Invasion of Toxoplasma gondii bradyzoites: Molecular dissection of the moving junction proteins and effective vaccination targets.

Toxoplasmosis is a neglected parasitic disease necessitating public health control. Host cell invasion by Toxoplasma occurs at different stages of the parasite's life cycle and is crucial for survival and establishment of infection. In tachyzoites, which are responsible for acute toxoplasmosis, invasion involves the formation of a molecular bridge between the parasite and host cell membranes, referred to as the moving junction (MJ). The MJ is shaped by the assembly of AMA1 and RON2, as part of a complex involving additional RONs. While this essential process is well characterized in tachyzoites, the invasion process remains unexplored in bradyzoites, which form cysts and are responsible for chronic toxoplasmosis and contribute to the dissemination of the parasite between hosts. Here, we show that bradyzoites invade host cells in an MJ-dependent fashion but differ in protein composition from the tachyzoite MJ, relying instead on the paralogs AMA2 and AMA4. Functional characterization of AMA4 reveals its key role for cysts burden during the onset of chronic infection, while being dispensable for the acute phase. Immunizations with AMA1 and AMA4, alone or in complex with their rhoptry neck respective partners RON2 and RON2L1, showed that the AMA1-RON2 pair induces strong protection against acute and chronic infection, while the AMA4-RON2L1 complex targets more selectively the chronic form. Our study provides important insights into the molecular players of bradyzoite invasion and indicates that invasion of cyst-forming bradyzoites contributes to cyst burden. Furthermore, we validate AMA-RON complexes as potential vaccine candidates to protect against toxoplasmosis.

A toolbox for conditional control of gene expression in apicomplexan parasites.

Apicomplexan parasites encompass diverse pathogens for humans and animals, including the causative agents of malaria and toxoplasmosis, Plasmodium spp. and Toxoplasma gondii. Genetic manipulation of these parasites has become central to explore parasite biology, unravel gene function and identify new targets for therapeutic strategies. Tremendous progress has been achieved over the past years with the advent of next generation sequencing and powerful genome editing methods. In particular, various methods for conditional gene expression have been developed in both Plasmodium and Toxoplasma to knockout or knockdown essential genes, or for inducible expression of master developmental regulators or mutant versions of proteins. Conditional gene expression can be achieved at three distinct levels. At the DNA level, inducible site-specific recombinases allow conditional genome editing. At the RNA level, regulation can be achieved during transcription, using stage-specific or regulatable promoters, or post-transcriptionally through alteration of mRNA stability or translation. At the protein level, several systems have been developed for inducible degradation or displacement of a protein of interest. In this review, we provide an overview of current systems for conditional control of gene expression in Plasmodium and Toxoplasma parasites, highlighting the advantages and limitations of each approach.

Hydroxamate-based compounds are potent inhibitors of Toxoplasma gondii HDAC biological activity.

Toxoplasmosis is a critical health issue for immune-deficient individuals and the offspring of newly infected mothers. It is caused by a unicellular intracellular parasite called Toxoplasma gondii that is found worldwide. Although efficient drugs are commonly used to treat toxoplasmosis, serious adverse events are common. Therefore, new compounds with potent anti-T. gondii activity are needed to provide better suited treatments. We have tested compounds designed to target specifically histone deacetylase enzymes. Among the 55 compounds tested, we identified three compounds showing a concentration of drug required for 50% inhibition (IC50) in the low 100 nM range with a selectivity index of more than 100. These compounds are not only active at inhibiting the growth of the parasite in vitro but also at preventing some of the consequences of the acute disease in vivo. Two of these hydroxamate based compound also induce a hyper-acetylation of the parasite histones while the parasitic acetylated tubulin level remains unchanged. These findings suggest that the enzymes regulating histone acetylation are potent therapeutic targets for the treatment of acute toxoplasmosis.

Cooperative binding of ApiAP2 transcription factors is crucial for the expression of virulence genes in Toxoplasma gondii.

