Profiling MHC II immunopeptidome of blood-stage malaria reveals that cDC1 control the functionality of parasite-specific CD4 T cells.

In malaria, CD4 Th1 and T follicular helper (TFH) cells are important for controlling parasite growth, but Th1 cells also contribute to immunopathology. Moreover, various regulatory CD4 T-cell subsets are critical to hamper pathology. Yet the antigen-presenting cells controlling Th functionality, as well as the antigens recognized by CD4 T cells, are largely unknown. Here, we characterize the MHC II immunopeptidome presented by DC during blood-stage malaria in mice. We establish the immunodominance hierarchy of 14 MHC II ligands derived from conserved parasite proteins. Immunodominance is shaped differently whether blood stage is preceded or not by liver stage, but the same ETRAMP-specific dominant response develops in both contexts. In naïve mice and at the onset of cerebral malaria, CD8α+ dendritic cells (cDC1) are superior to other DC subsets for MHC II presentation of the ETRAMP epitope. Using in vivo depletion of cDC1, we show that cDC1 promote parasite-specific Th1 cells and inhibit the development of IL-10+ CD4 T cells. This work profiles the P. berghei blood-stage MHC II immunopeptidome, highlights the potency of cDC1 to present malaria antigens on MHC II, and reveals a major role for cDC1 in regulating malaria-specific CD4 T-cell responses.

The structure of bradyzoite-specific enolase from Toxoplasma gondii reveals insights into its dual cytoplasmic and nuclear functions.

In addition to catalyzing a central step in glycolysis, enolase assumes a remarkably diverse set of secondary functions in different organisms, including transcription regulation as documented for the oncogene c-Myc promoter-binding protein 1. The apicomplexan parasite Toxoplasma gondii differentially expresses two nuclear-localized, plant-like enolases: enolase 1 (TgENO1) in the latent bradyzoite cyst stage and enolase 2 (TgENO2) in the rapidly replicative tachyzoite stage. A 2.75 Šresolution crystal structure of bradyzoite enolase 1, the second structure to be reported of a bradyzoite-specific protein in Toxoplasma, captures an open conformational state and reveals that distinctive plant-like insertions are located on surface loops. The enolase 1 structure reveals that a unique residue, Glu164, in catalytic loop 2 may account for the lower activity of this cyst-stage isozyme. Recombinant TgENO1 specifically binds to a TTTTCT DNA motif present in the cyst matrix antigen 1 (TgMAG1) gene promoter as demonstrated by gel retardation. Furthermore, direct physical interactions of both nuclear TgENO1 and TgENO2 with the TgMAG1 gene promoter are demonstrated in vivo using chromatin immunoprecipitation (ChIP) assays. Structural and biochemical studies reveal that T. gondii enolase functions are multifaceted, including the coordination of gene regulation in parasitic stage development. Enolase 1 provides a potential lead in the design of drugs against Toxoplasma brain cysts.

Nuclear glycolytic enzyme enolase of Toxoplasma gondii functions as a transcriptional regulator.

Apicomplexan parasites including Toxoplasma gondii have complex life cycles within different hosts and their infectivity relies on their capacity to regulate gene expression. However, little is known about the nuclear factors that regulate gene expression in these pathogens. Here, we report that T. gondii enolase TgENO2 is targeted to the nucleus of actively replicating parasites, where it specifically binds to nuclear chromatin in vivo. Using a ChIP-Seq technique, we provide evidence for TgENO2 enrichment at the 5' untranslated gene regions containing the putative promoters of 241 nuclear genes. Ectopic expression of HA-tagged TgENO1 or TgENO2 led to changes in transcript levels of numerous gene targets. Targeted disruption of TgENO1 gene results in a decrease in brain cyst burden of chronically infected mice and in changes in transcript levels of several nuclear genes. Complementation of this knockout mutant with ectopic TgENO1-HA fully restored normal transcript levels. Our findings reveal that enolase functions extend beyond glycolytic activity and include a direct role in coordinating gene regulation 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.

Cathepsin Cs are key for the intracellular survival of the protozoan parasite, Toxoplasma gondii.

