{"gene":"RH88_085440","gene_symbol":"","run_date":"2026-09-07T10:25:42","timeline":{"discoveries":[{"year":2000,"finding":"Cytochrome b of T. gondii was cloned and sequenced; atovaquone-resistant mutants carry mutations M129L and I254L within the Qo (ubiquinol-binding) domain, establishing that atovaquone interferes with electron transport at the cytochrome bc1 complex by targeting the Qo domain of cytochrome b. Structural modeling suggested cytochrome b may terminate within the mitochondrial membrane, differing from other homologues.","method":"Drug-resistant mutant generation, gene cloning and sequencing, structural modeling of Qo domain","journal":"Molecular and biochemical parasitology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct cloning and sequencing of resistant mutants with defined point mutations, replicated by subsequent studies; first complete mitochondrial gene described for T. gondii","pmids":["10802314"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1999,"finding":"A mutation in cytochrome b was identified in a decoquinate-resistant mutant of T. gondii, implicating cytochrome b as the target of decoquinate at the cytochrome bc1 complex.","method":"Drug-resistant mutant selection and cytochrome b gene sequencing","journal":"The Journal of eukaryotic microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single study, gene sequencing of resistant mutant; abstract not available for full method detail","pmids":["10519259"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1998,"finding":"Atovaquone at 0.03 µM totally inhibited respiration and disrupted mitochondrial membrane potential in T. gondii tachyzoites, and biochemical assays showed inhibition of the respiratory chain between cytochrome b and c1, consistent with bc1 complex inhibition at cytochrome b.","method":"In situ respiration assay using digitonin-permeabilized tachyzoites, mitochondrial membrane potential measurement with safranin O, TMPD/ascorbate rescue experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct biochemical assay with multiple substrates and inhibitor controls in intact parasites, single lab but multiple orthogonal readouts","pmids":["9813002"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2003,"finding":"Atovaquone binds to the ubiquinol oxidation (Qo) pocket of the cytochrome bc1 complex and interacts with the Rieske iron-sulfur protein; a L275F mutation in cytochrome b decreases atovaquone sensitivity without loss of activity, demonstrating that this residue contributes to species-differential drug binding. (Experiments performed in yeast as a surrogate for T. gondii and other pathogens.)","method":"Enzyme inhibition kinetics (Ki determination), EPR spectroscopy of Rieske center, site-directed mutagenesis (L275F) of cytochrome b in yeast, computed energy-minimized structural model","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution in isolated bc1 complex, mutagenesis validation, EPR spectroscopy, and structural modeling; yeast surrogate but directly informs T. gondii cytochrome b mechanism","pmids":["12791689"],"is_preprint":false,"attribution":[{"species":"Saccharomyces cerevisiae","strain":""}]},{"year":2017,"finding":"A T222P substitution in T. gondii cytochrome b confers 49-fold resistance to ELQ-316 and 19-fold resistance to antimycin, a well-characterized Qi site inhibitor, providing genetic evidence that ELQ-316 inhibits cytochrome bc1 at the Qi site of T. gondii cytochrome b.","method":"Drug-resistant mutant selection under ELQ-316 pressure, cytochrome b gene sequencing, cross-resistance testing with antimycin","journal":"Antimicrobial agents and chemotherapy","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via drug-resistance mutation with cross-resistance validation against a known Qi site inhibitor; single lab but two orthogonal drug resistance readouts","pmids":["27919897"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"finding":"Loss of TgMSH1 leads to accumulation of missense mutations in cytochrome b and reduced mitochondrial DNA content; despite cytochrome b mutations, atovaquone can still inhibit electron transport in Tgmsh1 knockout parasites (none of the mutations affect known atovaquone-sensitivity determinants). Further atovaquone culture of Tgmsh1 mutants leads to complete respiratory shutdown, revealing metabolic plasticity allowing parasites to survive without oxidative phosphorylation.","method":"Knockout of TgMSH1, whole mitochondrial genome sequencing, electron transport inhibition assay, atovaquone drug selection, growth assays with and without glucose","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO with defined molecular phenotype, sequencing of cytochrome b mutations, and functional electron transport assay; single lab","pmids":["29141004"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"Artemisinin-resistant T. gondii populations show dramatic amplification of the mitochondrial genome, including cytochrome b and cytochrome c oxidase I genes; treatment with high concentrations of artemisinin decreases mitochondrial membrane potential in wild-type parasites, a phenotype stably altered in resistant lines, implicating the mitochondrion (and cytochrome b-containing complexes) as a target of artemisinin action.","method":"In vitro resistance evolution, whole-genome sequencing, CRISPR/Cas9 mutagenesis, mitochondrial membrane potential measurement","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome sequencing of evolved lines with functional