25 ml of lysed parasites. Another 0.25 ml of lysed parasites 109

was used for flow cytometry. The YFP fluorescence allowed us to differentiate wild-type 110

and AurR parasites in the mixed populations to track their growth over serial passages. 111


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Host cell debris was removed by filtering of parasites with a 3 μm filter membrane. After 112

parasites were collected by centrifugation (400g for 10 min), the pellet was resuspended 113

in 1 ml PBS. The total number of parasites and the number of parasites expressing YFP 114

were quantified by flow cytometry using a FACSCalibur flow cytometer (Becton 115

Dickinson) and analyzed with Flowjo software (Tree Star). The result of three 116

independent experiments analyzing the replication fitness advantage between each 117

auranofin resistant clone and the wild-type parasites was analyzed with parametric 118

paired t-tests. A two-tailed p-value

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gDNA was sheared to bp in size using a Covaris S220 ultrasonicator 135

(shearing conditions: duty Cycle 10%, Cycles/ burst 200 and treatment time 55 sec) 136

(Covaris, MA). The size and the quality of the resulting DNA fragments were analyzed 137

with an Agilent DNA High Sensitivity chip. Ten nanograms of each sample’ s 138

fragmented DNA was end-repaired, poly adenylated and ligated to a 6bp DNA barcodes 139

(Bioo Scientific NextFlex DNA barcodes) using a PrepX Complete ILMN DNA Library kit 140

(Takara Bio, CA). The genomic library was built using the Apollo 324 system (Takara 141

Bio, CA) to generate 520 bps inserts. To enrich the DNA libraries, 8 cycles of PCR 142

amplification using a Kapa library amplification kit was used (cycling conditions: 98°C for 143

45 s, 8 cycles of 98°C for 15 sec, 60°C for 30 s 72° C for 30 s and then 72°C for 1 min) 144

in an Apollo 324 system. The ultimate DNA concentration was calibrated using Qubit 145

2.0 dsDNA HS Assay kit and analyze with an Agilent DNA High Sensitivity chip. Before 146

sequencing, we quantified our DNA libraries using a KAPA Library Quantification kit for 147

Illumina platforms (Roche). Each library was normalized to 2nM and then pooled 148

equimolar amounts in the final multiplex library that was sequenced on the Illumina 149

HiSeq 4000. 150

The ten auranofin resistant T. gondii clones and wild-type RH T.gondii were 151

sequenced with HiSeq 4000 paired end 100 reads, quality analyzed, adapter and quality 152

trimmed before alignment with the annotated reference genome of GT1 strain from 153

ToxoDB.org (published 7/19/2013)(22). After initial quality control with FASTQC, reads 154

were trimmed using Illumina adapter sequences with the error probability of 0.05 and 155

reads shorter than 20 bases were discarded. Single Nucleotide Variant (SNV) calling for 156

the haploid genome had a coverage threshold of >5 fold. Each of the 10 mutant clones 157


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were compared to wild-type RH variant track and shared variants were filtered out. Only 158

SNVs in the coding regions of the 10 mutant clones are reported. Only genes containing 159

SNVs with 100 % frequency and ≥50% of read count and read coverage were 160

considered in our final analysis. Genomic DNA from each mutant clone was used to 161

amplify the targeted SNV region of interest. The resulting amplicons ranged between 162

1.7-4.5kbp in size. PCR amplicons were purified prior to sequencing via a QiaQuick 163

PCR Purification Kit (Qiagen 28106). Amplicons were sequenced by Sanger 164

sequencing (Genewiz), using sequencing primers targeted to the SNV region (Table S1) 165

166

4. Generation of modified SOD2 lines: 167

We generated a T. gondii clone with a 3’ knock-in YFP tag to TgSOD2 using a 168

ligation independent cloning approach as previously described (17). The mitochondrial 169

distribution of our clone was confirmed by fluorescent microscopy using a Zeiss 170

Axioskop with Axiovision camera and software. We subsequently introduced the L201P 171

mutation into the TgSOD2-YFP line using a modified CRISPR plasmid generated using 172

plasmid pSAG1::CAS9-U6::sgUPRT as the scaffold (23); Addgene # 54467). Our 173

plasmid had an additional DHFR-TM marker for pyrimethamine selection derived from 174

pLoxP-DHFR-mCherry ((24), Addgene # 70147), and the two gRNA sequences were 175

replaced with CGGTATACCGGACAGATCCG and TAGTTTCGCCCAGTTCAAGG, 176

selected by the Benchling software (Benchling.com). 177

The DNA sequence used for homology directed repair (HDR) was amplified with 178

the primers GATAGTGTGTGAAGAGCAGC and CTGCCGTTACCAACATGG from the 179

LIC-YFP-SOD2 plasmid generated above. PCR amplicon contained the L201P mutation 180