Cysteine proteases play key roles in apicomplexan invasion, organellar biogenesis, and intracellular survival. We have now characterized five genes encoding papain family cathepsins from Toxoplasma gondii, including three cathepsin Cs, one cathepsin B, and one cathepsin L. Unlike endopeptidases cathepsin B and L, T. gondii cathepsin Cs are exopeptidases and remove dipeptides from unblocked N-terminal substrates of proteins or peptides. TgCPC1 was the most highly expressed cathepsin mRNA in tachyzoites (by real-time PCR), but three cathepsins, TgCPC1, TgCPC2, and TgCPB, were undetectable in in vivo bradyzoites. The specific cathepsin C inhibitor, Gly-Phe-dimethylketone, selectively inhibited the TgCPCs activity, reducing parasite intracellular growth and proliferation. The targeted disruption of TgCPC1 does not affect the invasion and growth of tachyzoites as TgCPC2 is then up-regulated and may substitute for TgCPC1. TgCPC1 and TgCPC2 localize to constitutive secretory vesicles of tachyzoites, the dense granules. T. gondii cathepsin Cs are required for peptide degradation in the parasitophorous vacuole as the degradation of the marker protein, Escherichia coli beta-lactamase, secreted into the parasitophorous vacuole of transgenic tachyzoites was completely inhibited by the cathepsin C inhibitor. Cathepsin C inhibitors also limited the in vivo infection of T. gondii in the chick embryo model of toxoplasmosis. Thus, cathepsin Cs are critical to T. gondii growth and differentiation, and their unique specificities could be exploited to develop novel chemotherapeutic agents.

Functional and biophysical analyses of the class XIV Toxoplasma gondii myosin D.

The obligate intracellular parasite Toxoplasma gondii uses gliding motility to migrate across the biological barriers of the host and to invade cells. This unique form of locomotion requires an intact actin cytoskeleton and involves at least one motor protein (TgMyoA) that belongs to the class XIV of the myosin superfamily. TgMyoA is anchored in the inner membrane complex and is essential for the gliding motion, host cell invasion and egress of T. gondii tachyzoites. TgMyoD is the smallest T. gondii myosin and is structurally very closely related to TgMyoA. We show here that TgMyoD exhibits similar transient kinetic properties as the fast single-headed TgMyoA. To determine if TgMyoD also contributes to parasite gliding motility, the TgMyoD gene was disrupted by double homologous recombination. In contrast to TgMyoA, TgMyoD gene is dispensable for tachyzoite propagation and motility. Parasites lacking TgMyoD glide normally and their virulence is not compromised in mice. The fact that TgMyoD is predominantly expressed in bradyzoites explains the absence of a phenotype observed with myodko in tachyzoites and does not exclude a role of this motor in gliding that would be restricted to the cyst forming but nevertheless motile stage of the parasite.

Evolution of plant-like crystalline storage polysaccharide in the protozoan parasite Toxoplasma gondii argues for a red alga ancestry.

Single-celled apicomplexan parasites are known to cause major diseases in humans and animals including malaria, toxoplasmosis, and coccidiosis. The presence of apicoplasts with the remnant of a plastid-like DNA argues that these parasites evolved from photosynthetic ancestors possibly related to the dinoflagellates. Toxoplasma gondii displays amylopectin-like polymers within the cytoplasm of the dormant brain cysts. Here we report a detailed structural and comparative analysis of the Toxoplasma gondii, green alga Chlamydomonas reinhardtii, and dinoflagellate Crypthecodinium cohnii storage polysaccharides. We show Toxoplasma gondii amylopectin to be similar to the semicrystalline floridean starch accumulated by red algae. Unlike green plants or algae, the nuclear DNA sequences as well as biochemical and phylogenetic analysis argue that the Toxoplasma gondii amylopectin pathway has evolved from a totally different UDP-glucose-based metabolism similar to that of the floridean starch accumulating red alga Cyanidioschyzon merolae and, to a lesser extent, to those of glycogen storing animals or fungi. In both red algae and apicomplexan parasites, isoamylase and glucan-water dikinase sequences are proposed to explain the appearance of semicrystalline starch-like polymers. Our results have built a case for the separate evolution of semicrystalline storage polysaccharides upon acquisition of photosynthesis in eukaryotes.

Amylopectin biogenesis and characterization in the protozoan parasite Toxoplasma gondii, the intracellular development of which is restricted in the HepG2 cell line.

