The mitochondrion of apicomplexan parasites is critical for parasite survival, although the full complement of proteins that localize to this organelle has not been defined. Here we undertake two independent approaches to elucidate the mitochondrial proteome of the apicomplexan Toxoplasma gondii. We identify approximately 400 mitochondrial proteins, many of which lack homologs in the animals that these parasites infect, and most of which are important for parasite growth. We demonstrate that one such protein, termed TgApiCox25, is an important component of the parasite cytochrome c oxidase (COX) complex. We identify numerous other apicomplexan-specific components of COX, and conclude that apicomplexan COX, and apicomplexan mitochondria more generally, differ substantially in their protein composition from the hosts they infect. Our study highlights the diversity that exists in mitochondrial proteomes across the eukaryotic domain of life, and provides a foundation for defining unique aspects of mitochondrial biology in an important phylum of parasites.

Iron–sulfur (Fe‐S) clusters are prosthetic groups on proteins that function in a range of enzymatic and electron transfer reactions. Fe‐S cluster synthesis is essential for the survival of all eukaryotes. Independent Fe‐S cluster biosynthesis pathways occur in the mitochondrion, plastid, and cytosolic compartments of eukaryotic cells. Little is known about the cytosolic Fe‐S cluster biosynthesis in apicomplexan parasites, the causative agents of diseases such as malaria and toxoplasmosis. NBP35 serves as a key scaffold protein on which cytosolic Fe‐S clusters assemble, and has a cytosolic localization in most eukaryotes studied thus far. Unexpectedly, we found that the NBP35 homolog of the apicomplexan Toxoplasma gondii (TgNBP35) localizes to the outer mitochondrial membrane, with mitochondrial targeting mediated by an N‐terminal transmembrane domain. We demonstrate that TgNBP35 is critical for parasite proliferation, but that, despite its mitochondrial localization, it is not required for Fe‐S cluster synthesis in the mitochondrion. Instead, we establish that TgNBP35 is important for the biogenesis of cytosolic Fe‐S proteins. Our data are consistent with TgNBP35 playing a central and specific role in cytosolic Fe‐S cluster biosynthesis, and imply that the assembly of cytosolic Fe‐S clusters occurs on the cytosolic face of the outer mitochondrial membrane in these parasites.

Diverse compounds target the Plasmodium falciparum Na+ pump PfATP4, with cipargamin and (+)-SJ733 the most clinically-advanced. In a recent clinical trial for cipargamin, recrudescent parasites emerged, with most having a G358S mutation in PfATP4. Here, we show that PfATP4G358S parasites can withstand micromolar concentrations of cipargamin and (+)-SJ733, while remaining susceptible to antimalarials that do not target PfATP4. The G358S mutation in PfATP4, and the equivalent mutation in Toxoplasma gondii ATP4, decrease the sensitivity of ATP4 to inhibition by cipargamin and (+)-SJ733, thereby protecting parasites from disruption of Na+ regulation. The G358S mutation reduces the affinity of PfATP4 for Na+ and is associated with an increase in the parasite’s resting cytosolic [Na+]. However, no defect in parasite growth or transmissibility is observed. Our findings suggest that PfATP4 inhibitors in clinical development should be tested against PfATP4G358S parasites, and that their combination with unrelated antimalarials may mitigate against resistance development.

Small-molecule inhibitors of PfATP4, a Plasmodium falciparum protein that is believed to pump Na+ out of the parasite while importing H+, are on track to become much-needed new antimalarial drugs. The spiroindolone cipargamin is poised to become the first PfATP4 inhibitor to reach the field, having performed strongly in Phase 1 and 2 clinical trials. Previous attempts to generate cipargamin-resistant parasites in the laboratory have yielded parasites with reduced susceptibility to the drug; however, the highest 50% inhibitory concentration reported to date is 24 nM. Here, we show that P. falciparum parasites can acquire a clinically-significant level of resistance to cipargamin that enables them to withstand micromolar concentrations of the drug. Independent experiments to generate high-level cipargamin resistance using different protocols and strains led to the same change each time - a G358S mutation in PfATP4. Parasites with this mutation showed high-level resistance not only to cipargamin, but also to the dihydroisoquinolone (+)-SJ733. However, for certain other (less clinically advanced) PfATP4-associated compounds the G358S mutation in PfATP4 conferred only moderate resistance or no resistance. The G358S mutation in PfATP4 did not affect parasite susceptibility to antimalarials that do not target PfATP4. The G358S mutation in PfATP4, and the equivalent mutation in the Toxoplasma gondii ATP4 homologue (G419S), decreased the sensitivity of the Na+-ATPase activity of ATP4 to inhibition by cipargamin and (+)-SJ733, and decreased the sensitivity of parasites expressing these ATP4 mutations to disruption of parasite Na+ regulation by cipargamin- and (+)-SJ733. The G358S mutation in PfATP4 reduced the affinity of the protein for Na+ and was associated with an increase in the parasite's resting cytosolic Na+ concentration; however, no significant defect in parasite growth rate was observed. Our findings suggest that codon 358 in pfatp4 should be monitored closely in the field as a molecular marker for cipargamin resistance, and that PfATP4 inhibitors in clinical development should be tested for their activity against PfATP4G358S parasites.

Background: Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disease characterized by development of autoantibodies and multiorgan involvement. Kidney involvement, termed lupus nephritis, has major impact on life expectancy. It is increasingly recognized that SLE is likely a common clinical manifestation of pathophysiologically diverse processes, and lupus nephritis has similarly been associated with several distinct immunological processes. We compared the immune cell phenotypes of individuals with SLE in the presence or absence of nephritis. Methods: Cryopreserved peripheral blood mononuclear cells from SLE patients with and without kidney involvement underwent flow cytometric analysis to identify major populations in T cells, B cells and myeloid lineages. Results: We compared the frequencies of lymphocyte populations in 69 SLE patients without nephritis, 20 SLE patients with nephritis, and 92 healthy blood donors. Patients with SLE and lupus nephritis (LN) had reduced marginal zone B cells (P < 0.0001 in SLE; P = 0.001 in LN), memory B cells (P = 0.002 in SLE; P = 0.001 in LN) and circulating T follicular helper (Tfh) memory cells (P < 0.0001 in SLE and LN) compared to healthy donors. Patients with lupus nephritis had increase Th2 (P < 0.0001) and T regulatory cells (P < 0.0001) compared to both SLE patients without nephritis and healthy donors. Conclusion: SLE patients with and without lupus nephritis have distinct immunologic differences that may reflect the unique pathophysiological processes contributing to disease manifestations. K E Y W O R D S immunoprofiling, lupus nephritis, systemic lupus erythematosus Key points • Patients with SLE had reduced marginal zone B cells, memory B cells and circulating T follicular helper cells compared to healthy blood donors. • Patients with lupus nephritis had increased Th2 and T regulatory cells compared to SLE patients without lupus nephritis and healthy blood donors.

We report a case of new-onset seropositive inflammatory arthritis following denosumab use in an elderly patient. This highlights the need to consider this rare adverse effect in patients who develop severe arthralgia and myalgia after denosumab.

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