mitochondrial assay; cytochrome b amplification is correlative but membrane potential assay provides mechanistic link; single lab","pmids":["31806760"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"finding":"Co-crystallographic and mutant yeast studies demonstrate that novel 4-(1H)-quinolone scaffolds bind to the cytochrome bc1 complex Qi site; T. gondii cysts highly express cytochrome b, and Qi-site inhibitors targeting cytochrome b are effective against both tachyzoites and bradyzoites in vitro and in vivo.","method":"Co-crystallography with bc1 complex, mutant yeast bc1 inhibition assays, in vitro parasite proliferation assays, murine infection model","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — co-crystallography and mutant yeast biochemistry support Qi site binding; parasite assays confirm functional relevance; single lab","pmids":["27412848"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"EGS"}]},{"year":2025,"finding":"Cryo-EM structures of the T. gondii III2-IV respiratory supercomplex and of complex III2 alone were determined. Cytochrome b is a core subunit of complex III2; atovaquone and ELQ-300 were visualized in the cytochrome b binding sites. ELQ-300 binds in a flipped orientation compared with related compounds in the mammalian enzyme, revealing parasite-specific interactions at the Qo/Qi sites. A supercomplex knockout showed that III2-IV is critical for parasite fitness but does not simply enhance electron transfer efficiency as proposed for mammals.","method":"Cryo-electron microscopy structure determination, drug soaking/co-structure with atovaquone and ELQ-300, knockout analysis of supercomplex subunit, parasite fitness assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structures with drug-bound complexes, mutagenesis-informed drug design, and genetic knockout with fitness readout; multiple orthogonal methods in a single rigorous study","pmids":["40389671"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2007,"finding":"Combined treatment of T. gondii with HDQ (an alternative NADH dehydrogenase inhibitor) and atovaquone (a cytochrome b Qo site inhibitor) resulted in synergistic inhibition (FIC = 0.16 nM), demonstrating that simultaneous blockade of ubiquinone/ubiquinol cycling at two mitochondrial sites is highly effective against parasite replication.","method":"Combination drug assay in T. gondii-infected cell cultures, fractional inhibitory concentration calculation","journal":"Antimicrobial agents and chemotherapy","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — pharmacological epistasis via combination assay establishing pathway position of cytochrome b Qo site; single lab, single method","pmids":["17242151"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"T. gondii cytochrome b is a mitochondrially encoded core subunit of the cytochrome bc1 complex (complex III), functioning in electron transport at the Qo (ubiquinol oxidation) and Qi (quinone reduction) sites; atovaquone binds the Qo domain (with resistance conferred by M129L/I254L mutations), endochin-like quinolones bind either site (Qi inhibition confirmed by T222P resistance), and cryo-EM structures of the T. gondii III2-IV supercomplex have revealed the molecular basis of parasite-selective drug binding, informing next-generation inhibitor design."},"narrative":{"mechanistic_narrative":"Toxoplasma gondii cytochrome b is a mitochondrially encoded core subunit of the cytochrome bc1 complex (complex III), where it catalyzes electron transport between ubiquinol and cytochrome c1 as part of the parasite's respiratory chain [PMID:9813002, PMID:40389671]. It carries two functionally distinct catalytic sites: a ubiquinol-oxidation (Qo) site targeted by atovaquone—resistance to which arises from M129L, I254L, and L275F substitutions clustered in the Qo domain [PMID:10802314, PMID:12791689]—and a quinone-reduction (Qi) site where endochin-like quinolones act, established genetically by a T222P substitution that confers cross-resistance to the classic Qi inhibitor antimycin [PMID:27919897, PMID:27412848]. Blocking the Qo site with atovaquone fully collapses respiration and mitochondrial membrane potential [PMID:9813002], and simultaneous blockade of cytochrome b together with an upstream NADH dehydrogenase is synergistic, underscoring cytochrome b's central position in ubiquinone/ubiquinol cycling [PMID:17242151]. Cryo-EM structures of the T. gondii III2-IV supercomplex resolved atovaquone and ELQ-300 bound within cytochrome b, revealing parasite-specific drug-binding geometries, while supercomplex disruption confirmed that this assembly is critical for parasite fitness [PMID:40389671]. Cytochrome b is also implicated as part of the mitochondrial target of artemisinin, whose activity correlates with amplification of the cytochrome b-containing mitochondrial genome and loss of membrane potential [PMID:31806760].","teleology":[{"year":1998,"claim":"It was unknown where in the T. gondii respiratory chain atovaquone acts; biochemical dissection localized the block to cytochrome b, establishing the bc1 complex as the drug target and cytochrome b as essential for respiration.","evidence":"In situ respiration and membrane-potential assays in digitonin-permeabilized tachyzoites with TMPD/ascorbate rescue","pmids":["9813002"],"confidence":"High","gaps":["Did not resolve which cytochrome b site (Qo vs Qi) is bound","No structural or residue-level information"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1999,"claim":"The