The obligate intracellular protozoan Toxoplasma gondii belongs to the phylum Apicomplexa, which is composed of numerous parasites causing major diseases such as malaria, toxoplasmosis and coccidiosis. The life cycle of T. gondii involves developmental processes from one stage to another with both asexual and sexual parasitic forms. Throughout their life cycle, some apicomplexan parasites accumulate a crystalline storage polysaccharide analogous to amylopectin within the cytoplasm. In T. gondii, both the slowly dividing encysted bradyzoites and the sporozoites of the sexual stage contain a high number of amylopectin granules (AG), while the rapidly replicating tachyzoites are devoid of amylopectin. It is thought that this storage polysaccharide may represent an energy reserve that could fuel the transition from one developmental stage to another one. At present, by comparison to glycogen and plant starch, little is known about the biosynthesis, structure and biological functions of amylopectin in T. gondii. Here, we describe an in vitro system allowing the production and purification of a large amount of amylopectin, which has been subjected to detailed biochemical and structural analyses. Our data indicate that T. gondii synthesizes a genuine amylopectin following changes in the environmental conditions and that this storage polysaccharide differs from glycogen and starch in terms of glucan chain length.

The expression of lactate dehydrogenase is important for the cell cycle of Toxoplasma gondii.

In Toxoplasma gondii, lactate dehydrogenase is encoded by two independent and developmentally regulated genes LDH1 and LDH2. These genes and their products have been implicated in the control of a metabolic flux during parasite differentiation. To investigate the significance of LDH1 and LDH2 in this process, we generated stable transgenic parasite lines in which the expression of these two expressed isoforms of lactate dehydrogenase was knocked down in a stage-specific manner. These LDH knockdown parasites exhibited variable growth rates in either the tachyzoite or the bradyzoite stage, as compared with the parental parasites. Their differentiation processes were impaired when the parasites were grown under in vitro conditions. In vivo studies in a murine model system revealed that tachyzoites of these parasite lines were unable to form significant numbers of tissue cysts and to establish a chronic infection. Most importantly, all mice that were initially infected with tachyzoites of either of the four LDH knockdown lines survived a subsequent challenge with tachyzoites of the parental parasites (10(4)), a dose that usually causes 100% mortality, suggesting that live vaccination of mice with the LDH knockdown tachyzoites can confer protection against T. gondii. Thus, we conclude that LDH expression is essential for parasite differentiation. The knockdown of LDH1 and LDH2 expression gave rise to virulence-attenuated parasites that were unable to exhibit a significant brain cyst burden in a murine model of chronic infection.

Developmentally regulated biosynthesis of carbohydrate and storage polysaccharide during differentiation and tissue cyst formation in Toxoplasma gondii.

Toxoplasma gondii belongs to the Apicomplexa phylum, which comprises protozoan parasites of medical and veterinary significance, responsible for a wide variety of diseases in human and animals, including malaria, toxoplasmosis, coccidiosis and cryptosporidiosis. During infection in the intermediate host, T. gondii undergoes stage conversion between the rapidly replicating tachyzoite that is responsible for acute toxoplasmosis and the dormant or slowly dividing encysted bradyzoite. The tachyzoite-bradyzoite interconversion is central to the pathogenic process and is associated with the life-threatening recrudescence of infection observed in immunocompromised patients such as those suffering from AIDS. In chronic infections, the bradyzoites are located within tissue cysts found predominantly in brain and muscles. The tissue cyst is enclosed by a wall containing specific lectin binding sugars while the bradyzoites have accumulated large amounts of the storage polysaccharide of glucose, amylopectin. Our recent findings have identified several genes and proteins associated with amylopectin synthesis or degradation and glucose metabolism, including different isoforms of certain glycolytic enzymes, which are stage-specifically expressed during tachyzoite-bradyzoite interconversion. Here, we will discuss how the genes and enzymes involved in carbohydrate metabolisms are used as molecular and biochemical tools for the elucidation of molecular mechanisms controlling T. gondii stage interconversion and cyst formation.

Functional analysis of Toxoplasma gondii protease inhibitor 1.

We have characterized a Kazal family serine protease inhibitor, Toxoplasma gondii protease inhibitor 1 (TgPI-1), in the obligate intracellular parasite Toxoplasma gondii. TgPI-1 contains four inhibitor domains predicted to inhibit trypsin, chymotrypsin, and elastase. Antibodies against recombinant TgPI-1 detect two polypeptides, of 43 and 41 kDa, designated TgPI-1(43) and TgPI-1(41), in tachyzoites, bradyzoites, and sporozoites. TgPI-1(43) and TgPI-1(41) are secreted constitutively from dense granules into the excreted/secreted antigen fraction as well as the parasitophorous vacuole that T. gondii occupies during intracellular replication. Recombinant TgPI-1 inhibits trypsin, chymotrypsin, pancreatic elastase, and neutrophil elastase. Immunoprecipitation studies with anti-rTgPI-1 antibodies reveal that recombinant TgPI-1 forms a complex with trypsin that is dependent on interactions with the active site of the protease. TgPI-1 is the first anti-trypsin/chymotrypsin inhibitor to be identified in bradyzoites and sporozoites, stages of the parasite that would be exposed to proteolytic enzymes in the digestive tract of the host.