Toxoplasma gondii virulence depends on the expression of factors packed into specific organelles such as rhoptry and microneme. Although virulence factor expression is tightly regulated, the molecular mechanisms controlling their regulation remain poorly understood. ApiAP2 are a family of conserved transcription factors (TFs) that play an important role in regulating gene expression in apicomplexan parasites. TgAP2XI-5 is able to bind to transcriptionally active promoters of genes expressed during the S/M phase of the cell cycle, such as virulence genes (rhoptries and micronemes genes). We identified proteins interacting with TgAP2XI-5 including a cell cycle-regulated ApiAP2 TF, TgAP2X-5. Using an inducible knock-down strategy and RNA-seq, we demonstrated that the level of expression of number of virulence factors transcripts is affected by the disruption of TgAP2X-5 expression. While TgAP2X-5 disruption has mild effect on parasite invasion, it leads to the strain avirulence in mice. To better understand the molecular mechanisms at stake, we investigated the binding of TgAP2XI-5 at promoters in the TgAP2X-5 mutant strain in a genome-wide assay. We show that disruption of TgAP2X-5 expression leads to defects in TgAP2XI-5 binding to multiple rhoptry gene promoters. Taken together, these data suggest a cooperative contribution of two ApiAP2 TF in the regulation of virulence genes in T. gondii.

A coiled-coil protein is required for coordination of karyokinesis and cytokinesis in Toxoplasma gondii.

Toxoplasma gondii is a unicellular eukaryotic pathogen that belongs to the Apicomplexa phylum, which encompasses some of the deadliest pathogens of medical and veterinary importance. The centrosome is key to the organisation and coordination of the cell cycle and division of apicomplexan parasites. The T. gondii centrosome possesses a particular bipartite structure (outer and inner cores). One of the main roles of the centrosome is to ensure proper coordination of karyokinesis. However, how these 2 events are coordinated is still unknown in T. gondii, for which the centrosome components are poorly described. To gain more insights into the biology and the composition of the T. gondii centrosome, we characterised a protein that resides at the interface of the outer and inner core centrosomes. TgCep530 is a large coiled-coil protein with an essential role in the survival of the parasite. Depletion of this protein leads to the accumulation of parasites lacking nuclei and disruption of the normal cell cycle. Lack of TgCep530 results in a discoordination between the nuclear cycle and the budding cycle that yields fully formed parasites without nuclei. TgCep530 has a crucial role in the coordination of karyokinesis and cytokinesis.

An evolutionary conserved zinc finger protein is involved in Toxoplasma gondii mRNA nuclear export.

Apicomplexan parasites are responsible for some of the most deadly parasitic diseases affecting humans and livestock. There is an urgent need for new medicines that will target apicomplexan-specific pathways. We characterized a Toxoplasma gondii C2H2 zinc finger protein, named TgZNF2, which is conserved among eukaryotes. We constructed an inducible KO strain (iKO-TgZNF2) for this gene where the tgznf2 gene expression is repressed in the presence of a tetracycline analog (ATc). We showed that the iKO-TgZNF2 parasites are unable to proliferate after depletion of the TgZNF2 protein. Complementation with a full length copy of the gene restores the phenotype Moreover, the homolog of this protein in the related apicomplexan Plasmodium falciparum was shown to efficiently rescue the phenotype, suggesting that this pathway is likely conserved among apicomplexan parasites. We demonstrated that the iKO-mutant lacking TgZNF2 are arrested during the cell cycle during the G1 phase. We identified potential protein partners of this protein among which are spliceosomal complex and mRNA nuclear export components. We confirmed that TgZNF2 is able to bind in vivo to transcripts but splicing is not perturbed in the ATc-treated parasites. Instead, we demonstrated that TgZNF2 depletion leads to the sequestration of polyA+ mRNAs in the nucleus while ribosomal RNAs are not affected. We discovered a conserved protein with specific apicomplexan functional properties that is essential for the survival of T. gondii. TgZNF2 may be crucial to ensure the correct polyA+ mRNA nuclear export, a function that is conserved in P. falciparum.

Characterization of a nuclear pore protein sheds light on the roles and composition of the Toxoplasma gondii nuclear pore complex.

