Biology subjects

Sheiner, L.

Publications and source records attributed to Sheiner, L..

14 recordsLinked to original sources

bioRxiv2026

Proteome-wide crosslinking mass spectrometry reveals novel components of essential complexes in Toxoplasma

Protein-protein interactions underpin nearly all cellular processes, yet systematic definition of these networks remains limited outside a few model organisms. As a result, the architectures of essential complexes in many divergent lineages remain poorly characterized. Here we developed a high-coverage crosslinking mass spectrometry framework to map the proteome-wide interactome of the model apicomplexan parasite Toxoplasma gondii. From 29,624 crosslinked peptide pairs, we resolved a network of 2,859 protein-protein interactions that we integrated with structural modeling to resolve interaction interfaces. We identified and validated previously unrecognized components of essential protein complexes, including a structurally distinct ATP synthase subcomplex containing a highly divergent, apicomplexan-specific subunit essential for parasite fitness. Beyond revealing unexpected diversification of core mitochondrial machinery, these findings provide a general strategy to define the molecular architecture of divergent organisms and represent a foundational resource for hypothesis generation, structural inference, and discovery of lineage-specific vulnerabilities in pathogen biology.

microbiology↗

bioRxiv2026

Toxoplasma gondii Fatty Acid Elongases are Important for Virulence and Persistence

Toxoplasma gondii is an apicomplexan parasite which can infect a diverse range of warm-blooded host species. This promiscuous lifestyle requires metabolic flexibility yet our understanding of the metabolic pathways in this parasite remains incomplete. This gap includes the endoplasmic reticulum localised fatty acid elongation (FAE) pathway, which is responsible for synthesis of long (LCFA) and very long chain fatty acids (VLCFA), thus contributing to critical functions such as lipid storage and membrane biogenesis. Analyses of FAE in the commonly studied type I T. gondii strain revealed its contribution to the production of LCFA and VLCFA in vitro. However, the cystogenic type II T. gondii, which is responsible for most toxoplasmosis cases worldwide, was not studied. These type II parasites can readily differentiate between virulent and immune-evasive stages whereas type I cannot. This ability to readily transition between life stages, which are metabolically fundamentally different, prompts the question of whether the FAE pathway may play different roles in their distinct phenotype. Here we address this question, first confirming the role of the FAE enzymes, TgELO-A & B, in the production of LCFA and VLCFA in a type II T. gondii strain. Next, we demonstrated that their deletion leads to defects in replication and invasion when grown under lipid-restricted conditions. The mutants were further shown to be attenuated in mice as demonstrated by extended survival. Importantly, FAE enzyme deletion caused reduced brain cyst burdens. These findings highlight the importance of FAE for parasite survival under metabolic stress and in vivo. ImportanceToxoplasma gondii infects a wide range of hosts and persists for life in multiple tissues, making metabolic flexibility essential for its success. Fatty acid elongation (FAE), mediated by elongase (ELO) enzymes, enables the parasite to modify both de novo synthesised and host-scavenged fatty acids to support membrane biogenesis, lipid storage, and adaptation to changing nutrient environments. Previous studies in the type I strain established the importance of FAE for parasite metabolism, but its role in the clinically relevant, cystogenic type II lineage--which causes most cases of human toxoplasmosis and forms long-lived, immune-evasive bradyzoite cysts-- remained unknown. Here, we address this gap by characterising type II ELO mutants and show that disruption of FAE remodels the parasite lipidome, impairs fitness under fatty acid-limiting conditions, and reduces virulence and chronic persistence in mice. These findings identify FAE as a key metabolic pathway underpinning adaptation and persistence during chronic infection.

microbiology↗

bioRxiv2026

Doxycycline inhibits both apicoplast and mitochondrial translation in apicomplexan parasites