Evidence for nuclear localisation of two stage-specific isoenzymes of enolase in Toxoplasma gondii correlates with active parasite replication.

The protozoan parasite Toxoplasma gondii has a complex life cycle involving the developmental transition between the asexual exo-enteric stages (tachyzoites and bradyzoites) and the coccidian (sexual and asexual) forms (schizonts, macrogametes and microgametes). Previous work has established the stage-specific expression of certain proteins including two glycolytic isoenzymes of enolase and lactate dehydrogenase in T. gondii. Here we describe the expression and subcellular localisation of the two isoforms of enolase (ENO1 and ENO2) and lactate dehydrogenase (LDH1 and LDH2) in vivo using immunocytochemistry. In mice, proliferating parasites in the lung expressed ENO2 and LDH1 and were characterised as tachyzoites by the presence of a tachyzoite specific surface antigen (SAG1). In contrast, ENO1 and LDH2 were expressed by bradyzoites present in tissue cysts in the brain characterised by the presence of the bradyzoite specific antigen (BAG1). During stage conversion (tachyzoite/bradyzoite), the isoenzyme changes occur at an early stage when the bradyzoites are actively proliferating and thus may not simply be reflecting reduced metabolic needs. When the coccidian stages were examined in the cat intestine, they were negative for SAG1, BAG1, LDH2 and ENO1 but were similar to the tachyzoite in strongly expressing LDH1 and ENO2. The isoenzymes LDH1 and LDH2 were exclusively expressed in the cytoplasm. In contrast, it was observed that the strongest labelling for both ENO1 and ENO2 was observed in the nucleus with less intense but specific cytoplasmic staining. Immunoelectron microscopy confirmed the cytoplasmic location of LDH and the predominantly nuclear location of enolase. During early intracellular proliferation and development, all stages of the life cycle (tachyzoite, bradyzoite and coccidian stages) exhibited very strong nuclear labelling for enolase but this was markedly reduced in mature parasites to levels below that seen in the cytoplasm. The specific nuclear localisation of enolases appears to be associated with nuclear activity (transcription and/or division) and may play some part in the control of gene regulation during parasite proliferation and differentiation in addition to its role in glycolysis.

The SAG5 locus of Toxoplasma gondii encodes three novel proteins belonging to the SAG1 family of surface antigens.

We have identified three novel Toxoplasma gondii proteins showing close structural similarity to molecules of the SAG1 family, a group of glycosylphosphatidylinositol-anchored surface antigens expressed by the invasive stages of T. gondii. The novel proteins, denominated SAG5A, SAG5B and SAG5C, are encoded by tandemly arrayed and tightly clustered genes containing no introns. The 367 amino acid-long SAG5B and SAG5C are 97.5% identical to each other, whereas SAG5A (362 amino acids) consists of a C-terminal domain sharing 98% identity with SAG5B and SAG5C, and an N-terminal domain whose identity to the other SAG5 polypeptides is only 42%. Expression analysis of the T. gondii strains RH (virulent) and 76 K (avirulent) showed that all members of the SAG5 cluster are transcribed in T. gondii tachyzoites and bradyzoites. However, immunoblot studies on the RH strain revealed that the synthesis of SAG5A does not occur in tachyzoites and is possibly controlled at the post-transcriptional level. On the contrary, SAG5B and SAG5C were detected by immunoblot in tachyzoite lysates and found to migrate in the 40-45 kDa range under reducing conditions or at approximately 34 kDa under unreduced conditions. Triton X-114 partitioning of tachyzoite protein lysates treated with phosphatidylinositol-specific phospholipase C indicated that SAG5B and SAG5C are glycosylphosphatidylinositol-anchored membrane-associated molecules. Consistently, immunofluorescence analysis of transformed tachyzoites over-expressing SAG5B or SAG5C showed that these molecules are targeted to the parasite surface. The characterisation of the SAG5 locus sheds further light on the complex repertoire of SAG1-related genes in T. gondii, that now comprises 14 highly homologous members and five distantly related genes belonging to the SAG2 family.