Biology subjects

Soldati-Favre, D.

Publications and source records attributed to Soldati-Favre, D..

16 recordsLinked to original sources

bioRxiv2026

A transporter essential for apical secretory organelles homeostasis in Toxoplasma gondii

Apicomplexan parasites rely on the rhoptries and micronemes to coordinate egress, motility, and host cell invasion. In Toxoplasma gondii, mechanistic insights into the control of these organelles lipid composition remain sparse. Although nearly 60 T. gondii Major Facilitator Superfamily (MFS) are predicted, many remain functionally uncharacterized. The MFS protein HAP12 was previously annotated as an apicoplast protein contributing to invasion and organelle inheritance. Based on localization and phylogenetic analyses, we show that HAP12 (renamed TFP5), is predominantly associated with the Golgi, with additional signal in the ELC and apical compartments, without convincing evidence of a major apicoplast-associated pool. Furthermore, TFP5 clusters with a subgroup of MFS transporter facilitator proteins (TFPs) involved in the morphological integrity of secretory apical organelles. TFP5 depletion results in defective microneme secretion and pronounced alterations in rhoptry morphology and positioning. Structural analyses revealed that TFP5 is strongly similar to MFSD2-like lipid transporters, while lipidomic analyses showed substantial changes in parasite lipid composition following TFP5 depletion. Together, these findings support a model in which TFP5 regulates lipid homeostasis required for the function of apical secretory organelles, thereby critically contributing to parasite virulence.

microbiology↗

bioRxiv2026

MyoL-Dependent Coupling of Preconoidal Rings to Conoid Is Required for Motility in Toxoplasma gondii

Apicomplexan parasites are unified by their apical complex, a structure of cytoskeletal elements and secretory organelles. At its core lies the conoid, made of spiraling tubulin fibers essential for parasite motility and invasion. In Toxoplasma gondii, conoid extrusion and retraction through the apical polar ring is powered by Myosin H, ultimately regulating gliding motility. The conoid is capped by three preconoidal rings (PCRs), critical for extrusion and motility as they anchor formin 1, which produces filamentous actin needed for both processes. We demonstrate here that PCR composition and stability differ between daughter and mature cells and that Pcr2 and Pcr3 adopt a half-ring localization. The non-functional head containing Myosin-like protein MyoL, a previously unrecognized PCR component, is essential for gliding motility and invasion. Its depletion partially detaches PCRs from the conoid, highlighting its role as a mechanical tether. Targeted mutagenesis identifies the neck and tail regions of MyoL as critical for localization and function. Together, these findings illuminate the architecture and functional organization of the PCR in T. gondii, a structure conserved across several Apicomplexa.

microbiology↗

bioRxiv2025

An in vivo fitness gene of Toxoplasma, MIC11, is essential for PLP1-mediated egress from host cells

After invasion and replication, intracellular pathogens must egress from infected host cells. Toxoplasma gondii facilitates this process by permeabilizing host cells by releasing perforin-like protein 1 (PLP1) through induced microneme secretion. However, the precise mechanism of host cell permeabilization remains enigmatic. Here, we identified the secretory microneme protein MIC11 as a key factor for membrane disruption. A CRISPR-based in vivo screen revealed several genes including MIC11 as an essential gene for virulence. Deletion of MIC11 resulted in severe defects in both membrane rupture and egress. Scanning mutagenesis identified functional motifs in MIC11, and mechanistic analyses demonstrated that MIC11 directly associates with PLP1, regulating its activity in membrane disruption. The MIC11 paralogue MIC22 compensated for MIC11 deletion, suggesting a conserved mechanism of egress in the feline-restricted stages of T. gondii. The discovery of MIC11 advances the understanding of how parasites disrupt host cells to facilitate rapid egress and successful dissemination.

microbiology↗

bioRxiv2024

RNG2 tethers the conoid to the apical polar ring in Toxoplasma gondii: a key mechanism in parasite motility and invasion

In Toxoplasma gondii, the conoid is a dynamic organelle composed of spiraling tubulin fibers that extrudes during egress, gliding motility, and invasion. This organelle traverses the apical polar ring (APR) in response to calcium waves and plays a critical role in controlling parasite motility. While the actomyosin-dependent extrusion of the conoid is beginning to be understood, the mechanism by which it is anchored apically to the APR remains unclear. RNG2, a protein localized at both the conoid and the APR, has emerged as a key candidate for this function. By combining iterative ultrastructure expansion microscopy and immunoelectron microscopy we discovered that RNG2 forms 22 tethers between the APR and the conoid. The unique biochemical properties of RNG2, including several proteolytic processing events and its ability to form concatenations, enable it to function as a dynamic bridge between these structures. Conditional depletion of RNG2 resulted in the conoid organelle detaching from the APR without compromising the integrity of its structure, thereby confirming RNG2 essential tethering role. Although microneme secretion remains normal, parasites lacking RNG2 were unable to move and impaired in rhoptry discharge, highlighting the conoids crucial role in parasite motility and invasion. RNG2 is a pivotal protein that ensures conoid functionality in Coccidia.