molecular target of decoquinate in T. gondii was unknown; a resistance mutation in cytochrome b implicated the bc1 complex, extending cytochrome b as a shared antiparasitic drug target.","evidence":"Selection of a decoquinate-resistant mutant and cytochrome b gene sequencing","pmids":["10519259"],"confidence":"Medium","gaps":["Abstract-level detail only; exact mutation and site not defined","No enzymatic confirmation of altered drug binding"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2000,"claim":"The T. gondii cytochrome b sequence and the residues controlling atovaquone action were undefined; cloning of the gene from atovaquone-resistant mutants pinpointed M129L and I254L in the Qo domain, establishing atovaquone as a Qo-site inhibitor.","evidence":"Drug-resistant mutant generation, gene cloning/sequencing, and Qo-domain structural modeling","pmids":["10802314"],"confidence":"High","gaps":["Model predicted an unusual membrane-terminating topology not experimentally validated at the time","Qi site not addressed"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2003,"claim":"The physical basis of atovaquone binding and species-selectivity was unresolved; enzyme kinetics, EPR, and mutagenesis in a yeast surrogate showed atovaquone occupies the Qo pocket and engages the Rieske iron-sulfur protein, with L275F reducing sensitivity while preserving activity.","evidence":"bc1 inhibition kinetics, EPR of the Rieske center, site-directed mutagenesis in yeast, and structural modeling","pmids":["12791689"],"confidence":"High","gaps":["Performed in yeast, not native T. gondii enzyme","No high-resolution structure of the T. gondii binding site at this stage"],"attribution":[{"species":"Saccharomyces cerevisiae","strain":""}]},{"year":2007,"claim":"It was unclear how Qo-site inhibition combines with other respiratory blockades; a synergy assay with an NADH dehydrogenase inhibitor demonstrated that dual disruption of ubiquinone/ubiquinol cycling is highly potent, placing cytochrome b at a strategic pharmacological node.","evidence":"Combination drug assay with HDQ and atovaquone and fractional inhibitory concentration calculation in infected cultures","pmids":["17242151"],"confidence":"Medium","gaps":["Single lab, single combination readout","No direct measurement of enzyme-level interaction"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"claim":"Whether cytochrome b could be targeted at the Qi site against dormant stages was unknown; co-crystallography and parasite assays showed 4-(1H)-quinolones bind the Qi site and kill both tachyzoites and bradyzoites, which highly express cytochrome b.","evidence":"Co-crystallography with bc1, mutant yeast inhibition assays, in vitro proliferation assays, and a murine infection model","pmids":["27412848"],"confidence":"Medium","gaps":["Structural work used surrogate bc1, not T. gondii enzyme","Qi binding not yet confirmed genetically in T. gondii"],"attribution":[{"species":"Toxoplasma gondii","strain":"EGS"}]},{"year":2017,"claim":"Direct genetic evidence for a Qi-site inhibitor of T. gondii cytochrome b was missing; a T222P substitution conferring cross-resistance to ELQ-316 and the known Qi inhibitor antimycin established ELQ action at the Qi site.","evidence":"ELQ-316 resistance selection, cytochrome b sequencing, and antimycin cross-resistance testing","pmids":["27919897"],"confidence":"High","gaps":["Structural placement of T222P relative to the Qi pocket not resolved here","Effect on native enzyme kinetics not quantified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"claim":"The stability and mutability of the mitochondrial genome encoding cytochrome b was unclear; loss of TgMSH1 caused cytochrome b missense accumulation and revealed metabolic plasticity allowing survival without oxidative phosphorylation, while atovaquone still inhibited electron transport since mutations spared known sensitivity residues.","evidence":"TgMSH1 knockout, whole mitochondrial genome sequencing, electron transport assays, and glucose-dependence growth assays","pmids":["29141004"],"confidence":"Medium","gaps":["Functional consequence of individual cytochrome b missense mutations not dissected","Mechanism of MSH1-dependent mitochondrial DNA maintenance not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"claim":"Whether the cytochrome b-containing mitochondrion is an artemisinin target was unknown; artemisinin-resistant lines amplified the mitochondrial genome (including cytochrome b) and stably altered drug-induced membrane-potential loss, implicating mitochondrial respiratory complexes in artemisinin action.","evidence":"In vitro resistance evolution, whole-genome sequencing, CRISPR/Cas9 mutagenesis, and membrane-potential measurement","pmids":["31806760"],"confidence":"Medium","gaps":["Cytochrome b amplification is correlative rather than a defined mechanism","Direct artemisinin binding to cytochrome b not shown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"claim":"The native architecture of the T. gondii respiratory assembly and the structural basis of parasite-selective drug binding were unresolved; cryo-EM of the III2-IV supercomplex placed cytochrome b as a core subunit, visualized atovaquone and ELQ-300 in their sites (ELQ-300 in a flipped orientation versus mammalian enzyme), and knockout showed the supercomplex is critical for fitness.","evidence":"Cryo-EM structures of III2-IV and III2, drug co-structures, supercomplex-subunit knockout, and fitness