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(CTA->CCA) generated with Q5 Site-Directed Mutagenesis kit (New England Biolab), as 181

with the following modifications. To screen for positive CRISPR-Cas9 clones containing 182

L201P, two silent mutations were created to generate new and unique restriction 183

endonuclease sites close to Leucine 201: AflI on Leucine 180 (CTC->CTT, 62 bps 184

upstream of L201P) and XcmI on Serine 208 (TCA->TCT, 22 bps downstream of 185

L201P). The PCR amplicon contained two additional deletions corresponding to the 186

gRNA sites on endogenous SOD2 gene, in order to avoid SpCas9 protein cleaving the 187

amplicon for HDR. The sequence agagacccggcgg (including the PAM sequence cgg) 188

was deleted on the HDR sequence corresponding to gRNA #1, and sequence 189

cacttaacagagggtc (including the PAM sequence agg) was deleted on the HDR 190

sequence corresponding to gRNA #2. 191

The L201P point mutation was introduced into the TgSOD2.YFP line through 192

CRISPR-Cas9 (Figure S1). Transfection of the CRISPR-CAS9 plasmid and the HDR 193

DNA was carried out as published previously, with minor modifications (23). Briefly, 107 194

of freshly lysed parasites with SOD2-YFP were filtered and spun down at 400xg and 195

18⁰C for 10 min. The pellet was resuspended with Cytomix buffer (10 mM KPO4, 120 196

mM KCl, 5 mM MgCl2, 25 mM HEPES, 2 mM EDTA, 2µM ATP and 5µM glutathione) up 197

to 800µl, including 7.5µg of CRISPR plasmid and 1.5µg of HDR DNA. The mixture was 198

transfected using the ECM 630 Electro Cell Manipulator (BTX). Parasites were 199

incubated into T25 flasks after electroporation and switched to 2 μM pyrimethamine 200

selection 24 hours later. Transfected pools remained in pyrimethamine selection for 3 201

days, and then switched to D10 media without selection for one more day before single 202

cell cloning. The gDNA for isolated clones were collected by the DNeasy Blood and 203


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Tissue kit (Qiagen) and used to PCR amplify SOD2 with either primers SOD2 F/AmpR 204

(for SOD2-YFP) or primers SOD E2/SOD R (for SOD2 only, Table S1). PCR amplicons 205

were digested with XcmI for screening and positive clones were sequence verified. 206

207

5. Measurement of ROS accumulation in T. gondii: 208

To expose our parasites to oxidative stress, we harvested freshly lysed parasites 209

and treated 5x104 parasites with 500 μM hydrogen peroxide (H2O2) for 30 min at 37⁰C, 210

5% CO2, with or without 1 μM auranofin After incubation, ROS was detected using 211

H2DCDFC (Invitrogen). Each sample was incubated with 1 µM of H2DCDFC and 212

examined under a Spectramax i5, (Molecular Devices California US; multimode 213

microplate reader SoftMax Pro 7.0.2 Software) multimode microplate reader (excitation 214

494 nm; emission 522 nm) at 37⁰C for 15 minutes. For excitation, a single flash from a 215

UV Xenon lamp was used for each well, and emission signals were recorded with a 216

sensitivity setting of 100. The values are presented as relative fluorescence units. We 217

compared ROS accumulation of each representative auranofin resistant T. gondii clone 218

to that of wild-type parasites by an unpaired t-test in three independent experiments. A 219

two-tailed p-value

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RNR (PDB 3HND) (31). Clustal Omega was used to align the amino acid sequences 227

and EMBOSS was used to calculate percent identity and similarity. Information on MS 228

peptide coverage was obtained from the ToxoDB database (22). 229

230

RESULTS 231

1. AurR T. gondii lines have a growth advantage over wild type parasites: 232

To verify auranofin resistance of the selected clones, we adapted a previously 233

described competition assay (21). This was used in place of a normal dose-response 234

assay because at auranofin concentrations higher than 3 μM, the fibroblast monolayer 235

begins to disrupt (data not shown) making the plaque assays not feasible. To confirm 236

resistance to auranofin in the AurR T. gondii lines, we assessed their growth in 237

competition with YFP-expressing RH strain parasites in control media and in media with 238

2.5-3.0 μM auranofin. In competition assays, the inoculating parasite population is 239

composed of 50% YFP-expressing wild type (sensitive) parasites and 50% of an AurR 240

line verified by flow cytometry. The relative contribution of the individual populations was 241

measured after each cycle of lytic growth in serial culture. If a parasite line increases to 242