Doxycycline is a tetracycline-class antibiotic used for malarial prophylaxis and as an occasional partner drug in malaria treatment. Doxycyclines antimalarial mechanism of action is attributed to inhibiting the prokaryotic 70S ribosomes of the apicoplast, a non-photosynthetic plastid present in most apicomplexan species including Plasmodium and Toxoplasma. At lower concentrations (<5 M) doxycycline exhibits a delayed death phenotype, typical of inhibitors of apicoplast housekeeping processes. Unlike other delayed death drugs (e.g. clindamycin), doxycycline has rapid schizonticidal activity at higher concentrations (>10 M) via an unknown and apicoplast-independent mechanism. In other eukaryotes, and plausibly in Plasmodium and Toxoplasma, doxycycline inhibits mitochondrial 70S ribosomes. Here we use a mass spectrometry approach to assess steady state and turnover for apicoplast DNA encoded proteins in Plasmodium falciparum. We directly show that these proteins decrease in both abundance and synthesis following treatment with doxycycline and clindamycin. High concentrations of doxycycline, but not clindamycin, also reduced the abundance of a mitochondrial-encoded protein. Proteins encoded by the mitochondrial genome are required for the formation of complexes III and IV in the electron transport chain. We show a reduction in abundance and activity of complex IV following doxycycline treatment in Toxoplasma gondii. High doxycycline concentrations also disrupt oxidative phosphorylation in both P. falciparum and T. gondii, likely as a consequence of impaired complex III and IV formation. For the first time we directly measure apicoplast translation inhibition and characterise doxycycline as a mitochondrial translation inhibitor of P. falciparum and T. gondii.

microbiology↗

bioRxiv2026

Screening of purine nucleoside analogues against intracellular Toxoplasma gondii.

Toxoplasmosis remains a world-wide public health concern, especially for the immunocompromised. Although this population segment is increasing due to therapeutic interventions, organ transplants and infections including HIV, treatment relies almost exclusively on sulfadoxine and pyrimethamine, antifolates developed against malaria but with only moderate efficacy against acute toxoplasmosis and no effect on the chronic stage. Here we explore whether 7-substituted analogues of 7-deazaadenosine (tubercidin) that have shown remarkable efficacy against other protozoan pathogens, might also show anti-toxoplasmic activity. Tubercidin and a series of eleven 7-substituted analogues including 2-deoxy and 3-deoxyribofuranoses was tested against intracellular Toxoplasma gondii tachyzoites. The test compounds yielded EC50 values between 0.012 and 1.72 {micro}M, well below those of the control drug sulfadiazine (11.9 {micro}M) and the previously identified purine analogue adenosine arabinoside (Ara-A; 11.4 {micro}M). The tubercidin analogues displayed at most moderate toxicity to HFF cells, with the most efficacious compound, 7-(3,4-di-Cl-phenyl)-3-deoxytubercidin (FH8513) reaching a selectivity index of >2500. These nucleosides are most likely taken up by T. gondii through one of the four Equilibrative Nucleoside Transporters (ENTs) encoded by the parasites. However, deletion of TgENT2 and/or TgENT3 had no effect on the EC50 values, and deletion of TgAT1 actually sensitised the tachyzoites to most of the tubercidin analogues. We propose that these nucleosides are internalised through the TgENT1 uridine transporter and that the sensitisation in {Delta}TgAT1 cells is the result of reduced uptake of adenosine that competes with the tubercidin analogues for metabolic enzymes such as adenosine kinase.

microbiology↗

bioRxiv2025

Complexome profile of mitochondrial complexes in the myzozoan parasite of oysters, Perkinsus marinus

Mitochondrial complexes, such as the mitochondrial electron transport chain (mETC), the F1Fo-ATP synthase and the mitochondrial ribosome, are centrally important for mitochondrial function. Recently, an unexpected diversity in the composition of these complexes across unicellular eukaryotic lineages, with the apicomplexan parasites featuring prominently, has been revealed. However, whether the observed enlarged and divergent mitochondrial complexes are conserved across the wider Myzozoan lineage has not been investigated. Here, using a complexome profiling proteomic approach, we have characterised the composition of the mitochondrial complexes of the myzozoan parasite of oysters, Perkinsus marinus. We show that it shares with Plasmodium and Toxoplasma huge ATP synthase and mETC complexes. Moreover, the Perkinsus mitoribosome possesses many of the divergent features recently discovered in apicomplexans, such as an expanded subunit repertoire and the inclusion of RAP-domain and ApiAP2-like proteins as ribosomal proteins. Our study reveals the divergent subunit composition of mitochondrial complexes is an ancestral and highly conserved feature of Myzozoa.

microbiology↗

bioRxiv2024

Structure of the II2-III2-IV2 mitochondrial supercomplex from the para-site Perkinsus marinus