assays","pmids":["40389671"],"confidence":"High","gaps":["Functional role of the supercomplex arrangement beyond fitness not fully explained","Dynamics of Qo/Qi catalysis not captured by static structures"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"How individual cytochrome b mutations quantitatively alter native enzyme catalysis versus drug binding, and the precise mechanism by which mitochondrial genome amplification confers artemisinin resistance, remain open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No kinetic dissection of native T. gondii cytochrome b with defined mutations","Artemisinin-cytochrome b relationship remains correlative"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["Cytochrome bc1 complex (complex III / III2)","III2-IV respiratory supercomplex"],"partners":["RIESKE IRON-SULFUR PROTEIN","CYTOCHROME C1","COMPLEX IV (CYTOCHROME C OXIDASE, WITHIN III2-IV SUPERCOMPLEX)"],"other_free_text":["Qo (ubiquinol oxidation) site: atovaquone binding; resistance via M129L, I254L, L275F [#0, #3]","Qi (quinone reduction) site: endochin-like quinolone and antimycin binding; T222P resistance [#4, #7]","Mitochondrially encoded; genome amplification associated with artemisinin resistance [#6]","Missense mutations accumulate upon TgMSH1 loss without necessarily affecting atovaquone sensitivity determinants [#5]"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"10802314","id":"PMC_10802314","title":"Characterization of cytochrome b from Toxoplasma gondii and Q(o) domain mutations as a mechanism of atovaquone-resistance.","date":"2000","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/10802314","citation_count":124,"is_preprint":false},{"pmid":"12791689","id":"PMC_12791689","title":"Molecular basis for atovaquone binding to the cytochrome bc1 complex.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12791689","citation_count":113,"is_preprint":false},{"pmid":"18212105","id":"PMC_18212105","title":"In vitro susceptibility of various genotypic strains of Toxoplasma gondii to pyrimethamine, sulfadiazine, and atovaquone.","date":"2008","source":"Antimicrobial agents and chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/18212105","citation_count":106,"is_preprint":false},{"pmid":"9813002","id":"PMC_9813002","title":"Respiration and oxidative phosphorylation in the apicomplexan parasite Toxoplasma gondii.","date":"1998","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9813002","citation_count":91,"is_preprint":false},{"pmid":"17242151","id":"PMC_17242151","title":"Growth inhibition of Toxoplasma gondii and Plasmodium falciparum by nanomolar concentrations of 1-hydroxy-2-dodecyl-4(1H)quinolone, a high-affinity inhibitor of alternative (type II) NADH dehydrogenases.","date":"2007","source":"Antimicrobial agents and chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/17242151","citation_count":66,"is_preprint":false},{"pmid":"25512210","id":"PMC_25512210","title":"Molecular characterisation of Sarcocystis lutrae n. sp. and Toxoplasma gondii from the musculature of two Eurasian otters (Lutra lutra) in Norway.","date":"2014","source":"Parasitology research","url":"https://pubmed.ncbi.nlm.nih.gov/25512210","citation_count":60,"is_preprint":false},{"pmid":"17826334","id":"PMC_17826334","title":"Modeling the molecular basis of atovaquone resistance in parasites and pathogenic fungi.","date":"2007","source":"Trends in parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/17826334","citation_count":52,"is_preprint":false},{"pmid":"27919897","id":"PMC_27919897","title":"Genetic Evidence for Cytochrome b Qi Site Inhibition by 4(1H)-Quinolone-3-Diarylethers and Antimycin in Toxoplasma gondii.","date":"2017","source":"Antimicrobial agents and chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/27919897","citation_count":49,"is_preprint":false},{"pmid":"30117728","id":"PMC_30117728","title":"Targeted Structure-Activity Analysis of Endochin-like Quinolones Reveals Potent Qi and Qo Site Inhibitors of Toxoplasma gondii and Plasmodium falciparum Cytochrome bc1 and Identifies ELQ-400 as a Remarkably Effective Compound against Acute Experimental Toxoplasmosis.","date":"2018","source":"ACS infectious diseases","url":"https://pubmed.ncbi.nlm.nih.gov/30117728","citation_count":40,"is_preprint":false},{"pmid":"27412848","id":"PMC_27412848","title":"New paradigms for understanding and step changes in treating active and chronic, persistent apicomplexan infections.","date":"2016","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/27412848","citation_count":37,"is_preprint":false},{"pmid":"31806760","id":"PMC_31806760","title":"Evolution of resistance in vitro reveals mechanisms of artemisinin activity in Toxoplasma gondii.","date":"2019","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/31806760","citation_count":35,"is_preprint":false},{"pmid":"21251932","id":"PMC_21251932","title":"Design of anti-parasitic and anti-fungal hydroxy-naphthoquinones that are less susceptible to drug resistance.","date":"2011","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/21251932","citation_count":31,"is_preprint":false},{"pmid":"23358734","id":"PMC_23358734","title":"Characterisation of full-length mitochondrial copies and partial nuclear copies (numts) of the cytochrome b and cytochrome c oxidase subunit I genes of Toxoplasma gondii, Neospora caninum, Hammondia heydorni and Hammondia triffittae (Apicomplexa: Sarcocystidae).","date":"2013","source":"Parasitology