>50% of the population, it exhibits a growth advantage over the competing line. In the 243

absence of auranofin, AurR lines grew comparably or less well than the parental RH line 244

that does not express YFP (Figure 1A). However, in the presence of 2.5 μM auranofin, 245

all AurR lines displayed significant growth advantage over YFP-expressing wild-type RH 246

parasites (Figure 1B). The observations indicated that day 6 is the optimal day to 247

capture the growth advantage of resistant lines under auranofin selection because YFP-248

expressing wild-type parasites were eliminated from the culture. Given these results, we 249


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measured the ability of a larger set of AurR lines to compete with wild-type YFP-250

expressing RH strain parasites in the absence or presence of auranofin by quantifying 251

their contribution to the culture at 6 days (Figure 2). These results show that 9 of the 10 252

AurR lines outcompete the sensitive YFP-expressing RH line in the presence of 2.5 μM 253

auranofin. Line 6 has a distinct profile: it grows slowly in both control and auranofin 254

media, suggesting that it may evade auranofin-mediated killing by protracted replication. 255

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2. AurR lines do not harbor mutations in the thioredoxin reductase gene: 257

Considering that thioredoxin reductase (1-4) is the proposed target of auranofin, 258

we hypothesized that resistance in T. gondii would develop through mutations to the 259

parasite thioredoxin reductase gene (TgTrxR, TGGT1_309730). Therefore, we amplified 260

and sequenced the TgTrxR gene in 53 lines derived from parallel (independent) 261

selections (not shown). We did not detect mutations (SNVs) in the thioredoxin reductase 262

gene of any of the lines, indicating that auranofin resistance does not arise through 263

modifications to TgTrxR that directly influence auranofin binding or TgTrxR enzyme 264

activity. 265

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3. AurR lines accumulate less ROS: 267

The anti-parasitic activity of auranofin relies on its ability to induce accumulation 268

of ROS in parasites (4). We exposed wild type RH strain parasites and four 269

representative AurR lines to hydrogen peroxide (H2O2) for 30 minutes before measuring 270

ROS accumulation with dichloro-fluorescein (H2DCDFC). All AurR lines displayed 271


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decreased accumulation of ROS relative to the parental RH line in both the absence 272

and presence of auranofin (Figure 3). 273

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4. AurR lines harbor diverse mutations that introduce point mutations: 275

Given that we did not observe mutations in T. gondii thioredoxin reductase, we 276

performed whole genome sequencing (WGS) analysis of 10 independent T. gondii AurR 277

lines. Our goal was to determine whether these T. gondii parasites harbor a common 278

mutation(s) that underlies resistance to auranofin. Analysis of coding sequences with 279

100% SNVs frequency and at least 50-fold coverage identified a variety of ~5 non-280

synonymous SNVs in the exons of each line. Selected SNVs observed in the WGS 281

were validated by PCR amplification and Sanger sequencing (primers in Table S1). We 282

chose to specifically investigate the AurR line 8 to consider which SNV was most likely 283

to underlie auranofin resistance. Line 8 has 12 SNVs that introduce amino acid 284

substitutions to diverse protein coding genes (Table 1). We used the community 285

annotated ToxoDB database and BLAST analysis to investigate these loci. In particular, 286

the availability of RNA-seq datasets permitted us to evaluate whether loci were 287

expressed in the tachyzoite stage. In order to assess the significance of expressed loci, 288

we used BLAST analysis to look for protein homologs and significant motifs and 289

collected RNA-seq values and CRISPR screen scores for available loci. This 290

information led us to focus on 2 of the loci (TGGT1_316330 and TGGT1_294640) as 291

most likely resistance conferring. These loci encode a superoxide dismutase (SOD2) 292

and the large subunit (R1) of ribonucleotide reductase (RNR). Both enzymes have 293


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strong fitness conferring effects in the deposited CRISPR screen data and well-294

conserved metabolic roles. 295

RNR removes the 2'-hydroxyl group of the ribose ring of nucleoside diphosphates 296

to form deoxyribonucleotides from ribonucleotides which are used in DNA synthesis 297

while SOD inactivates damaging superoxide (O2−) radicals by converting them into 298

molecular oxygen and hydrogen peroxide. Since both TgRNR and TgSOD2 are 299

conserved proteins, we threaded the amino acid sequence onto homologous crystal 300

structures to locate the position of the amino acid substitutions. TgSOD2 is a Fe-type 301

SOD and was previously demonstrated to localize to the tachyzoite mitochondrion (32, 302