The nuclear pore is a key structure in eukaryotes regulating nuclear-cytoplasmic transport as well as a wide range of cellular processes. Here, we report the characterization of the first Toxoplasma gondii nuclear pore protein, named TgNup302, which appears to be the orthologue of the mammalian Nup98-96 protein. We produced a conditional knock-down mutant that expresses TgNup302 under the control of an inducible tetracycline-regulated promoter. Under ATc treatment, a substantial decrease of TgNup302 protein in inducible knock-down (iKD) parasites was observed, causing a delay in parasite proliferation. Moreover, the nuclear protein TgENO2 was trapped in the cytoplasm of ATc-treated mutants, suggesting that TgNup302 is involved in nuclear transport. Fluorescence in situ hybridization revealed that TgNup302 is essential for 18S RNA export from the nucleus to the cytoplasm, while global mRNA export remains unchanged. Using an affinity tag purification combined with mass spectrometry, we identified additional components of the nuclear pore complex, including proteins potentially interacting with chromatin. Furthermore, reverse immunoprecipitation confirmed their interaction with TgNup302, and structured illuminated microscopy confirmed the NPC localization of some of the TgNup302-interacting proteins. Intriguingly, facilitates chromatin transcription complex (FACT) components were identified, suggesting the existence of an NPC-chromatin interaction in T. gondii. Identification of TgNup302-interacting proteins also provides the first glimpse at the NPC structure in Apicomplexa, suggesting a structural conservation of the NPC components between distant eukaryotes.

The Toxoplasma gondii inhibitor-2 regulates protein phosphatase 1 activity through multiple motifs.

Toxoplasma gondii has a complex life cycle characterized by multiple differentiation steps that are essential for its survival in both human and definitive feline host. Several studies have demonstrated the importance of phosphorylations by protein kinases during the life cycle of T. gondii. However, very little is known about protein phosphatases and their regulators in the parasite. We report the molecular and functional characterization of the T. gondii ortholog of the inhibitor-2 protein, designated TgI2. We show that TgI2 encompasses conserved motifs involved in the interaction and modulation of the phosphatase activity of T. gondii protein phosphatase 1, named TgPP1. We show that a specific combination of motifs is involved in binding and/or inhibition of the TgPP1 activity. We show here that the TgI2 protein is a potent inhibitor of TgPP1 phosphatase activity. TgI2 SILK and RVxF motifs are critical for regulating the activity of TgPP1, a feature that is common with the higher eukaryotes inhibitor-2 protein.

Toxoplasma transcription factor TgAP2XI-5 regulates the expression of genes involved in parasite virulence and host invasion.

Gene regulation in apicomplexan parasites, a phylum containing important protozoan parasites such as Plasmodium and Toxoplasma, is poorly understood. The life cycle of Toxoplasma gondii is complex, with multiple proliferation and differentiation steps, of which tachyzoite proliferation is the most relevant to pathogenesis in humans and animals. Tachyzoites express invasion and virulence factors that are crucial for their survival and manipulation of host cell functions. The expression of those factors is tightly controlled during the tachyzoite cell cycle to permit their correct packaging in newly formed apical secretory organelles named micronemes and rhoptries in the daughter cells. However, little is known about the factors that control the expression of genes encoding the virulence factors present in these parasite-specific secretory organelles. We report that the plant-like nuclear factor TgAP2XI-5 targets more than 300 gene promoters and actively controls the transcription of these genes. Most of these target genes, including those that are essential for parasite virulence, showed a peak of expression in the S and M phases of the cell cycle. Furthermore, we identified the cis-regulatory element recognized by TgAP2XI-5 and demonstrated its ability to actively drive gene transcription. Our results demonstrated that TgAP2XI-5 is a novel DNA sequence-specific transcription factor associated with promoter activation. TgAP2XI-5 may regulate gene transcription of crucial virulence factors in T. gondii.

The Toxoplasma nuclear factor TgAP2XI-4 controls bradyzoite gene expression and cyst formation.