research","url":"https://pubmed.ncbi.nlm.nih.gov/23358734","citation_count":30,"is_preprint":false},{"pmid":"32626661","id":"PMC_32626661","title":"Potent Tetrahydroquinolone Eliminates Apicomplexan Parasites.","date":"2020","source":"Frontiers in cellular and infection microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/32626661","citation_count":26,"is_preprint":false},{"pmid":"32540978","id":"PMC_32540978","title":"Orally Bioavailable Endochin-Like Quinolone Carbonate Ester Prodrug Reduces Toxoplasma gondii Brain Cysts.","date":"2020","source":"Antimicrobial agents and chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/32540978","citation_count":26,"is_preprint":false},{"pmid":"30562391","id":"PMC_30562391","title":"Occurrence of tissue cyst forming coccidia in Magellanic penguins (Spheniscus magellanicus) rescued on the coast of Brazil.","date":"2018","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/30562391","citation_count":21,"is_preprint":false},{"pmid":"32161765","id":"PMC_32161765","title":"Activities of Endochin-Like Quinolones Against in vitro Cultured Besnoitia besnoiti Tachyzoites.","date":"2020","source":"Frontiers in veterinary science","url":"https://pubmed.ncbi.nlm.nih.gov/32161765","citation_count":16,"is_preprint":false},{"pmid":"10519259","id":"PMC_10519259","title":"Cytochrome b mutation identified in a decoquinate-resistant mutant of Toxoplasma gondii.","date":"1999","source":"The Journal of eukaryotic microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/10519259","citation_count":12,"is_preprint":false},{"pmid":"40389671","id":"PMC_40389671","title":"Structure, assembly and inhibition of the Toxoplasma gondii respiratory chain supercomplex.","date":"2025","source":"Nature structural & molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/40389671","citation_count":10,"is_preprint":false},{"pmid":"38820542","id":"PMC_38820542","title":"Molecular prevalence, phylogeny and hematological impact of Toxoplasma gondii and Plasmodium spp. in common quails from Punjab, Pakistan.","date":"2024","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/38820542","citation_count":10,"is_preprint":false},{"pmid":"29141004","id":"PMC_29141004","title":"Lack of mitochondrial MutS homolog 1 in Toxoplasma gondii disrupts maintenance and fidelity of mitochondrial DNA and reveals metabolic plasticity.","date":"2017","source":"PloS 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Institute","url":"https://pubmed.ncbi.nlm.nih.gov/42058244","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":15142,"output_tokens":2905,"usd":0.0445,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8811,"output_tokens":4416,"usd":0.077228,"stage2_stop_reason":"end_turn"},"total_usd":0.121728,"stage1_batch_id":"msgbatch_018xTxyBPcQFK9jdgubtxgsj","stage2_batch_id":"msgbatch_01UiG5BTaJP7BvKKL6RLeyv2","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"Cytochrome b of T. gondii was cloned and sequenced; atovaquone-resistant mutants carry mutations M129L and I254L within the Qo (ubiquinol-binding) domain, establishing that atovaquone interferes with electron transport at the cytochrome bc1 complex by targeting the Qo domain of cytochrome b. Structural modeling suggested cytochrome b may terminate within the mitochondrial membrane, differing from other homologues.\",\n      \"method\": \"Drug-resistant mutant generation, gene cloning and sequencing, structural modeling of Qo domain\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct cloning and sequencing of resistant mutants with defined point mutations, replicated by subsequent studies; first complete mitochondrial gene described for T. gondii\",\n      \"pmids\": [\"10802314\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"A mutation in cytochrome b was identified in a decoquinate-resistant mutant of T. gondii, implicating cytochrome b as the target of decoquinate at the cytochrome bc1 complex.\",\n      \"method\": \"Drug-resistant mutant selection and cytochrome b gene sequencing\",\n      \"journal\": \"The Journal of eukaryotic microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single study, gene sequencing of resistant mutant; abstract not available for full method detail\",\n      \"pmids\": [\"10519259\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Atovaquone at 0.03 µM totally inhibited respiration and disrupted mitochondrial membrane potential in T. gondii tachyzoites, and biochemical assays showed inhibition of the respiratory chain between cytochrome b and c1, consistent with bc1 complex inhibition at cytochrome b.\",\n      \"method\": \"In situ respiration assay using digitonin-permeabilized tachyzoites, mitochondrial membrane potential measurement with safranin O, TMPD/ascorbate rescue experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct biochemical assay with multiple substrates and inhibitor controls in intact parasites, single lab but multiple orthogonal readouts\",\n      \"pmids\": [\"9813002\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Atovaquone binds to the ubiquinol oxidation (Qo) pocket of the cytochrome bc1 complex and interacts with the Rieske iron-sulfur protein; a L275F mutation in cytochrome b decreases atovaquone sensitivity without loss of activity, demonstrating that this residue contributes to species-differential drug binding. (Experiments performed in yeast as a surrogate for T. gondii and other pathogens.)