Biology subjects

Besteiro, S.

Publications and source records attributed to Besteiro, S..

11 recordsLinked to original sources

bioRxiv2026

ABCE1-dependent translational control links Fe-S cluster biogenesis to parasite growth and lipid homeostasis in Toxoplasma gondii

Toxoplasma gondii relies on tightly regulated protein synthesis to adapt to diverse host environments and to progress through its developmental stages. Here, we investigated the role of the ATP-binding cassette protein ABCE1, a broadly-conserved factor involved in ribosome recycling and translational control. Using a conditional knockdown approach, we demonstrate that depletion of TgABCE1 severely impairs parasite growth and disrupts global protein synthesis, confirming its essential role in maintaining translational capacity. TgABCE1 function depends on the incorporation of iron-sulfur (Fe-S) clusters, likely mediated by the cytosolic iron-sulfur assembly (CIA) pathway component HCF101. Depletion of TgABCE1 phenocopies the defects observed in TgHCF101-depleted parasites, supporting a functional link between these proteins. Notably, loss of TgABCE1 also disrupts lipid homeostasis, resulting in the accumulation of lipid droplets. Together, these findings uncover a critical link between translational regulation, Fe-S cluster biogenesis, and lipid homeostasis, highlighting the central role of proteostasis in parasite survival and development.

microbiology↗

bioRxiv2025

HIV-1 Tat favors the multiplication of Mycobacterium tuberculosis and toxoplasma by inhibiting clathrin-mediated endocytosis and autophagy

AbstractHIV-1 and Mycobacterium tuberculosis (Mtb) coinfections are a major public health problem but are not well characterized. HIV-1 Tat is secreted by infected cells, generating nanomolar concentrations of Tat in the sera of people living with HIV. Circulating Tat enters cells, binds to PI(4,5)P2 then undergoes palmitoylation, thereby becoming resident on this phosphoinositide. Here, we found that Tat favors the multiplication of Mtb in macrophages. Moreover, Tat renders zebrafish larvae more sensitive to mycobacterial infection. We found that Tat binding to PI(4,5)P2 and palmitoylation enable Tat to inhibit the recruitment of the AP-2 adaptor, thereby inhibiting clathrin-mediated endocytosis and in turn autophagy. This inhibition prevents the degradation of intracellular pathogens such as Mtb and opsonized Toxoplasma gondii, but also of lipid droplets, thereby facilitating the access of these pathogens to lipids. We thus identified a mechanism enabling HIV Tat to favor the multiplication of intracellular pathogens such as Mtb.

microbiology↗

bioRxiv2024

TgGloL is an atypical glyoxalase/VOC domain-containing apicoplast protein that is important for the growth of Toxoplasma

Glycolysis is a conserved metabolic pathway that converts glucose into pyruvate in the cytosol, producing ATP and NADH. In Toxoplasma gondii and several other apicomplexan parasites, some glycolytic enzymes have isoforms located in their plastid (called the apicoplast). In this organelle, glycolytic intermediates like glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) are imported from the cytosol and further metabolized, providing ATP, reducing power, and precursors for anabolic pathways such as isoprenoid synthesis. However, GAP and DHAP can spontaneously convert into methylglyoxal, a toxic by-product detoxified by the glyoxalase system, typically involving Glyoxalase-1 (Glo-1) and Glyoxalase-2 (Glo-2). In T. gondii, we identified an atypical protein, TgGloL, containing a Glo-1-like motif but with limited homology to typical Glo enzymes. TgGloL localizes to the apicoplast, and its conditional knockdown impairs parasite growth, indicating its importance. While a specific and direct role for TgGloL in methylglyoxal detoxification within the apicoplast remains unclear, it is crucial for maintaining organelle homeostasis and for overall parasite fitness.

microbiology↗

bioRxiv2024

HCF101 is a novel component of the CIA cytosolic iron-sulfur synthesis pathway in the human pathogen Toxoplasma gondii