Respiratory complexes have co-evolved into supercomplexes in different clades to sustain energy production at the basis of eukaryotic life. In this study, using cryogenic electron microscopy, we determined the 2.1 [A] resolution structure of a 104-subunit II2-III2-IV2 supercomplex from the parasite Perkinsus marinus, related to Apicomplexa, capable of complete electron transport from succinate to molecular oxygen. A feature of the parasite is the association of two copies of complex II via the apicomplexan subunit SDHG that interacts with both complexes III and IV and bridge the supercomplex. In the c1 state, we identified two protein factors, ISPR1 and ISPR2 bound on the surface of complex III, where Cytochrome c docks, acting as negative regulators. The acquisition of 15 specific subunits to complex IV results in its lateral offset, increasing the distance between the Cytochrome c electron donor and acceptor sites. The domain homologous to canonical mitochondria-encoded transmembrane subunit COX2 is made of three separate polypeptides encoded in the nucleus, and their correct assembly is a prerequisite for electron transport in the supercomplex. Subunits Cytochrome b and COX1 comprise a +2 frameshift introduced during protein synthesis by the mitoribosome. Among 114 modelled endogenous lipids, we detect a direct contribution to the formation of the divergent supercomplex and its functional sites, including assembly of CII and ubiquinone binding. Together, our findings expose the uniqueness of the principal components of bioenergetics in the mitochondria of parasites.

biochemistry↗

bioRxiv2024

The Toxoplasma gondii mitochondrial transporter ABCB7 is essential for cytosolic iron-sulfur cluster biogenesis and protein translation

Iron-sulfur (Fe-S) clusters are ubiquitous inorganic cofactors required for numerous essential cellular pathways. Since they cannot be scavenged from the environment, Fe-S clusters are synthesised de novo in cellular compartments such as the apicoplast, mitochondrion and cytosol. The cytosolic Fe-S cluster biosynthesis pathway relies on transport of an intermediate from the mitochondrial pathway. An ATP binding cassette (ABC) transporter called ABCB7 is responsible for this role in numerous commonly studied organisms, but its role in the medically important apicomplexan parasites has not yet been studied. Here we identify and characterise the Toxoplasma gondii ABCB7 homolog. Genetic depletion shows that it is essential for parasite growth, and that disruption triggers partial stage conversion. Characterisation of the knock-down line highlights a defect in cytosolic Fe-S cluster biogenesis leading to defects in protein translation and other pathways including DNA and RNA replication and metabolism. Our work provides support for a broad conservation of the connection between mitochondrial and cytosolic in Fe-S cluster biosynthesis and reveal its importance for parasite survival.

microbiology↗

bioRxiv2024

Plasmodium falciparum mitochondrial complex III, the target of atovaquone, is essential for progression to the transmissible sexual stages.

The Plasmodium mitochondrial electron transport chain (mETC) is responsible for essential metabolic pathways such as de novo pyrimidine synthesis and ATP synthesis. The mETC complex III (cytochrome bc1 complex) is responsible for transferring electrons from ubiquinol to cytochrome c and generating a proton gradient across the inner mitochondrial membrane, which is necessary for the function of ATP synthase. Recent studies revealed that the composition of Plasmodium complex III is divergent from human, highlighting its suitability as a target for specific inhibition. Indeed, complex III is the target of the clinically used anti-malarial atovaquone and of several inhibitors undergoing pre-clinical trials, yet its role in parasite biology have not been thoroughly studied. We provide evidence that the universally conserved subunit, PfRieske, and the new parasite subunit, PfC3AP2, are part of Plasmodium falciparum complex III (PfCIII), with the latter providing support for the prediction of its divergent composition. Using inducible depletion, we show that PfRieske, and therefore PfCIII as a whole, is essential for asexual blood stage parasite survival, in line with previous observations. We further found that depletion of PfCIII results in gametocyte maturation defect. These phenotypes are linked to defects in mitochondrial functions upon PfRieske depletion, including increased sensitivity to mETC inhibitors in asexual stages and decreased cristae abundance alongside abnormal mitochondrial morphology in gametocytes. This is the first study which explores the direct role of the PfCIII in gametogenesis via genetic disruption, paving the way for a better understanding of the role of mETC in the complex life cycle of these important parasites and providing further support for the focus of antimalarial drug development on this pathway.