\",\n      \"method\": \"Enzyme inhibition kinetics (Ki determination), EPR spectroscopy of Rieske center, site-directed mutagenesis (L275F) of cytochrome b in yeast, computed energy-minimized structural model\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution in isolated bc1 complex, mutagenesis validation, EPR spectroscopy, and structural modeling; yeast surrogate but directly informs T. gondii cytochrome b mechanism\",\n      \"pmids\": [\"12791689\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Saccharomyces cerevisiae\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"A T222P substitution in T. gondii cytochrome b confers 49-fold resistance to ELQ-316 and 19-fold resistance to antimycin, a well-characterized Qi site inhibitor, providing genetic evidence that ELQ-316 inhibits cytochrome bc1 at the Qi site of T. gondii cytochrome b.\",\n      \"method\": \"Drug-resistant mutant selection under ELQ-316 pressure, cytochrome b gene sequencing, cross-resistance testing with antimycin\",\n      \"journal\": \"Antimicrobial agents and chemotherapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via drug-resistance mutation with cross-resistance validation against a known Qi site inhibitor; single lab but two orthogonal drug resistance readouts\",\n      \"pmids\": [\"27919897\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Loss of TgMSH1 leads to accumulation of missense mutations in cytochrome b and reduced mitochondrial DNA content; despite cytochrome b mutations, atovaquone can still inhibit electron transport in Tgmsh1 knockout parasites (none of the mutations affect known atovaquone-sensitivity determinants). Further atovaquone culture of Tgmsh1 mutants leads to complete respiratory shutdown, revealing metabolic plasticity allowing parasites to survive without oxidative phosphorylation.\",\n      \"method\": \"Knockout of TgMSH1, whole mitochondrial genome sequencing, electron transport inhibition assay, atovaquone drug selection, growth assays with and without glucose\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO with defined molecular phenotype, sequencing of cytochrome b mutations, and functional electron transport assay; single lab\",\n      \"pmids\": [\"29141004\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Artemisinin-resistant T. gondii populations show dramatic amplification of the mitochondrial genome, including cytochrome b and cytochrome c oxidase I genes; treatment with high concentrations of artemisinin decreases mitochondrial membrane potential in wild-type parasites, a phenotype stably altered in resistant lines, implicating the mitochondrion (and cytochrome b-containing complexes) as a target of artemisinin action.\",\n      \"method\": \"In vitro resistance evolution, whole-genome sequencing, CRISPR/Cas9 mutagenesis, mitochondrial membrane potential measurement\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome sequencing of evolved lines with functional mitochondrial assay; cytochrome b amplification is correlative but membrane potential assay provides mechanistic link; single lab\",\n      \"pmids\": [\"31806760\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Co-crystallographic and mutant yeast studies demonstrate that novel 4-(1H)-quinolone scaffolds bind to the cytochrome bc1 complex Qi site; T. gondii cysts highly express cytochrome b, and Qi-site inhibitors targeting cytochrome b are effective against both tachyzoites and bradyzoites in vitro and in vivo.\",\n      \"method\": \"Co-crystallography with bc1 complex, mutant yeast bc1 inhibition assays, in vitro parasite proliferation assays, murine infection model\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — co-crystallography and mutant yeast biochemistry support Qi site binding; parasite assays confirm functional relevance; single lab\",\n      \"pmids\": [\"27412848\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"EGS\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM structures of the T. gondii III2-IV respiratory supercomplex and of complex III2 alone were determined. Cytochrome b is a core subunit of complex III2; atovaquone and ELQ-300 were visualized in the cytochrome b binding sites. ELQ-300 binds in a flipped orientation compared with related compounds in the mammalian enzyme, revealing parasite-specific interactions at the Qo/Qi sites. A supercomplex knockout showed that III2-IV is critical for parasite fitness but does not simply enhance electron transfer efficiency as proposed for mammals.\",\n      \"method\": \"Cryo-electron microscopy structure determination, drug soaking/co-structure with atovaquone and ELQ-300, knockout analysis of supercomplex subunit, parasite fitness assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structures with drug-bound complexes, mutagenesis-informed drug design, and genetic knockout with fitness readout; multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"40389671\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Combined treatment of T. gondii with HDQ (an alternative NADH dehydrogenase inhibitor) and atovaquone (a cytochrome b Qo site inhibitor) resulted in synergistic inhibition (FIC = 0.16 nM), demonstrating that simultaneous blockade of ubiquinone/ubiquinol cycling at two mitochondrial sites is highly effective against parasite replication.