33). SODs form dimers with shared coordination of two iron (FeIII) cofactors. The L201P 303

mutation is located near to the key iron-coordinating residues: H111, E249 and H160 304

(Figure 4A) and is located in an α-helix. Substitution of a proline at this site likely causes 305

the helix to bend, perhaps altering SOD enzymatic properties. TgRNR aligns well with 306

human ribonucleotide reductase 1 (61.7% identity and 78.2% similarity). However, there 307

is a 59 amino acid N-terminal extension to the Toxoplasma protein, with the conserved 308

sequence beginning with M60 (Figure 4B). This leader does not represent mis-309

annotation of the N-terminus of the protein: there are MS peptides mapping to this 310

sequence, especially in samples that are from monomethylarginine and 311

phosphopeptide-enriched datasets in ToxoDB. This is particularly interesting as arginine 312

methylation is a modification associated with proteins that have roles in transcriptional 313

regulation, RNA metabolism and DNA repair. RNR use free-radical chemistry to convert 314

ribonucleotides into deoxyribonucleotides. Significantly, ribose reduction requires 315

generation of a free radical and RNR requires electrons donated from thioredoxin. 316


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Moreover, superoxide has been shown to inactivate RNR and yeast lacking either 317

cytoplasmic or mitochondrial SODs have increased susceptibility to oxidative stress and 318

RNR inactivation. The L166Q mutation is in a solvent exposed loop (Figure 4C), that is 319

poorly conserved and not known to contribute to enzyme interactions. 320

321

4. Resistance to auranofin is not conferred by an isolated mutation in TgSOD2: 322

The anti-parasitic effect of auranofin appears to stem from its molecule of gold, 323

which is known to induce the accumulation of ROS in parasites (4, 34). Since we 324

hypothesized that a single mutation (SNV) suffices to generate resistance to auranofin, 325

we analyzed the effect of the TgSOD2 L201P mutation on auranofin sensitivity. SOD2 is 326

a bona fide enzymatic ROS scavenger and SOD2 is the sole mitochondrial superoxide 327

dismutase in tachyzoites. To analyze the role of mutant SOD2 in auranofin resistance, 328

we generated a T. gondii clone with a knock-in YFP tag for SOD2, that localizes to the 329

tachyzoite mitochondrion as previously reported (Figure 5A). Subsequently, we 330

generated two T. gondii SOD2.L201P lines: one lacking the YFP tag and one tagged 331

with a C-terminal YFP fusion (Figure S1). Both lines were corroborated by Sanger 332

sequencing. In the absence of Auranofin, both L201P lines grew less well than the 333

TgSOD2-YFP wild-type line but were more fit than AurR line 8 (Figure 5B). Previous 334

attempts to create SOD1 and SOD2 gene deletions failed (35) and SOD2 is a fitness-335

conferring locus in a genome-wide CRISPR/Cas screen (36). It is noteworthy that the 336

L201P mutation is associated with reduced fitness in the absence of superoxide stress 337

(control conditions). In the presence of auranofin, only line 8 grows well, indicating that 338

the single L201P mutation to TgSOD2 does not confer resistance. This may indicate 339


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that this substitution does not influence superoxide metabolism at all or that auranofin 340

resistance also requires the mutant TgRNR enzyme. It is unlikely that these enzymes 341

directly interact, as RNR enzymes are cytosolic while TgSOD2 is in the mitochondrial 342

compartment. However, the SOD2.L201P T. gondii line accumulated ROS similarly to 343

wild-type parasites, consistent with its auranofin-sensitive growth (Figure 5C). 344

345

Discussion: 346

Auranofin, is an FDA-approved drug for treatment of rheumatoid arthritis. More 347

recently it has been proposed to be a promising anti-parasitic agent with activity against 348

diverse parasites with public health significance. We previously demonstrated that 349

auranofin decreases the parasite burden during a systemic infection with T. gondii using 350

a chicken embryo model of acute toxoplasmosis (13). Our preliminary observations 351

suggest that auranofin exerts a dual effect: it modulates both parasite proliferation and 352

host immunopathology. Thioredoxin reductase has been implicated to be the molecular 353

target of auranofin in parasites. Herein, we report isolation and characterization of 354

auranofin-resistant T. gondii lines. None of our resistant lines has mutations in the 355

thioredoxin reductase gene. This may indicate that this enzyme is not the molecular 356

target of auranofin in T. gondii; alternately, it may indicate that thioredoxin reductase 357

cannot tolerate resistance-conferring mutations or is not the sole significant target in this 358

organism. Each of our auranofin resistant T. gondii lines carry several mutations 359