microbiology↗

bioRxiv2024

N -acetylglucosamine supplementation fails to bypass the critical acetylation of glucosamine-6-phosphate required for Toxoplasma gondii replication and invasion

The cell surface of Toxoplasma gondii is rich in glycoconjugates which hold diverse and vital functions in the lytic cycle of this obligate intracellular parasite. Additionally, the cyst wall of bradyzoites, that shields the persistent form responsible for chronic infection from the immune system, is heavily glycosylated. Formation of glycoconjugates relies on activated sugar nucleotides, such as uridine diphosphate N-acetylglucosamine (UDP- GlcNAc). The Glucosamine-phosphate-N-acetyltransferase (GNA1) generates N- acetylglucosamine-6-phosphate critical to produce UDP-GlcNAc. Here, we demonstrate that downregulation of T. gondii GNA1 results in a severe reduction of UDP-GlcNAc and a concomitant drop in glycosylphosphatidylinositol (GPI), leading to impairment of the parasites ability to invade and replicate in the host cell. Surprisingly, attempts to rescue this defect through exogenous GlcNAc supplementation fail to completely restore these essential functions. In depth metabolomic analyses elucidate diverse causes underlying the failed rescue: utilization of GlcNAc is inefficient under glucose-replete conditions and fails to restore UDP-GlcNAc levels in GNA1-depleted parasites. In contrast, GlcNAc- supplementation under glucose-deplete conditions fully restores UDP-GlcNAc levels but fails to rescue the defects associated with GNA1 depletion. Our results underscore the essentiality of GlcN6P acetylation in governing T. gondii replication and invasion and highlight the potential of the evolutionary divergent GNA1 in Apicomplexa as a target for the development of much-needed new therapeutic strategies.

microbiology↗

bioRxiv2023

Structural and regulatory insights into the glideosome-associated connector from Toxoplasma gondii

The phylum of Apicomplexa groups intracellular parasites that employ substratedependent gliding motility to invade host cells, egress from the infected cells and cross biological barriers. The glideosome associated connector (GAC) is a conserved protein essential to this process. GAC facilitates the association of actin filaments with surface transmembrane adhesins and the efficient transmission of the force generated by myosin translocation of actin to the cell surface substrate. Here, we present the crystal structure of Toxoplasma gondii GAC and reveal a unique, supercoiled armadillo repeat region that adopts a closed ring conformation. Characterisation of the membrane binding interface within the C-terminal PH domain as well as an N-terminal fragment necessary for association with F-actin suggest that GAC adopts multiple conformations. A multi-conformational model for assembly of GAC within the glideosome is proposed.

biochemistry↗

bioRxiv2022

Nanoscopy of organelles and tissues with iterative ultrastructure expansion microscopy (iU-ExM)

Expansion microscopy is an approach of super-resolution fluorescence microscopy that does not yet achieve the precision of nanoscopy techniques such as single-molecule light microscopy (SMLM). Here, we developed an iterative ultrastructure expansion microscopy approach (iU-ExM), which now matches the SMLM resolution as demonstrated using standard references such as the nuclear pores. Applicable to both cells and tissues, iU-ExM allows a broad research community to access high precision super-resolution microscopy.

cell biology↗

bioRxiv2022

Conoid extrusion serves as gatekeeper for entry of glideosome components into the pellicular space to control motility and invasion in Apicomplexa

Members of the Apicomplexa are defined by apical cytoskeletal structures and secretory or-ganelles, tailored for motility and invasion. Gliding is powered by actomyosin-dependent rearward translocation of apically secreted transmembrane adhesins. In Toxoplasma gondii, the conoid, composed of a cone of spiraling tubulin fibers and apposed preconoidal rings (PCRs), is an enigmatic, dynamic organelle of undefined function. Here we mapped five new components of the PCRs and deduce that the structure serves as a pivotal hub for actin polymerization and glideosome assembly. F-actin produced by Formin1 on the PCRs is used by Myosin H to generate the force for conoid extrusion. A set of B-box-type zinc finger domain containing proteins conserved in Apicomplexa is indispensable for PCRs formation, conoid extrusion and motility in Toxoplasma and Plasmodium. Conoid dynamics directs the flux of F-actin to the pellicular space, acting as dynamic gatekeeper to tightly control parasite motility during invasion and egress.