\",\n      \"method\": \"Combination drug assay in T. gondii-infected cell cultures, fractional inhibitory concentration calculation\",\n      \"journal\": \"Antimicrobial agents and chemotherapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — pharmacological epistasis via combination assay establishing pathway position of cytochrome b Qo site; single lab, single method\",\n      \"pmids\": [\"17242151\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"T. gondii cytochrome b is a mitochondrially encoded core subunit of the cytochrome bc1 complex (complex III), functioning in electron transport at the Qo (ubiquinol oxidation) and Qi (quinone reduction) sites; atovaquone binds the Qo domain (with resistance conferred by M129L/I254L mutations), endochin-like quinolones bind either site (Qi inhibition confirmed by T222P resistance), and cryo-EM structures of the T. gondii III2-IV supercomplex have revealed the molecular basis of parasite-selective drug binding, informing next-generation inhibitor design.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"Toxoplasma gondii cytochrome b is a mitochondrially encoded core subunit of the cytochrome bc1 complex (complex III), where it catalyzes electron transport between ubiquinol and cytochrome c1 as part of the parasite's respiratory chain [#2, #8]. It carries two functionally distinct catalytic sites: a ubiquinol-oxidation (Qo) site targeted by atovaquone—resistance to which arises from M129L, I254L, and L275F substitutions clustered in the Qo domain [#0, #3]—and a quinone-reduction (Qi) site where endochin-like quinolones act, established genetically by a T222P substitution that confers cross-resistance to the classic Qi inhibitor antimycin [#4, #7]. Blocking the Qo site with atovaquone fully collapses respiration and mitochondrial membrane potential [#2], and simultaneous blockade of cytochrome b together with an upstream NADH dehydrogenase is synergistic, underscoring cytochrome b's central position in ubiquinone/ubiquinol cycling [#9]. Cryo-EM structures of the T. gondii III2-IV supercomplex resolved atovaquone and ELQ-300 bound within cytochrome b, revealing parasite-specific drug-binding geometries, while supercomplex disruption confirmed that this assembly is critical for parasite fitness [#8]. Cytochrome b is also implicated as part of the mitochondrial target of artemisinin, whose activity correlates with amplification of the cytochrome b-containing mitochondrial genome and loss of membrane potential [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"It was unknown where in the T. gondii respiratory chain atovaquone acts; biochemical dissection localized the block to cytochrome b, establishing the bc1 complex as the drug target and cytochrome b as essential for respiration.\",\n      \"evidence\": \"In situ respiration and membrane-potential assays in digitonin-permeabilized tachyzoites with TMPD/ascorbate rescue\",\n      \"pmids\": [\"9813002\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve which cytochrome b site (Qo vs Qi) is bound\", \"No structural or residue-level information\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"The molecular target of decoquinate in T. gondii was unknown; a resistance mutation in cytochrome b implicated the bc1 complex, extending cytochrome b as a shared antiparasitic drug target.\",\n      \"evidence\": \"Selection of a decoquinate-resistant mutant and cytochrome b gene sequencing\",\n      \"pmids\": [\"10519259\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Abstract-level detail only; exact mutation and site not defined\", \"No enzymatic confirmation of altered drug binding\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"The T. gondii cytochrome b sequence and the residues controlling atovaquone action were undefined; cloning of the gene from atovaquone-resistant mutants pinpointed M129L and I254L in the Qo domain, establishing atovaquone as a Qo-site inhibitor.\",\n      \"evidence\": \"Drug-resistant mutant generation, gene cloning/sequencing, and Qo-domain structural modeling\",\n      \"pmids\": [\"10802314\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Model predicted an unusual membrane-terminating topology not experimentally validated at the time\", \"Qi site not addressed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"The physical basis of atovaquone binding and species-selectivity was unresolved; enzyme kinetics, EPR, and mutagenesis in a yeast surrogate showed atovaquone occupies the Qo pocket and engages the Rieske iron-sulfur protein, with L275F reducing sensitivity while preserving activity.\",\n      \"evidence\": \"bc1 inhibition kinetics, EPR of the Rieske center, site-directed mutagenesis in yeast, and structural modeling\",\n      \"pmids\": [\"12791689\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Performed in yeast, not native T. gondii enzyme\", \"No high-resolution structure of the T. gondii binding site at this stage\"],\n      \"attribution\": [{\"species\": \"Saccharomyces cerevisiae\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"It was unclear how Qo-site inhibition combines with other respiratory blockades; a synergy assay with an NADH dehydrogenase inhibitor demonstrated that dual disruption of ubiquinone/ubiquinol cycling is highly potent, placing cytochrome b at a strategic pharmacological node.\",\n      \"evidence\": \"Combination drug assay with HDQ and atovaquone and fractional inhibitory concentration calculation in infected cultures\",\n      \"pmids\": [\"17242151\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab, single combination readout\", \"No direct measurement of enzyme-level interaction\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Whether cytochrome b could be targeted at the Qi site against dormant stages was unknown; co-crystallography and parasite assays showed 4-(1H)-quinolones bind the Qi site and kill both tachyzoites and bradyzoites, which highly express cytochrome b.