Several key cellular functions depend on proteins harboring an iron-sulfur (Fe-S) cofactor. As these Fe-S proteins localize to several subcellular compartments, they require a dedicated machinery for cofactor assembly. For instance, in plants and algae there are Fe-S cluster synthesis pathways localizing to the cytosol, but also present in the mitochondrion and in the chloroplast, two organelles of endosymbiotic origin. Toxoplasma gondii is a plastid-bearing parasitic protist responsible for a pathology affecting humans and other warm-blooded vertebrates. We have characterized the Toxoplasma homologue of HCF101, originally identified in plants as a protein transferring Fe-S clusters to photosystem I subunits in the chloroplast. Contrarily to plants, we have shown that HCF101 does not localize to the plastid in parasites, but instead is an important component of the cytosolic Fe-S assembly (CIA) pathway which is vital for Toxoplasma. While the CIA pathway is widely conserved in eukaryotes, it is the first time the involvement of HCF101 in this pan-eukaryotic machinery is established. Moreover, as this protein is essential for parasite viability and absent from its mammalian hosts, it constitutes a novel and promising potential drug target.

microbiology↗

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↗

bioRxiv2023

Iron depletion has different consequences on the growth and survival of Toxoplasma gondii strains

Toxoplasma gondii is an obligate intracellular parasite that is responsible for a pathology called toxoplasmosis which is primarily affecting immunocompromised individuals and developing fetuses. The parasite is able to scavenge essential nutrients from its host to support its own growth and survival. Among them, iron is one of the most important elements needed to sustain basic cellular functions, as it is involved in a number of key metabolic processes, including oxygen transport, redox balance and electron transport. We have evaluated the effects of an iron chelator on the development of several parasite strains and found that they differed in their ability to tolerate iron depletion. The growth of parasites usually associated with a model of acute toxoplasmosis was strongly impacted by iron depletion, while cystogenic strains were less sensitive as they were able to convert into persisting developmental forms which are associated with the chronic form of the disease. Ultrastructural and biochemical characterization of the impact of iron depletion on the parasites also highlighted striking changes in both in their metabolism and the one of the host, with a marked accumulation of lipid droplets and perturbation of lipid homeostasis. Overall, our study demonstrates that although acute iron depletion has an important effect on the growth of T. gondii, it has a more profound impact on actively dividing parasites, while less metabolically-active parasite forms may be able to avoid some of the most detrimental consequences.

microbiology↗

bioRxiv2023

The apicoplast is important for the viability and persistence of Toxoplasma gondii bradyzoites

Toxoplasma gondii is responsible for toxoplasmosis, a disease that can be serious when contracted during pregnancy, but can also be a threat for immunocompromised individuals. Acute infection is associated with the tachyzoite form that spreads rapidly within the host. However, under stress conditions, some parasites can differentiate into cyst-forming bradyzoites, residing mainly in the central nervous system, retina and muscle. Because this latent form of the parasite is resistant to all currently available treatments, and is central to persistence and transmission of the parasite, new specific therapeutic strategies targeting this developmental stage need to be discovered. T. gondii contains a plastid of endosymbiotic origin called the apicoplast, which is an appealing drug target because it is essential for tachyzoite viability and contains several key metabolic pathways that are largely absent from the mammalian host. Its function in bradyzoites, however, is unknown. Our objective was thus to study the contribution of the apicoplast to the viability and persistence of bradyzoites during chronic toxoplasmosis. We have used complementary strategies based on stage-specific promoters to generate conditional bradyzoite mutants of essential apicoplast genes. Our results show that specifically targeting the apicoplast in both in vitro or in vivo-differentiated bradyzoites leads to a loss of long-term bradyzoite viability, highlighting the importance of this organelle for this developmental stage. This validates the apicoplast as a potential area to look for new therapeutic targets in bradyzoites, with the aim to interfere with this currently incurable parasite stage. Significance StatementIn its intermediate hosts, the parasite Toxoplasma gondii can persist as a cyst-contained developmental form that might reactivate and cause severe pathologies. Importantly, this form is resistant to current anti-parasitic drugs. T. gondii harbors a plastid of endosymbiotic origin called the apicoplast, containing important and potentially druggable metabolic pathways, but whose contribution to the fitness and viability of persistent parasites has never been assessed. We have generated conditional mutants specifically affected for the homeostasis of the apicoplast in cyst-contained parasites and showed that this organelle is important for persistence of these particular developmental forms. Our work thus validates the apicoplast as a relevant drug target in the context of chronic T. gondii infection.