cell biology↗

bioRxiv2023

Pyrimidine salvage in Toxoplasma gondii as a target for new treatment

Toxoplasmosis is a common protozoan infection that can have severe outcomes in the immunocompromised and during pregnancy, but treatment options are limited. Recently, nucleotide metabolism has received much attention as a target for new antiprotozoal agents and here we focus on pyrimidine salvage by Toxoplasma gondii as a drug target. Whereas uptake of [3H]-cytidine and particularly [3H]-thymidine was at most marginal, [3H]-uracil and [3H]-uridine were readily taken up. Kinetic analysis of uridine uptake was consistent with a single transporter with a Km of 3.3 {+/-} 0.8 {micro}M, which was inhibited by uracil with high affinity (Ki = 1.15 {+/-} 0.07 {micro}M) but not by thymidine or 5-methyluridine, showing that the 5-Me group is incompatible with uptake by T. gondii. Conversely, [3H]-uracil transport displayed a Km of 2.05 {+/-} 0.40 {micro}M, not significantly different from the uracil Ki on uridine transport, and was inhibited by uridine with a Ki 2.44 {+/-} 0.59 {micro}M, also not significantly different from the experimental uridine Km. The reciprocal, complete inhibition, displaying Hill slopes of approximately [~]1, strongly suggest that uridine and uracil share a single transporter with similarly high affinity for both, and we designate it uridine/uracil transporter 1 (TgUUT1). While TgUUT1 excludes 5-methyl substitutions, the smaller 5F substitution was tolerated as 5F-uracil inhibited uptake of [3H]-uracil with a Ki of 6.80 {+/-} 2.12 {micro}M (P > 0.05 compared to uracil Km). Indeed, we found that 5F-Uridine, 5F-uracil and 5F,2-deoxyuridine were all potent antimetabolites against T. gondii with EC50 values well below that of the current first line treatment, sulfadiazine. In vivo evaluation also showed that 5F-uracil and 5F,2-deoxyuridine were similarly effective as sulfadiazine against acute toxoplasmosis. Our preliminary conclusion is that TgUUT1 mediates potential new anti-toxoplasmosis drugs with activity superior to the current treatment.

microbiology↗

bioRxiv2020

Depletion of voltage-dependent anion channel (VDAC) of Toxoplasma gondii affects multiple mitochondrial functions, but not calcium signalling.

The Voltage Dependent Anion channel (VDAC) is a ubiquitous channel in the outer membrane of the mitochondrion with multiple roles in protein, metabolite and small molecule transport. In mammalian cells, VDAC, as part of a larger complex including the inositol triphosphate receptor, has been shown to have a role in mediating contact between the mitochondria and ER. We identify VDAC of the pathogenic apicomplexan Toxoplasma gondii and demonstrate its importance for parasite growth. We show that VDAC is involved in protein import and metabolite transfer to the mitochondria, but does not appear to modulate calcium (Ca2+) signalling. Further, depletion of VDAC resulted in significant morphological changes of the mitochondrion and ER, suggesting a role in mediating contacts between these organelles in T. gondii.

cell biology↗

bioRxiv2020

Complexome profile of Toxoplasma gondii mitochondria identifies a divergent cytochrome bc1 complex

The mitochondrial electron transport chain (mETC) and F1Fo-ATP synthase are of central importance for energy and metabolism in eukaryotic cells. The Apicomplexa, important pathogens of humans causing diseases such as toxoplasmosis and malaria, depend on their mETC in every known stage of their complicated life cycles. Here, using a complexome profiling proteomic approach, we have characterised the Toxoplasma mETC complexes and F1Fo-ATP synthase. We identified and assigned 60 proteins to complexes II, IV and F1Fo-ATP synthase of Toxoplasma, of which 16 have not been identified previously. Notably, our complexome profile elucidates the composition of the Toxoplasma complex III, the target of clinically used drugs such as atovaquone. We identified two new homologous subunits and two new parasite-specific subunits, one of which is broadly conserved in myzozoans. We demonstrate all four proteins are essential for complex III stability and parasite growth, and show their depletion leads to decreased mitochondrial potential, supporting their role as complex III subunits. Our study highlights the divergent subunit composition of the apicomplexan mETC complexes and sets the stage for future structural and drug discovery studies. Author SummaryApicomplexan parasites, such as Toxoplasma and Plasmodium, cause diseases of global importance, such as toxoplasmosis and malaria. The mitochondrial electron transport chain (mETC) and F1Fo-ATP synthase, which provide the parasite with energy and important metabolites, are essential for parasite function. Here, using a proteomic technique called complexome profiling, we report the composition of the Toxoplasma mETC and F1Fo-ATP synthase. In particular, we reveal the compositions of complexes II and III for the first time. Complex III is an important drug target, yet its full protein composition was unknown. We identify new parasite-specific complex III subunits and demonstrate that they are essential for parasite survival and for proper functioning of the mETC. Our study highlights the divergent nature of the apicomplexan mETC and F1Fo-ATP synthase.