Toxoplasma gondii undergoes many phenotypic changes during its life cycle. The recent identification of AP2 transcription factors in T. gondii has provided a platform for studying the mechanisms controlling gene expression. In the present study, we report that a recombinant protein encompassing the TgAP2XI-4 AP2 domain was able to specifically bind to a DNA motif using gel retardation assays. TgAP2XI-4 protein is localized in the parasite nucleus throughout the tachyzoite life cycle in vitro, with peak expression occurring after cytokinesis. We found that the TgAP2XI-4 transcript level was higher in bradyzoite cysts isolated from brains of chronically infected mice than in the rapidly replicating tachyzoites. A knockout of the TgAP2XI-4 gene in both T. gondii virulent type I and avirulent type II strains reveals its role in modulating expression and promoter activity of genes involved in stage conversion of the rapidly replicating tachyzoites to the dormant cyst forming bradyzoites. Furthermore, mice infected with the type II KO mutants show a drastically reduced brain cyst burden. Thus, our results validate TgAP2XI-4 as a novel nuclear factor that regulates bradyzoite gene expression during parasite differentiation and cyst formation.

Toxoplasma gondii sequesters centromeres to a specific nuclear region throughout the cell cycle.

Members of the eukaryotic phylum Apicomplexa are the cause of important human diseases including malaria, toxoplasmosis, and cryptosporidiosis. These obligate intracellular parasites produce new invasive stages through a complex budding process. The budding cycle is remarkably flexible and can produce varied numbers of progeny to adapt to different host-cell niches. How this complex process is coordinated remains poorly understood. Using Toxoplasma gondii as a genetic model, we show that a key element to this coordination is the centrocone, a unique elaboration of the nuclear envelope that houses the mitotic spindle. Exploiting transgenic parasite lines expressing epitope-tagged centromeric H3 variant CenH3, we identify the centromeres of T. gondii chromosomes by hybridization of chromatin immunoprecipitations to genome-wide microarrays (ChIP-chip). We demonstrate that centromere attachment to the centrocone persists throughout the parasite cell cycle and that centromeres localize to a single apical region within the nucleus. Centromere sequestration provides a mechanism for the organization of the Toxoplasma nucleus and the maintenance of genome integrity.

Two ancient membrane pores mediate mitochondrial-nucleus membrane contact sites.

Coordination between nucleus and mitochondria is essential for cell survival, and thus numerous communication routes have been established between these two organelles over eukaryotic cell evolution. One route for organelle communication is via membrane contact sites, functional appositions formed by molecular tethers. We describe a novel nuclear-mitochondrial membrane contact site in the protozoan Toxoplasma gondii. We have identified specific contacts occurring at the nuclear pore and demonstrated an interaction between components of the nuclear pore and the mitochondrial protein translocon, highlighting them as molecular tethers. Genetic disruption of the nuclear pore or the TOM translocon components, TgNup503 or TgTom40, respectively, result in contact site reduction, supporting their potential involvement in this tether. TgNup503 depletion further leads to specific mitochondrial morphology and functional defects, supporting a role for nuclear-mitochondrial contacts in mediating their communication. The discovery of a contact formed through interaction between two ancient mitochondrial and nuclear complexes sets the ground for better understanding of mitochondrial-nuclear crosstalk in eukaryotes.

A potent HDAC inhibitor blocks Toxoplasma gondii tachyzoite growth and profoundly disrupts parasite gene expression.

INTRODUCTION: Toxoplasmosis is a major health issue worldwide, especially for immune-deficient individuals and the offspring of newly infected mothers. It is caused by a unicellular intracellular parasite called Toxoplasma gondii. Although the drugs commonly used to treat toxoplasmosis are efficient, they present serious side effects and adverse events are common. Therefore, there is a need for the discovery of new compounds with potent anti-Toxoplasma gondii activity. METHODS: This study tested compounds designed to target enzymes that are involved in the epigenetic regulation of gene expression. RESULTS: Among the most active compounds, an HDAC inhibitor showing an IC50 of 30 nM with a selectivity index above 100 was identified. MC1742 was active at inhibiting the growth of the parasite in vitro but also at preventing the consequences of the acute disease in vivo. This compound induced hyper-acetylation of histones, while the acetylated tubulin level remained unchanged. After MC1742 treatment, the parasite expression profile was profoundly changed with the activation of genes preferentially expressed in the sexual stages that are normally repressed in the tachyzoite stage. CONCLUSIONS: These findings suggest that this compound disturbs the Toxoplasma gondii gene expression program, inducing parasite death.