microbiology↗

bioRxiv2021

Toxoplasma gondii phosphatidylserine flippase complex ATP2B-CDC50.4 critically participates in microneme exocytosis

Regulated microneme secretion governs motility, host cell invasion and egress in the obligate intracellular apicomplexans. Intracellular calcium oscillations and phospholipid dynamics critically regulate micronemes exocytosis. Despite its importance for the lytic cycle of these parasites, molecular mechanistic details about exocytosis are still missing. Some members of the P4-ATPases act as flippases, changing the phospholipid distribution by translocation from the outer to the inner leaflet of the membrane. Here, the localization and function of the repertoire of P4-ATPases was investigated across the lytic cycle of Toxoplasma gondii. Of relevance, ATP2B and the non-catalytic subunit cell division control protein 50.4 (CDC50.4) form a stable heterocomplex at the parasite plasma membrane, essential for microneme exocytosis. This complex is responsible for flipping phosphatidylserine (PS), which presumably acts as a lipid mediator for the organelle fusion with the plasma membrane. DOC2.1, a previously described key egress and invasion factor, is shown here to be affected in its function in egress upon mutation on residues putatively involved in calcium binding. This study points toward the importance of PS in microneme exocytosis and unveils subtle differences in the signaling cascades leading to organelle secretion between intracellular and extracellular parasites to ensure egress and invasion, respectively. Author SummaryBiological membranes display diverse functions, including membrane fusion, which are conferred by a defined composition and organization of proteins and lipids. Apicomplexan parasites possess specialized secretory organelles (micronemes), implicated in motility, invasion and egress from host cells. Microneme exocytosis is already known to depends on phosphatidic acid for its fusion with the plasma membrane. Here we identify a type P4-ATPase and its CDC50 chaperone (ATP2B-CDC50.4) that act as flippase and contribute to the enrichment of phosphatidylserine (PS) in the inner leaflet of the parasite plasma membrane. PS and the previously described C2-containing protein DOC2.1 differentially participate in microneme exocytosis in the context of environmental changes. Overall, our results shed light on the importance membrane homeostasis and lipid composition in controlling microneme secretion.

cell biology↗

bioRxiv2021

Organization and composition of apicomplexan kinetochores reveals plasticity in chromosome segregation across parasite modes of division

Kinetochores are multiprotein assemblies directing mitotic spindle attachment and chromosome segregation. In apicomplexan parasites, most known kinetochore components and associated regulators are apparently missing, suggesting a minimal structure with limited control over chromosome segregation. In this study, we use interactomics combined with deep homology searches to identify six divergent eukaryotic components, in addition to a set of eight apicomplexan kinetochore proteins (AKiTs) that bear no detectable sequence similarity to known proteins. The nanoscale organization of the apicomplexan kinetochore includes four subdomains, each displaying different evolutionary rates across the phylum. Functional analyses confirm AKiTs are essential for mitosis and reveal architectures parallel to biorientation at metaphase. Furthermore, we identify a homolog of MAD1 at the apicomplexan kinetochore, suggesting conserved spindle assembly checkpoint signaling. Finally, we show unexpected plasticity in kinetochore composition and segregation throughout the parasite lifecycle, indicating diverse requirements to maintain fidelity of chromosome segregation across apicomplexan modes of division.

microbiology↗

bioRxiv2021

Pantothenate biosynthesis is critical for chronic infection by the neurotropic parasite Toxoplasma gondii

Coenzyme A (CoA) is an essential molecule acting in metabolism, post-translational modification, and regulation of gene expression. While all organisms synthesize CoA, many, including humans, are unable to produce its precursor, pantothenate. Intriguingly, like most plants, fungi and bacteria, parasites of the coccidian subgroup of Apicomplexa, including the human and animal pathogen Toxoplasma gondii, possess all the enzymes required for de novo synthesis of pantothenate. Here, the importance of CoA and pantothenate biosynthesis for the acute and chronic stages of T. gondii infection was dissected through genetic, biochemical and metabolomic approaches, revealing that CoA synthesis is essential for T. gondii tachyzoites, due to the parasites inability to salvage CoA or intermediates of the pathway. In contrast, de novo pantothenate synthesis was only partially active in T. gondii tachyzoites, making the parasite reliant on Pan uptake. However, Pan synthesis proved to be crucial for the establishment of chronic infection, offering a promising target for intervention against the persistent stage of T. gondii.