cell biology↗

bioRxiv2020

Lipoic acid biosynthesis is essential for Plasmodium falciparum transmission and influences redox response and carbon metabolism of parasite asexual blood stages

Malaria is still one of the most important global infectious diseases. Emergence of drug resistance and a shortage of new efficient anti-malarials continue to hamper a malaria eradication agenda. Malaria parasites are highly sensitive to changes in redox environment. Understanding the mechanisms regulating parasite redox could contribute to the design of new drugs. Malaria parasites have a complex network of redox regulatory systems housed in their cytosol, in their mitochondrion and in their plastid (apicoplast). While the roles of enzymes of the thioredoxin and glutathione pathways in parasite survival have been explored, the antioxidant role of -lipoic acid (LA) produced in the apicoplast has not been tested. We analysed the effects of LA depletion on mutant Plasmodium falciparum lacking the apicoplast lipoic acid protein ligase B (lipB). Our results showed a change in expression of redox regulators in the apicoplast and the cytosol. We further detected a change in parasite central carbon metabolism, with LA depletion influencing glycolysis and tricarboxylic acid cycle activity. Importantly, abrogation of LipB impacted P. falciparum mosquito development, preventing oocyst maturation and production of infectious sporozoite stages, thus flagging LA biosynthesis as a potential target for the development of new transmission drugs.

cell biology↗

bioRxiv2019

In silico screen identifies a new Toxoplasma gondii mitochondrial ribosomal protein essential for mitochondrial translation

Apicomplexan parasites cause diseases such as malaria and toxoplasmosis. The apicomplexan mitochondrion shows striking differences from common model organisms, including in fundamental processes such as mitochondrial translation. Despite evidence that mitochondrial translation is essential for parasites survival, it is largely understudied. Progress has been restricted by the absence of functional assays to detect apicomplexan mitochondrial translation, a lack of knowledge of proteins involved in the process and the inability to identify and detect mitoribosomes. Using mRNA expression patterns, 279 candidate mitochondrial housekeeping components were identified in Toxoplasma. 11 were validated, including the mitoribosomal small subunit protein 35 (TgmS35). TgmS35 tagging enabled the detection of a macromolecular complex corresponding to the mitoribosomal small subunit for the first time in apicomplexans. A new analytical pipeline detected defects in mitochondrial translation upon TgmS35 depletion, while other mitochondrial functions remain unaffected. Our work lays a foundation for the study of apicomplexan mitochondrial translation. Abbreviated summaryThe apicomplexan mitochondrion is divergent and essential yet poorly studied. Mitochondrial translation is predicted to utilize ribosomes assembled from fragmented rRNA but this was never shown. Knowing the mitochondrial protein content is critical for these studies. We identified 11 new mitochondrial proteins via in-silico searches. Tagging and depletion of a mitoribosomal small subunit protein enabled the first detection of a macromolecular ribosomal complex, and provided proof of principle for our new mitochondrial translation analytic pipeline.

cell biology↗

bioRxiv2018

Engineering Brain Parasites for Intracellular Delivery of Therapeutic Proteins

Protein therapy has the potential to alleviate many neurological diseases; however, delivery mechanisms for the central nervous system (CNS) are limited, and intracellular delivery poses additional hurdles. To address these challenges, we harnessed the protist parasite Toxoplasma gondii, which can migrate into the CNS and secrete proteins into cells. Using a fusion protein approach, we engineered T. gondii to secrete therapeutic proteins for human neurological disorders. We tested two secretion systems, generated fusion proteins that localized to T. gondiis secretory organelles and assessed their intracellular targeting in various mammalian cells including neurons. We show that T. gondii expressing GRA16 fused to the Rett syndrome protein MeCP2 deliver a fusion protein that mimics the endogenous MeCP2, binding heterochromatic DNA in neurons. This demonstrates the potential of T. gondii as a therapeutic protein vector, which could provide either transient or chronic, in situ synthesis and delivery of intracellular proteins to the CNS.

synthetic biology↗