Deciphering the Role of Protein Phosphatases in Apicomplexa: The Future of Innovative Therapeutics?

Parasites belonging to the Apicomplexa phylum still represent a major public health and world-wide socioeconomic burden that is greatly amplified by the spread of resistances against known therapeutic drugs. Therefore, it is essential to provide the scientific and medical communities with innovative strategies specifically targeting these organisms. In this review, we present an overview of the diversity of the phosphatome as well as the variety of functions that phosphatases display throughout the Apicomplexan parasites' life cycles. We also discuss how this diversity could be used for the design of innovative and specific new drugs/therapeutic strategies.

Separate To Operate: the Centriole-Free Inner Core of the Centrosome Regulates the Assembly of the Intranuclear Spindle in Toxoplasma gondii.

Centrosomes are the main microtubule-organizing center of the cell. They are normally formed by two centrioles, embedded in a cloud of proteins known as pericentriolar material (PCM). The PCM ascribes centrioles with their microtubule nucleation capacity. Toxoplasma gondii, the causative agent of toxoplasmosis, divides by endodyogeny. Successful cell division is critical for pathogenesis. The centrosome, one of the microtubule organizing centers of the cell, plays central roles in orchestrating the temporal and physical coordination of major organelle segregation and daughter cell formation during endodyogeny. The Toxoplasma centrosome is constituted by multiple domains: an outer core, distal from the nucleus; a middle core; and an inner core, proximal to the nucleus. This modular organization has been proposed to underlie T. gondii's cell division plasticity. However, the role of the inner core remains undeciphered. Here, we focus on understanding the function of the inner core by finely studying the localization and role of its only known molecular marker; TgCep250L1. We show that upon conditional degradation of TgCep250L1 parasites are unable to survive. Mutants exhibit severe nuclear segregation defects. In addition, the rest of the centrosome, defined by the position of the centrioles, disconnects from the nucleus. We explore the structural defects underlying these phenotypes by ultrastructure expansion microscopy. We show that TgCep250L1's location changes with respect to other markers, and these changes encompass the formation of the mitotic spindle. Moreover, we show that in the absence of TgCep250L1, the microtubule binding protein TgEB1, fails to localize at the mitotic spindle, while unsegregated nuclei accumulate at the residual body. Overall, our data support a model in which the inner core of the T. gondii centrosome critically participates in cell division by directly impacting the formation or stability of the mitotic spindle. IMPORTANCE Toxoplasma gondii parasites cause toxoplasmosis, arguably the most widespread and prevalent parasitosis of humans and animals. During the clinically relevant stage of its life cycle, the parasites divide by endodyogeny. In this mode of division, the nucleus, containing loosely packed chromatin and a virtually intact nuclear envelope, parcels into two daughter cells generated within a common mother cell cytoplasm. The centrosome is a microtubule-organizing center critical for orchestrating the multiple simultaneously occurring events of endodyogeny. It is organized in two distinct domains: the outer and inner cores. We demonstrate here that the inner core protein TgCEP250L1 is required for replication of T. gondii. Lack of TgCEP250L1 renders parasites able to form daughter cells, while unable to segregate their nuclei. We determine that, in the absence of TgCEP250L1, the mitotic spindle, which is responsible for karyokinesis, does not assemble. Our results support a role for the inner core in nucleation or stabilization of the mitotic spindle in T. gondii.

Primary brain cell infection by Toxoplasma gondii reveals the extent and dynamics of parasite differentiation and its impact on neuron biology.

Toxoplasma gondii is a eukaryotic parasite that forms latent cysts in the brain of immunocompetent individuals. The latent parasite infection of the immune-privileged central nervous system is linked to most complications. With no drug currently available to eliminate the latent cysts in the brain of infected hosts, the consequences of neurons' long-term infection are unknown. It has long been known that T. gondii specifically differentiates into a latent form (bradyzoite) in neurons, but how the infected neuron responds to the infection remains to be elucidated. We have established a new in vitro model resulting in the production of mature bradyzoite cysts in brain cells. Using dual, host and parasite RNA-seq, we characterized the dynamics of differentiation of the parasite, revealing the involvement of key pathways in this process. Moreover, we identified how the infected brain cells responded to the parasite infection revealing the drastic changes that take place. We showed that neuronal-specific pathways are strongly affected, with synapse signalling being particularly affected, especially glutamatergic synapse signalling. The establishment of this new in vitro model allows investigating both the dynamics of parasite differentiation and the specific response of neurons to long-term infection by this parasite.