microbiology↗

bioRxiv2021

Revisiting the role of Toxoplasma gondii ERK7 in the maintenance and stability of the apical complex

Toxoplasma gondii ERK7 is known to contribute to the integrity of the apical complex and to be involved only in the final step of the conoid biogenesis. In the absence of ERK7, mature parasites lose their conoid complex and are unable to glide, invade or egress from host cells. In contrast to a previous report, we show here that depletion of ERK7 phenocopies the depletion of the apical cap proteins AC9 or AC10. The absence of ERK7 leads to the loss of the apical polar ring, the disorganization of the basket of subpellicular microtubules and an impairment in micronemes secretion. Ultra-expansion microscopy (U-ExM) coupled to NHS-Ester staining on intracellular parasites offers an unprecedented level of resolution and highlights the disorganization of the rhoptries as well as the dilated plasma membrane at the apical pole in the absence of ERK7. Comparative proteomics analysis of wild-type and ERK7 or AC9 depleted parasites led to the disappearance of known, predicted, as well as putative novel components of the apical complex. In contrast, the absence of ERK7 led to an accumulation of microneme proteins, resulting from the defect in exocytosis of the organelles. ImportanceThe conoid is an enigmatic, dynamic organelle positioned at the apical tip of the coccidian subgroup of the Apicomplexa, close to the apical polar ring (APR) from which the subpellicular microtubules (SPTMs) emerge and at the site of microneme and rhoptry secretory organelles exocytosis. In Toxoplasma gondii, the conoid protrudes concomitantly to microneme secretion, during egress, motility and invasion. The conditional depletion of the apical cap structural protein AC9 or AC10 leads to a disorganization of SPTMs as well as the loss of APR and conoid that result in microneme secretion defect and block in motility, invasion and egress. We show here that depletion of the kinase ERK7 phenocopies completely AC9 and AC10 mutants. Moreover, the combination of ultrastructure expansion microscopy with an NHS ester staining revealed that ERK7 depleted parasites exhibit a dilated apical plasma membrane and a mis-positioning of the rhoptry organelles.

microbiology↗

bioRxiv2020

Naturally acquired blocking human monoclonal antibodies to Plasmodium vivax reticulocyte binding protein 2b

Plasmodium vivax preferentially invades reticulocytes and recognition of these cells is mediated by P. vivax Reticulocyte Binding Protein 2b (PvRBP2b) binding to human Transferrin receptor 1 (TfR1) and Transferrin (Tf). Longitudinal cohort studies in Papua New Guinea, Thailand and Brazil show that PvRBP2b antibodies are correlated with protection against P. vivax infection and disease. Here, we isolated and characterized anti-PvRBP2b human monoclonal antibodies from two individuals in Cambodia with natural P. vivax infection. These antibodies bind with high affinities and map to different regions of PvRBP2b. Several human antibodies blocked PvRBP2b binding to reticulocytes and inhibited complex formation with human TfR1-Tf. We describe different structural mechanisms for functional inhibition, including either steric hindrance with TfR1-Tf or the reticulocyte membrane. These results show that naturally acquired human antibodies against PvRBP2b can inhibit its function which is important for P. vivax invasion.

microbiology↗

bioRxiv2020

Two alveolin network proteins are essential for the subpellicular microtubules assembly and conoid anchoring to the apical pole of mature Toxoplasma gondii

Toxoplasma gondii belongs to the coccidian sub-group of Apicomplexa that possess an apical complex harboring a conoid, made of unique tubulin polymer fibers. This enigmatic and dynamic organelle extrudes in extracellular invasive parasites and is associated to the apical polar ring (APR), a microtubule-organizing center for the 22 subpellicular microtubules (SPMTs). The SPMTs are linked to the Inner Membrane Complex (IMC), a patchwork of flattened vesicles, via an intricate network of small filaments composed of alveolins proteins. Here, we capitalize on super-resolution techniques including stimulated emission depletion (STED) microscopy and ultrastructure expansion microscopy (U-ExM) to localize the Apical Cap protein 9 (AC9) and its close partner AC10, identified by BioID, to the alveolin network and intercalated between the SPMTs. Conditional depletion of AC9 or AC10 using the Auxin-induced Degron (AiD) system uncovered a severe loss of fitness. Parasites lacking AC9 or AC10 replicate normally but are defective in microneme secretion and hence fail to invade and egress from infected cells. Remarkably, a series of crucial apical complex proteins (MyoH, AKMT, FRM1, CPH1, ICMAP1 and RNG2) are lost in the mature parasites although they are still present in the forming daughter cells. Electron microscopy on intracellular or deoxycholate-extracted parasites revealed that the mature parasite mutants are conoidless. Closer examination of the SPMTs by U-ExM highlighted the disassembly of the SPMTs in the apical cap region that is presumably at the origin of the catastrophic loss of APR and conoid. AC9 and AC10 are two critical components of the alveolin network that ensure the integrity of the whole apical complex in T. gondii and likely other coccidians.