(SNVs). Importantly, resistant lines accumulate less reactive oxygen species (ROS) 360

than wild-type parasites. 361


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There are several approaches to target identification in drug discovery. In T. 362

gondii, chemical mutagenesis is a well-established and robust approach (i.e. (15, 19) to 363

identify the molecular targets of candidate anti-T. gondii drugs. This approach is also 364

relevant to anticipating mechanisms of drug resistance. We isolated auranofin-resistant 365

T. gondii lines derived from the RH strain. Resistant lines have increased replication 366

relative to wild type RH parasites in competition assays carried out in the presence of 367

auranofin. Lines with ≤5 accumulated SNVs (lines 1-4) appear to have a growth 368

advantage over wild-type parasites in the absence auranofin. This probably reflects a 369

fitness defect induced by expression of YFP in wild type parasites (21) ; these lines 370

likely grow comparably to non-YFP expressing wild type parasites without auranofin 371

present. In contrast, resistant lines that have >5 SNVs each compete poorly with wild 372

type parasites in the absence of selection, suggesting that accumulation of SNVs 373

comes at a cost to parasite fitness. An alternative explanation to the isolation of multiple 374

SNVs in each resistant clone is that auranofin interferes with many processes and 375

development of resistance requires alterations to more than one target. 376

Auranofin contains a molecule of gold in a 3,4,5-Triacetyloxy-6-sulfanyl-oxan-2-yl 377

methyl ethanoate scaffold and has been proposed to be a pro-drug (37) that delivers 378

gold to dithiol groups, like those found in proteins that bear thioredoxin-containing 379

domains (38). Although auranofin inhibits mammalian thioredoxin reductase (39), the 380

source of its anti-inflammatory activity remains ill-defined and may reflect inhibition of 381

multiple targets. For example, macrophages decrease production of nitric oxide and 382

pro-inflammatory cytokines in response to auranofin, which also inhibits 383

cyclooxygenase-2 -dependent prostaglandin E2 production (40). Previous studies using 384


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the parasite enzymes trypanothione reductase and glutathione-thioredoxin reductase 385

incubated with auranofin demonstrated that co-crystals harbor gold bound to reactive 386

cysteines in these proteins (2, 3, 41) In addition, S. mansoni worms with decreased 387

expression of thioredoxin glutathione reductase (SmTGR) exhibit diminished parasite 388

viability upon exposure to auranofin. E. histolytica treated with auranofin upregulate 389

transcripts of a gene encoding a protein resembling an arsenite-inducible RNA-390

associated protein (AIRAP) (4). Interestingly, both arsenite and auranofin are inhibitors 391

of thioredoxin reductase, implying that EhTrxR is a target of auranofin. Studies in both 392

S. mansoni and E. histolytica corroborated the interaction of thioredoxin reductase with 393

auranofin by mobility shift assays or other in vitro measures (4, 14). In these cases, the 394

approach involved target validation guided by the chemical properties of gold rather 395

than target identification through genetic resistance. 396

It was surprising to not identify changes to the thioredoxin reductase gene, given 397

previous studies that implicate antioxidant enzymes containing thioredoxin or 398

thioredoxin-like domains as a target for auranofin in parasites. However, although S. 399

mansoni and E. histolytica thioredoxin reductase antioxidant systems are essential for 400

survival, a previous study indicates that T. gondii thioredoxin reductase is not essential 401

(42). It should be noted that infection with 106 TgTrxR knockout parasites increases 402

survival in mice by approximately 2 weeks relative to the parental RH line (42). In vitro, 403

the null line exhibits reduced antioxidant capacity, invasion efficiency, and proliferation. 404

These observations are in-line with the CRISPR fitness score for thioredoxin reductase 405

(-1.98) which places it as fitness-conferring but not essential. 406


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Among the significant SNVs found in our auranofin resistant T. gondii clones, we 407

directed our attention at a point mutation to the superoxide dismutase 2 (SOD2) gene. 408

SOD2 is a bona fide enzymatic antioxidant system that catalyzes the dismutation of 409

superoxide anions into hydrogen peroxide and oxygen. The Toxoplasma genome 410

contains three SOD genes. TgSOD1 is expressed in the cytoplasm of tachyzoites and 411

TgSOD2 is located in the mitochondrion; TgSOD3 is specifically expressed in oocysts. 412

TgSOD2 is a critical enzyme for tachyzoite survival: previous efforts to generate a 413