microbiology↗

bioRxiv2022

Disruption of the plastid-hosted iron-sulfur cluster biogenesis pathway in Toxoplasma gondii has pleiotropic effects irreversibly impacting parasite viability

Like many other apicomplexan parasites, Toxoplasma gondii contains a plastid harbouring key metabolic pathways, including the SUF pathway that is involved in the biosynthesis of iron-sulfur clusters. These cofactors are key for a variety of proteins involved in important metabolic reactions, potentially including plastidic pathways for the synthesis of isoprenoid and fatty acids. It was shown previously that impairing the NFS2 cysteine desulfurase, involved in the first step of the SUF pathway, leads to an irreversible killing of intracellular parasites. However, the metabolic impact of disrupting the pathway remained unexplored. We have generated another mutant of the pathway, deficient for the SUFC ATPase, and we have investigated in details the phenotypic consequences of TgNFS2 and TgSUFC depletion on parasite homeostasis. Our analysis confirms that Toxoplasma SUF mutants are severely and irreversibly impacted in growth: cell division and membrane homeostasis are particularly affected. Lipidomic analysis suggests a defect in apicoplast-generated fatty acids, along with a simultaneous increase in scavenging of host-derived lipids. However, addition of exogenous lipids did not allow full restauration of growth, suggesting other more important cellular functions were impacted in addition to fatty acid synthesis. For instance, we have shown that the SUF pathway is also key for generating isoprenoid-derived precursors necessary for the proper targeting of GPI-anchored proteins as well as for the parasite gliding motility. Thus, plastid-generated iron-sulfur clusters support the functions of proteins involved in several vital downstream cellular pathways, which implies the SUF machinery may be explored for discovering new potential anti-Toxoplasma targets.

microbiology↗

bioRxiv2021

Differential contribution of two organelles of endosymbiotic origin to iron-sulfur cluster synthesis in Toxoplasma

Iron-sulfur (Fe-S) clusters are one of the most ancient and ubiquitous prosthetic groups, and they are required by a variety of proteins involved in important metabolic processes. Apicomplexan parasites have inherited different plastidic and mitochondrial Fe-S clusters biosynthesis pathways through endosymbiosis. We have investigated the relative contributions of these pathways to the fitness of Toxoplasma gondii, an apicomplexan parasite causing disease in humans, by generating specific mutants. Phenotypic analysis and quantitative proteomics allowed us to highlight notable differences in these mutants. Both Fe-S cluster synthesis pathways are necessary for optimal parasite growth in vitro, but their disruption leads to markedly different fates: impairment of the plastidic pathway leads to a loss of the organelle and to parasite death, while disruption of the mitochondrial pathway trigger differentiation into a stress resistance stage. This highlights that otherwise similar biochemical pathways hosted by different sub-cellular compartments can have very different contributions to the biology of the parasites, which is something to consider when exploring novel strategies for therapeutic intervention. Author summaryToxoplasma gondii is a ubiquitous unicellular parasite that harbours two organelles of endosymbiotic origin: the mitochondrion, and a relict plastid named the apicoplast. Each one of these organelles contains its own machinery for synthesizing iron-sulfur clusters, which are important protein co- factors. In this study, we show that interfering with either the mitochondrial or the plastidic iron- sulfur cluster synthesizing machinery has a profound impact on parasite growth. However, while disrupting the plastidic pathway led to an irreversible loss of the organelle and subsequent death of the parasite, disrupting the mitochondrial pathway led to conversion of the parasites into a stress resistance form. We used a comparative quantitative proteomic analysis of the mutants, combined with experimental validation, to provide mechanistic clues into these different phenotypic outcomes. Although the consequences of disrupting each pathway were manifold, our data highlighted distinct changes at the metabolic level. For instance, the plastidic iron-sulfur cluster synthesis pathway is likely important for maintaining the lipid homeostasis of the parasites, while the mitochondrial pathway is clearly crucial for maintaining their respiratory capacity. Interestingly, we have discovered that other mutants severely impacted for mitochondrial function, in particular the respiratory chain, are able to survive and initiate conversion to the stress resistance form. This illustrates a different capacity for T. gondii to adapt for survival in response to distinct metabolic dysregulations.