TgAP2IX-5 is a key transcriptional regulator of the asexual cell cycle division in Toxoplasma gondii.

Apicomplexan parasites have evolved efficient and distinctive strategies for intracellular replication where the timing of emergence of the daughter cells (budding) is a decisive element. However, the molecular mechanisms that provide the proper timing of parasite budding remain unknown. Using Toxoplasma gondii as a model Apicomplexan, we identified a master regulator that controls the timing of the budding process. We show that an ApiAP2 transcription factor, TgAP2IX-5, controls cell cycle events downstream of centrosome duplication. TgAP2IX-5 binds to the promoter of hundreds of genes and controls the activation of the budding-specific cell cycle expression program. TgAP2IX-5 regulates the expression of specific transcription factors that are necessary for the completion of the budding cycle. Moreover, TgAP2IX-5 acts as a limiting factor that ensures that asexual proliferation continues by promoting the inhibition of the differentiation pathway. Therefore, TgAP2IX-5 is a master regulator that controls both cell cycle and developmental pathways.

An analytical pipeline for genomic representations used for cytosine methylation studies.

MOTIVATION: Representations of the genome can be generated by the selection of a subpopulation of restriction fragments using ligation-mediated PCR. Such representations form the basis for a number of high-throughput assays, including the HELP assay to study cytosine methylation. We find that HELP data analysis is complicated not only by PCR amplification heterogeneity but also by a complex and variable distribution of cytosine methylation. To address this, we created an analytical pipeline and novel normalization approach that improves concordance between microarray-derived data and single locus validation results, demonstrating the value of the analytical approach. A major influence on the PCR amplification is the size of the restriction fragment, requiring a quantile normalization approach that reduces the influence of fragment length on signal intensity. Here we describe all of the components of the pipeline, which can also be applied to data derived from other assays based on genomic representations.

Epigenomic modifications predict active promoters and gene structure in Toxoplasma gondii.

Mechanisms of gene regulation are poorly understood in Apicomplexa, a phylum that encompasses deadly human pathogens like Plasmodium and Toxoplasma. Initial studies suggest that epigenetic phenomena, including histone modifications and chromatin remodeling, have a profound effect upon gene expression and expression of virulence traits. Using the model organism Toxoplasma gondii, we characterized the epigenetic organization and transcription patterns of a contiguous 1% of the T. gondii genome using custom oligonucleotide microarrays. We show that methylation and acetylation of histones H3 and H4 are landmarks of active promoters in T. gondii that allow us to deduce the position and directionality of gene promoters with >95% accuracy. These histone methylation and acetylation "activation" marks are strongly associated with gene expression. We also demonstrate that the pattern of histone H3 arginine methylation distinguishes certain promoters, illustrating the complexity of the histone modification machinery in Toxoplasma. By integrating epigenetic data, gene prediction analysis, and gene expression data from the tachyzoite stage, we illustrate feasibility of creating an epigenomic map of T. gondii tachyzoite gene expression. Further, we illustrate the utility of the epigenomic map to empirically and biologically annotate the genome and show that this approach enables identification of previously unknown genes. Thus, our epigenomics approach provides novel insights into regulation of gene expression in the Apicomplexa. In addition, with its compact genome, genetic tractability, and discrete life cycle stages, T. gondii provides an important new model to study the evolutionarily conserved components of the histone code.

Toxoplasma gondii and Cryptosporidium parvum lack detectable DNA cytosine methylation.

Epigenetic factors play a role in the expression of virulence traits in Apicomplexa. Apicomplexan genomes encode putative DNA cytosine methylation enzymes. To assess the presence of cytosine methylation of Toxoplasma gondii and Cryptosporidium parvum DNA, we used mass spectrometry analysis and confirmed that these organisms lack detectable methylcytosine in their DNA.