Real-Time Analysis of Mitochondrial Electron Transport Chain Function in Toxoplasma gondii Parasites Using a Seahorse XFe96 Extracellular Flux Analyzer

AbstractThe mitochondrion is critical for the survival of apicomplexan parasites. Several major anti-parasitic drugs, such as atovaquone and endochin-like quinolones, act through inhibition of the mitochondrial electron transport chain at the coenzyme Q:cytochrome c oxidoreductase complex (Complex III). Despite being an important drug target, the protein composition of Complex III of apicomplexan parasites has not been elucidated. Here, we undertake a mass spectrometry-based proteomic analysis of Complex III in the apicomplexan Toxoplasma gondii. Along with canonical subunits that are conserved across eukaryotic evolution, we identify several novel or highly divergent Complex III components that are conserved within the apicomplexan lineage. We demonstrate that one such subunit, which we term TgQCR11, is critical for parasite proliferation, mitochondrial oxygen consumption and Complex III activity, and establish that loss of this protein leads to defects in Complex III integrity. We conclude that the protein composition of Complex III in apicomplexans differs from that of the mammalian hosts that these parasites infect.Author summaryApicomplexan parasites cause numerous diseases in humans and animals, including malaria (Plasmodium species) and toxoplasmosis (Toxoplasma gondii). The coenzyme Q:cytochrome c oxidoreductase protein complex (Complex III) performs a central role in the mitochondrial electron transport chain of many eukaryotes. Despite being the target of several major anti-apicomplexan drugs, the protein composition of Complex III in apicomplexans was previously unknown. Our work identifies novel proteins in Complex III of apicomplexans, one of which is critical for complex function and integrity. Our study highlights divergent features of Complex III in apicomplexans, and provides a broader understanding of Complex III evolution in eukaryotes. Our study also provides important insights into what sets this major drug target apart from the equivalent complex in host species.

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AB0031 T HELPER 17 CELLS WERE SIGNIFICANTLY DECREASED BY MITOCHONDRIAL ELECTRON TRANSPORT CHAIN COMPLEX INHIBITOR IN PATIENTS WITH RHEUMATOID ARTHRITIS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.3441 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 1318.2-1318

Author(s):

H. R. Lee ◽

S. J. Yoo ◽

J. Kim ◽

I. S. Yoo ◽

C. K. Park ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Electron Transport ◽

Disease Activity ◽

Th17 Cells ◽

Electron Transport Chain ◽

Chain Complex ◽

Complex Iii ◽

Mitochondrial Electron Transport Chain ◽

Transport Chain ◽

Healthy Control

Background:Reactive oxygen species (ROS) and T helper 17 (TH17) cells have been known to play an important role in the pathogenesis of rheumatoid arthritis (RA). However, the interrelationship between ROS and TH17 remains unclear in RAObjectives:To explore whether ROS affect TH17 cells in peripheral blood mononuclear cells (PBMC) of RA patients, we analyzed ROS expressions among T cell subsets following treatment with mitochondrial electron transport chain complex inhibitors.Methods:Blood samples were collected from 40 RA patients and 10 healthy adult volunteers. RA activity was divided according to clinical parameter DAS28. PBMC cells were obtained from the whole blood using lymphocyte separation medium density gradient centrifugation. Following PBMC was stained with Live/Dead stain dye, cells were incubated with antibodies for CD3, CD4, CD8, and CD25. After fixation and permeabilization, samples were stained with antibodies for FoxP3 and IL-17A. MitoSox were used for mitochondrial specific staining.Results:The frequency of TH17 cells was increased by 4.83 folds in moderate disease activity group (5.1>DAS28≥3.2) of RA patients compared to healthy control. Moderate RA activity patients also showed higher ratio of TH17/Treg than healthy control (3.57 folds). All RA patients had elevated expression of mitochondrial specific ROS than healthy control. When PBMC cells were treated with 2.5uM of antimycin A (mitochondrial electron transport chain complex III inhibitor) for 16 h, the frequency of TH17 cells was significantly decreased.Conclusion:The mitochondrial electron transport chain complex III inhibitor markedly downregulated the frequency of TH17 cells in moderate disease activity patients with RA. These findings provide a novel approach to regulate TH17 function in RA through mitochondrial metabolism related ROS production.References:[1]Szekanecz, Z., et al., New insights in synovial angiogenesis. Joint Bone Spine, 2010. 77(1): p. 13-9.[2]Prevoo, M.L., et al., Modified disease activity scores that include twenty-eight-joint counts. Development and validation in a prospective longitudinal study of patients with rheumatoid arthritis. Arthritis Rheum, 1995. 38(1): p. 44-8.Disclosure of Interests:None declared

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