TgSOD2 knockout line were not successful and its CRISPR fitness score for (-4.09) 414

indicates that it is strongly fitness conferring and likely essential (35). We hypothesized 415

that the L201P mutation in TgSOD2 might increase its capacity to convert superoxide 416

into hydrogen peroxide and oxygen to confer auranofin resistance. To test this 417

hypothesis, we generated several lines that harbor knock-in tag to wild type SOD2 and 418

engineered both YFP-tagged and untagged lines that bear the L201P mutation. 419

Consistent with previous characterization of TgSOD2 (32), both wild type and L201P 420

TgSOD2-YFP lines exhibit a mitochondrial distribution. However, neither the tagged or 421

untagged L201P TgSOD2 lines exhibit increased growth in 2.5 uM auranofin, indicating 422

that the single TgSOD2 mutation cannot confer resistance to this level of auranofin or 423

reduce accumulation of ROS in our assay. 424

In addition to the L201P TgSOD2 mutation, AurR line 8 harbors a mutation in the 425

large subunit of TgRNR, an essential enzyme which generates deoxyribonucleotides 426

that are required for genome duplication and parasite replication. TgRNR has a 59 427

amino acid N-terminal extension relative to other RNRs. Peptides from this extension 428

are represented in phosphorylated and monomethylated MS datasets, suggesting that 429


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20

this domain may regulate RNR activity. RNRs perform free-radical chemistry, require 430

electrons donated from thioredoxin and are susceptible to superoxide inactivation. 431

Although the L166Q mutation is in an area that is not known to contribute to enzyme 432

interactions, it may play a role diminishing TgRNR susceptibility to superoxide 433

inactivation. While this mutation may indirectly contribute to resistance to superoxide, 434

RNR enzymes do not directly contribute to redox homeostasis and the observed 435

reduced accumulation of ROS in the AurR lines. 436

Our previous and current findings suggest that auranofin decreases T. gondii viability 437

through oxidative stress (13). AurR lines exhibit an increased ability to scavenge ROS. 438

The genetic studies presented here did not identify a consistent mechanism for 439

acquisition of resistance by point mutations to a specific protein target. Our future 440

studies will examine whether there are transcriptional, translational or post-translational 441

changes that contribute to auranofin resistance in these lines. For example, difference-442

gel electrophoresis and mass spectrometry have been used to identify differentially 443

expressed proteins in sulfadiazine-resistant T. gondii isolates. While it was 444

disappointing to not identify a single molecular target for auranofin in T. gondii in our 445

studies, this result likely indicates that an enhanced ability to scavenge ROS in the 446

presence of auranofin requires multiple changes to the parasite genome. Therefore, if 447

auranofin is repurposed as an anti-parasitic treatment, it would likely present an 448

increased threshold to resistance in clinical contexts. 449

450


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42. Xue J, Jiang W, Chen Y, Gong F, Wang M, Zeng P, Xia C, Wang Q, Huang K. 593 2017. Thioredoxin reductase from Toxoplasma gondii: an essential virulence 594 effector with antioxidant function. FASEB J 31:4447-4457. 595

596

597


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FIGURES: 598

599

Figure 1. AurR lines have a growth advantage under selective pressure with auranofin. 600

(A) In the absence of auranofin, individual resistant lines grow as well or less well than 601

the parental RH strain. (B) In the presence of 3 µM auranofin, wild-type parasites are 602

eliminated by day 6 (red box). 603

604


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605

606

Figure 2. Resistant T. gondii lines outcompete wild type parasites under auranofin 607

selection. AurR lines are represented as percentage of the total population of parasites 608

after 6 days in competition with YFP-expressing wild type (sensitive) parasites, no drug 609

media (white) and 2.5 uM auranofin (black). Mean difference between parasite 610

population in the absence of auranofin and in the presence of auranofin: 40.6; SD 14.6; 611

two-tailed unpaired t-test p

27

614

615

Figure 3. Auranofin-resistant T. gondii parasites accumulate less ROS. Freshly lysed 616

wild-type parasites were treated with 500 μM hydrogen peroxide (H2O2) for 30 min and 617

accumulation of reactive oxygen species (ROS) was assayed with dichloro-fluorescein 618

H2DCDFC. Auranofin-resistant clones accumulated less ROS after H2O2 treatment 619

compared to the wild-type RH parasites in the presence of H2O2 (white) or H2O2 + 1 µM 620

auranofin (black). (*) p

28

623

Figure 4. Location of substitutions in TgSOD2 and TgRNR. (A) SOD forms dimers that 624

coordinately bind to iron cofactors at their interface. The Toxoplasma amino acid 625

sequence was threaded onto a Plasmodium knowlesi protein structure (PDB 2AWP) to 626

create a model for TgSOD2. The mutation at position 201 (red) is near to the end of an 627