\",\n      \"evidence\": \"Co-crystallography with bc1, mutant yeast inhibition assays, in vitro proliferation assays, and a murine infection model\",\n      \"pmids\": [\"27412848\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural work used surrogate bc1, not T. gondii enzyme\", \"Qi binding not yet confirmed genetically in T. gondii\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"EGS\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Direct genetic evidence for a Qi-site inhibitor of T. gondii cytochrome b was missing; a T222P substitution conferring cross-resistance to ELQ-316 and the known Qi inhibitor antimycin established ELQ action at the Qi site.\",\n      \"evidence\": \"ELQ-316 resistance selection, cytochrome b sequencing, and antimycin cross-resistance testing\",\n      \"pmids\": [\"27919897\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural placement of T222P relative to the Qi pocket not resolved here\", \"Effect on native enzyme kinetics not quantified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"The stability and mutability of the mitochondrial genome encoding cytochrome b was unclear; loss of TgMSH1 caused cytochrome b missense accumulation and revealed metabolic plasticity allowing survival without oxidative phosphorylation, while atovaquone still inhibited electron transport since mutations spared known sensitivity residues.\",\n      \"evidence\": \"TgMSH1 knockout, whole mitochondrial genome sequencing, electron transport assays, and glucose-dependence growth assays\",\n      \"pmids\": [\"29141004\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of individual cytochrome b missense mutations not dissected\", \"Mechanism of MSH1-dependent mitochondrial DNA maintenance not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Whether the cytochrome b-containing mitochondrion is an artemisinin target was unknown; artemisinin-resistant lines amplified the mitochondrial genome (including cytochrome b) and stably altered drug-induced membrane-potential loss, implicating mitochondrial respiratory complexes in artemisinin action.\",\n      \"evidence\": \"In vitro resistance evolution, whole-genome sequencing, CRISPR/Cas9 mutagenesis, and membrane-potential measurement\",\n      \"pmids\": [\"31806760\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cytochrome b amplification is correlative rather than a defined mechanism\", \"Direct artemisinin binding to cytochrome b not shown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"The native architecture of the T. gondii respiratory assembly and the structural basis of parasite-selective drug binding were unresolved; cryo-EM of the III2-IV supercomplex placed cytochrome b as a core subunit, visualized atovaquone and ELQ-300 in their sites (ELQ-300 in a flipped orientation versus mammalian enzyme), and knockout showed the supercomplex is critical for fitness.\",\n      \"evidence\": \"Cryo-EM structures of III2-IV and III2, drug co-structures, supercomplex-subunit knockout, and fitness assays\",\n      \"pmids\": [\"40389671\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional role of the supercomplex arrangement beyond fitness not fully explained\", \"Dynamics of Qo/Qi catalysis not captured by static structures\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How individual cytochrome b mutations quantitatively alter native enzyme catalysis versus drug binding, and the precise mechanism by which mitochondrial genome amplification confers artemisinin resistance, remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No kinetic dissection of native T. gondii cytochrome b with defined mutations\", \"Artemisinin-cytochrome b relationship remains correlative\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008121\", \"supporting_discovery_ids\": [2, 8], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0009055\", \"supporting_discovery_ids\": [2, 9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005743\", \"supporting_discovery_ids\": [2, 8], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0005750\", \"supporting_discovery_ids\": [0, 8], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0006122\", \"supporting_discovery_ids\": [2, 3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0022904\", \"supporting_discovery_ids\": [2, 9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\n      \"Cytochrome bc1 complex (complex III / III2)\",\n      \"III2-IV respiratory supercomplex\"\n    ],\n    \"partners\": [\n      \"Rieske iron-sulfur protein\",\n      \"cytochrome c1\",\n      \"complex IV (cytochrome c oxidase, within III2-IV supercomplex)\"\n    ],\n    \"other_free_text\": [\n      \"Qo (ubiquinol oxidation) site: atovaquone binding; resistance via M129L, I254L, L275F [#0, #3]\",\n      \"Qi (quinone reduction) site: endochin-like quinolone and antimycin binding; T222P resistance [#4, #7]\",\n      \"Mitochondrially encoded; genome amplification associated with artemisinin resistance [#6]\",\n      \"Missense mutations accumulate upon TgMSH1 loss without necessarily affecting atovaquone sensitivity determinants [#5]\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"RH88","gene_id":"TGRH88_085440","name":null,"product":"cytochrome b","uniprot_acc":null,"chromosome":"JAAUHK010000041","start_pos":47240}]}