microbiology↗

bioRxiv2019

Transcriptome analysis of Plasmodium berghei during exo-erythrocytic development

The complex life cycle of malaria parasites requires well-orchestrated stage specific gene expression. In the vertebrate host the parasites grow and multiply by schizogony in two different environments: within erythrocytes and within hepatocytes. Whereas erythrocytic parasites are rather well-studied in this respect, relatively little is known about the exo-erythrocytic stages. In an attempt to fill this gap, we performed genome wide RNA-seq analyses of various exo-erythrocytic stages of Plasmodium berghei including sporozoites, samples from a time-course of liver stage development and detached cells, which contain infectious merozoites and represent the final step in exo-erythrocytic development. The analysis represents the completion of the transcriptome of the entire life cycle of P. berghei parasites with temporal detailed analysis of the liver stage allowing segmentation of the transcriptome across the progression of the life cycle. We have used these RNA-seq data from different developmental stages to cluster genes with similar expression profiles, in order to infer their functions. A comparison with published data of other parasite stages confirmed stage-specific gene expression and revealed numerous genes that are expressed differentially in blood and exo-erythrocytic stages. One of the most exo-erythrocytic stage-specific genes was PBANKA_1003900, which has previously been annotated as a "gametocyte specific protein". The promoter of this gene drove high GFP expression in exo-erythrocytic stages, confirming its expression profile seen by RNA-seq. The comparative analysis of the genome wide mRNA expression profiles of erythrocytic and different exo-erythrocytic stages improves our understanding of gene regulation of Plasmodium parasites and can be used to model exo-erythrocytic stage metabolic networks and identify differences in metabolic processes during schizogony in erythrocytes and hepatocytes.

microbiology↗

bioRxiv2017

Toxoplasma gondii microneme proteins 1 and 4 bind to Toll-like receptors 2 and 4 N-glycans triggering innate immune response

Infection of host cells by Toxoplasma gondii is an active process, which is regulated by secretion of microneme (MICs) and rhoptry proteins (ROPs and RONs) from specialized organelles in the apical pole of the parasite. MIC1, MIC4 and MIC6 assemble into an adhesin complex, secreted on the parasite surface and function to promote infection competency. MIC1 and MIC4 are known to bind terminal sialic acid residues and galactose residues, respectively and to induce IL-12 production from splenocytes. Here we show that rMIC1- and rMIC4-stimulated dendritic cells and macrophages to produce proinflammatory cytokines, and they do so by engaging TLR2 and TLR4. This process depends on sugar recognition, since point mutations in the carbohydrate-recognition domains (CRD) of rMIC1 and rMIC4 inhibit innate immune cells activation. HEK cells transfected with TLR2 glycomutants were selectively unresponsive to MICs. Following in vitro infection, parasites lacking MIC1 or MIC4, as well as expressing MIC proteins with point mutations in their CRD, failed to induce wild-type (WT) levels of IL-12 secretion by innate immune cells. However, only MIC1 was shown to impact systemic levels of IL-12 and IFN-{gamma} in vivo. Together, our data show that MIC1 and MIC4 interact physically with TLR2 and TLR4 N-glycans to trigger IL-12 responses, and MIC1 is playing a significant role in vivo by altering T. gondii infection competency and murine pathogenesis.\n\nAUTHOR SUMMARYToxoplasmosis is caused by the protozoan Toxoplasma gondii, belonging to the Apicomplexa phylum. This phylum comprises important parasites able to infect a broad diversity of animals, including humans. A particularity of T. gondii is its ability to invade virtually any nucleated cell of all warm-blooded animals through an active process, which depends on the secretion of adhesin proteins. These proteins are discharged by specialized organelles localized in the parasite apical region, and termed micronemes and rhoptries. We show in this study that two microneme proteins from T. gondii utilize their adhesion activity to stimulate innate immunity. These microneme proteins, denoted MIC1 and MIC4, recognize specific sugars on receptors expressed on the surface of mammalian immune cells. This binding activates these innate immune cells to secrete cytokines, which promotes efficient host defense mechanisms against the parasite and regulate their pathogenesis. This activity promotes a chronic infection by controlling parasite replication during acute infection.

immunology↗