α-helix; substitution of a proline for leucine at this location likely causes the helix to 628

bend. This mutation is also proximal to key iron-coordinating residues H111, E249 and 629

H160. (B) Alignment of the Toxoplasma and human RNR sequences indicates that 630

TgRNR has a novel 59 amino acid N-terminal extension. MS peptides (yellow highlight) 631

in ToxoDB map to this sequence with S and T phosphorylated, and R monomethylated 632

(underlined). (C) The Toxoplasma amino acid sequence for RNR was threaded onto the 633

human RNR structure (PDB 3HND) to create a model for TgRNR. The L166Q mutation 634


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29

is in a solvent exposed loop, that is poorly conserved and not known to contribute to 635

enzyme interactions. 636

637


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30

638

639

Figure 5: A L201P mutation in TgSOD2 does not alter localization of TgSOD2 or 640

confer resistance to auranofin. (A) Wild type and mutant SOD2-YFP exhibit a 641

mitochondrial distribution. (B) Growth competition assays indicate that the L201P 642

mutation is associated with reduced fitness in the absence of auranofin (control 643

conditions, white bars). In the presence of auranofin (black bars), only line 8 shows 644


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31

growth advantage, indicating that the single L201P mutation to TgSOD2 does not confer 645

resistance. (C) In the absence of auranofin, the SOD2.L201P line accumulated ROS 646

similarly to wild type parasites, consistent with its auranofin-sensitive growth; however, it 647

accumulated less ROS in the presence of auranofin 648

649

650


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Table 1: SNVs in AurR line 8

Rank Mutation Locus Predicted protein CRISPR

*RNA-seq

Motif Index

1 L201P TGGT1_316330 Superoxide dismutase SOD2

-4.09 76.99 SOD motif 2.50

2 L166Q TGGT1_294640 Ribonucleoside-diphosphate reductase

-4.79 40.9 R1 subunit, ribonucleotide reductase

2.29

3 Y95C TGGT1_233460 SAG-related sequence SRS29B

-0.07 1809.46 Major surface antigen p30 2.10

4 Y637H TGGT1_268870 Tetracopeptide repeat-containing

-1.66 38.52 Predicted dehydrogenase 1.81

5 P1357-S1358Δ

TGGT1_310350 PGAP1 family protein -2.41 11.67 alpha/beta-hydrolase/PGAP1-like protein 1.45

6 D487G TGGT1_289820 TBC domain-containing protein

-1.14 15.49 Rab-GTPase-TBC domain 1.25

7 L606L TGGT1_271290 Hypothetical protein -2.02 8.1 Alternative splicing regulator 1.21

8 F424L TGGT1_234250 Hypothetical protein -2.02 6.39 SWAP/MAEBL 1.11

9 A574G TGGT1_290910 Hypothetical protein -1.39 7.63 No putative conserved domain 1.03

10 D1983del TGGT1_211310 Hypothetical protein -0.69 12.86 RCC1 repeat 0.95

11 F563S TGGT1_260490 Hypothetical protein -1.62 5.34 Herpes BLLF1 0.94

12 P66P TGGT1_225340 Hypothetical protein 0.73 24.06 No putative conserved domain

N/A

*Transcriptome Gregory series at 2 hrs RH (log 2 FPKM+1). FPKM= Fragments Per Kilobase Of Exon Per Million Fragments Mapped N/A: Not calculated due to dispensable (positive) CRISPR phenotype index index Formula=Sum of Log10 of (-CRISPR phenotype index)+log10 (RNAseq expression)

651

Table 1. SNVs in AurR line 8 652


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33

653

SUPPLEMENTAL FIGURES 654

655

Figure S1: CRISPR-Cas9 strategy for generating the T. gondii SOD2.L201P line. TgSOD2-YFP parasites incorporating 656

the LIC plasmid (blue line) undergo CRISPR-Cas9 mediated homologous directed repair (HDR). The DNA amplicon used 657

for HDR contains the L201P mutation (gold vertical line in exon 3) and two silent and unique restriction sites AflI and XcmI 658

(black vertical lines in exon 3), which allow for screening of positive clones. Sequences corresponding to the two gRNA 659

were deleted from the DNA for HDR (Δg1 and Δg2, golden triangles). Each of the gRNA was present twice in TgSDO2-660


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YFP parasites (g1a/b, g2a/b; red font), resulting in two T. gondii SOD2.L201P lines. For HDR after g1a/g2a cuts, a T. 661

gondii SOD2.YFP.L201P line is generated (lower left). For HDR after g1a/g2b cuts, a T. gondii SOD2.L201P line without 662