microbiology↗

bioRxiv2020

Toxoplasma TgATG9 is critical for autophagy and long-term persistence in tissue cysts

Many of the worlds warm-blooded species are chronically infected with Toxoplasma gondii tissue cysts, including up to an estimated one third of the global human population. The cellular processes that permit long-term parasite persistence within the cyst are largely unknown, not only for T. gondii but also for related coccidian parasites that impact human and animal health. A previous study revealed an accumulation of autophagic material in the lysosome-like Vacuolar Compartment (VAC) of chronic stage bradyzoites lacking functional cathepsin L protease (TgCPL) activity. Furthermore, it was shown that TgCPL knockout bradyzoites have compromised viability, indicating the turnover of autophagic material could be necessary for bradyzoite survival. However, the extent to which autophagy itself contributes to bradyzoite development and fitness remained unknown. Herein we show that genetic ablation of TgATG9 substantially reduces canonical autophagy and compromises bradyzoite viability. Transmission electron microscopy revealed structural abnormalities occurring in {Delta}atg9 bradyzoites, including disorganization of the inner membrane complex and plasma membrane, the occurrence of multiple nuclei within a single bradyzoite cell, as well as various anomalies associated with the VAC. TgATG9-deficient bradyzoites accumulated significantly less undigested material in the VAC upon inhibition of TgCPL activity, suggesting that autophagy contributes material to the VAC for degradation. Intriguingly, abnormal mitochondria networks were observed in TgATG9-deficient bradyzoites. They were thin and elongated and often adopted a horseshoe conformation. Some abnormal mitochondrial structures were found to contain numerous different cytoplasmic components and organelles. Bradyzoite fitness was found to be drastically compromised, both in vitro and in mice, with very few brain cysts identified in mice 5 weeks post-infection. Taken together, our data suggests that TgATG9, and by extension autophagy, is critical for cellular homeostasis in bradyzoites and is necessary for long-term persistence within the cyst of this coccidian parasite.

microbiology↗

bioRxiv2017

Characterisation of two Toxoplasma PROPPINs homologous to Atg18/WIPI suggests they have evolved distinct specialised functions

Toxoplasma gondii is a parasitic protist possessing a limited set of proteins involved in the autophagy pathway, a self-degradative machinery for protein and organelle recycling. This distant eukaryote has even repurposed part of this machinery, centered on protein ATG8, for a non-degradative function related to the maintenance of the apicoplast, a parasite-specific organelle. However, some evidence also suggest Toxoplasma is able to generate autophagic vesicles upon stress, and that some autophagy-related proteins, such as ATG9, might be involved solely in the canonical autophagy function. Here, we have characterised two Toxoplasma proteins containing WD-40 repeat that can bind lipids for their recruitment to vesicular structures upon stress. They belong to the PROPPIN family and are homologues to ATG18/WIPI, which are known to be important for the autophagic process. We conducted a functional analysis of these two Toxoplasma PROPPINs. One of them is dispensable for normal in vitro growth, although it may play a role for parasite survival in specific stress conditions or for parasite fitness in the host, through a canonical autophagy-related function. The other, however, seems important for parasite viability in normal growth conditions and could be primarily involved in a non-canonical function. These divergent roles for two proteins from the same family illustrate the functional versatility of the autophagy-related machinery in Toxoplasma.

microbiology↗