YFP or LIC plasmid (blue) is generated (lower right). Positive clones were screened with XcmI digest before sequence 663

verification. 664

665


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Name Gene ID* Sequence Function

TrxR 12F 309730 GGACCTATGAGTTGCCTCTCTG TrxR amplification 5’ half TrxR R10 309730 GTCAAATCCCAATTCGCGGAG TrxR amplification, 5’ half TrxR For 8 309730 GTCTGCTCTCTCTGTTTCGCAAG TrxR amplification, 3’ half TrxR Rev 9 309730 CCTCCACATCCTCCAGAAGC TrxR amplification, 3’ half 5TrxR 1R 309730 CGAGTTTTCTAGAGCGTGTGC TrxR sequencing, 5' half, exon 1 5TrxR 1F 309730 CATCCCTGTGCACTTTCC TrxR sequencing, 5' half, exons 2-3 5TrxR 3F 309730 GTGGATACCCTCGTATAATATGC TrxR sequencing, 5' half, exons 4-5 TrxR For 8 309730 GTCTGCTCTCTCTGTTTCGCAAG TrxR sequencing, 3' half, exons 6-7 3TrxR 2F 309730 GCTTCGTTTGATTGCCTGTC TrxR sequencing, 3' half, exons 8-9 3TrxR 3F 309730 CATCTTGTTCCTCCAACCTCTG TrxR sequencing, 3' half, exon 10 3TrxR 4F 309730 GATGTCTGCTTTCGTCTTCC TrxR sequencing, 3' half, exon 11 SOD LIC3 316330 TACTTCCAATCCAATTTACGAAGGTGAGGAGCA

TTCG SOD2 amplification with LIC casette

SOD LIC5 316330 TCCTCCACTTCCAATTTTAGCGTTGCTTTCAAGTGCTTTCACC

SOD2 amplification with LIC casette

SOD2 RE Ins F 316330 TGATAAGCTAGCCAGTGATCT Site directed mutagenesis for NheI SOD2 RE Ins R 316330 TCTCCATCTCCCCCTGAT Site directed mutagenesis for NheI SOD2 SNP F 316330 TTTTCCAAACCAGCGGCAGGC Site directed mutagenesis for L201P SOD2 SNP R 316330 CTCATCCTTGAACTGGGC Site directed mutagenesis for L201P SOD Seq1 316330 CTTTCGAATCCTGAAACACC Validation of SOD2 SNV SOD Seq2 316330 GTCATGTGTGTTTGCCCCGTAG Validation of SOD2 SNV SOD2 F 316330 GCAGAGACTCTTCGCTTTCAC Amplification of SOD2.YFP CRISPR clone AmpR N/A CCTTGAAGAAGATGGTGCGC Amplification of SOD2.YFP CRISPR clone SOD E2 316330 GCTTCATTCAAGGCACACC Amplification of SOD2 in CRISPR clone SOD R 316330 CTATCCAGGGTTCACGACG Amplification of SOD2 in CRISPR clone CDS F 300120 GTGTGTCTGCATCGTTTCC Aminotransferase amplification CDS R 300120 GTTCCTCTGAAGCATGTTTCC Aminotransferase amplification CDS E1 300120 CTCTGTTTGGTGTCTTTGACC Aminotransferase SNV sequencing COR F 253120 CTCAGCAATGTGACATGCTAGC Mandelonitrile lysase amplification COR R 253120 GTTCGACGGTGAAATGTGCTC Mandelonitrile lysase amplification COR E8 253120 CTTGCTACCATTTCTCCTCG Mandelonitrile lysase SNV MBL F 234250 GTGAACTCAGTGAAACCGC MAEBL amplification MBL R 234250 GCACTCTCGAGTTTTACGC MAEBL amplification MBL E2 234250 GGAGGATCAGGTTTATGAGTGC MAEBL SNV sequencing NAD F 244700 CGTCTCCTTCTCTCGATGG NAD(+)/NADH kinase amplification 666


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Table S1: Primers for SNV validation 667

668


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Acknowledgements 669

Special gratitude to Dr. Melissa Lodoen (UCI) for her advice and critical review of this 670

manuscript and to Edward Brignole (MIT) for his thoughts on the TgRNR mutation. RMA 671

was supported by NIHK08 5K08AI102989-04 and AMFDP RWJF 70642 (the views 672

expressed here do not necessarily reflect the views of the Foundation). This work was 673

made possible, in part, through access to the Genomics High Throughput Facility 674

Shared Resource of the Cancer Center Support Grant (P30CA-062203) at the 675

University of California, Irvine and NIH shared instrumentation grants 1S10RR025496-676

01, 1S10OD010794-01, and 1S10OD021718-01. 677

678

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