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The Toxoplasma inner membrane complex (IMC) is a unique organelle that plays critical roles in parasite motility, invasion, egress, and replication. The IMC is delineated into the apical, body, and basal regions, defined by proteins that localize to these distinct subcompartments. The IMC can be further segregated by proteins that localize specifically to the maternal IMC, the daughter bud IMC, or both. While the function of the maternal IMC has been better characterized, the precise roles of most daughter IMC components remain poorly understood. Here, we demonstrate that the daughter protein IMC29 plays an important role in parasite replication. We show that Δ imc29 parasites exhibit severe replication defects, resulting in substantial growth defects and loss of virulence. Deletion analyses revealed that IMC29 localization is largely dependent on the N-terminal half of the protein containing four predicted coiled-coil domains while IMC29 function requires a short C-terminal helical region. Using proximity labeling, we identify eight novel IMC proteins enriched in daughter buds, significantly expanding the daughter IMC proteome. We additionally report four novel proteins with unique localizations to the interface between two parasites or to the outer face of the IMC, exposing new subregions of the organelle. Together, this work establishes IMC29 as an important early daughter bud component of replication and uncovers an array of new IMC proteins that provides important insights into this organelle. IMPORTANCE The inner membrane complex (IMC) is a conserved structure across the Apicomplexa phylum, which includes obligate intracellular parasites that cause toxoplasmosis, malaria, and cryptosporidiosis. The IMC is critical for the parasite to maintain its intracellular lifestyle, particularly in providing a scaffold for daughter bud formation during parasite replication. While many IMC proteins in the later stages of division have been identified, components of the early stages of division remain unknown. Here, we focus on the early daughter protein IMC29, demonstrating that it is crucial for faithful parasite replication and identifying specific regions of the protein that are important for its localization and function. We additionally use proximity labeling to reveal a suite of daughter-enriched IMC proteins, which represent promising candidates to further explore this IMC subcompartment.

The apicoplast, which harbors key pathways involved in biosynthesis of vital metabolites, is a unique and essential nonphotosynthetic plastid organelle in apicomplexan parasites. Intriguingly, autophagy-related protein 8 (Atg8), a highly conserved eukaryotic protein, can localize to the outermost membrane of the apicoplast and modulate its inheritance in both Toxoplasma and Plasmodium parasites. The Atg8-Atg3 interaction plays a key role in Atg8 lipidation and localization, and our previously work in Toxoplasma has suggested that the core Atg8-family interacting motif (AIM) in TgAtg3, 239 FADI 242 , and the R27 residue of TgAtg8 contribute to TgAtg8-TgAtg3 interaction in vitro . However, little is known about the function of this interaction or its importance in tachyzoite growth in Toxoplasma gondii . Here, we generated two complemented cell lines, TgAtg3 F239A/I242A and TgAtg8 R27E , based on the TgAtg3 and TgAtg8 conditional knockdown cell lines, respectively. We found that both mutant complemented cell lines were severely affected in terms of tachyzoite growth and displayed delayed death upon conditional knockdown of endogenous TgAtg3 or TgAtg8. Intriguingly, both complemented lines appeared to be defective in TgAtg8 lipidation and apicoplast inheritance. Moreover, we showed that the interaction of TgAtg8 and TgAtg3 is critical for TgAtg8 apicoplast localization. In addition, we found that the TgAtg3 F239A/I242A complemented line exhibits an integral mitochondrial network upon ablation of endogenous TgAtg3, which is distinct from TgAtg3-depleted parasites with a fragmented mitochondrial network. Taken together, this work solidifies the contribution of the TgAtg8-TgAtg3 interaction to apicoplast inheritance and the growth of T. gondii tachyzoites. IMPORTANCE is a widespread intracellular parasite infecting a variety of warm-blooded animals, including humans. Current frontline treatment of toxoplasmosis suffers many drawbacks, including toxicity, drug resistance, and failure to eradicate tissue cysts, underscoring the need to identify novel drug targets for suppression or treatment of toxoplasmosis. TgAtg8 is thought to serve multiple functions in lipidation and is considered essential to the growth and development of both tachyzoites and bradyzoites. Here, we show that Toxoplasma gondii has adapted a conserved Atg8-Atg3 interaction, required for canonical autophagy in other eukaryotes, to function specifically in apicoplast inheritance. Our finding not only highlights the importance of TgAtg8-TgAtg3 interaction in tachyzoite growth but also suggests that this interaction is a promising drug target for the therapy of toxoplasmosis.Toxoplasma gondii has adapted a conserved Atg8-Atg3 interaction, required for canonical autophagy in other eukaryotes, to function specifically in apicoplast inheritance. Our finding not only highlights the importance of TgAtg8-TgAtg3 interaction in tachyzoite growth but also suggests that this interaction is a promising drug target for the therapy of toxoplasmosis.

Toxoplasma gondii ( T. gondii ) infection causes severe zoonotic toxoplasmosis, which threatens the safety of almost one-third of the human population globally. However, there is no effective protective vaccine against human toxoplasmosis. This necessitates anti- T. gondii vaccine development, which is a main priority of public health. In this study, we optimized the adjuvant system 04 (AS04), a vaccine adjuvant constituted by 3-O-desacyl-4'-monophosphoryl lipid A (a TLR4 agonist) and aluminum salts, by packing it within natural extracts of β-glucan particles (GPs) from Saccharomyces cerevisiae to form a GP-AS04 hybrid adjuvant system. Through a simple mixing procedure, we loaded GP-AS04 particles with the total extract (TE) of T. gondii lysate, forming a novel anti- T. gondii vaccine GP-AS04-TE. Results indicated that the hybrid adjuvant can efficiently and stably load antigens, mediate antigen delivery, facilitate the dendritic uptake of antigens, boost dendritic cell maturation and stimulation, and increase the secretion of pro-inflammatory cytokines. In the mouse inoculation model, GP-AS04-TE significantly stimulated the function of dendritic cells, induced a very strong TE-specific humoral and cellular immune response, and finally showed a strong and effective protection against toxoplasma chronic and acute infections. This work proves the potential of GP-AS04 for exploitation as a vaccine against a range of pathogens.

Our studies on novel cyst wall proteins serendipitously led us to the discovery that cyst wall and vacuolar matrix protein MAG1, first identified a quarter of a century ago, functions as a secreted immunomodulatory effector. MAG1 is a dense granular protein that is found in the parasitophorous vacuolar matrix in tachyzoite vacuoles and the cyst wall and matrix in bradyzoite vacuoles. In the current study, we demonstrated that MAG1 is secreted beyond the parasitophorous vacuole into the host cytosol in both tachyzoites and bradyzoites. Secretion of MAG1 gradually decreases as the parasitophorous vacuole matures, but prominent MAG1 puncta are present inside host cells even at 4 and 6 days following infection. During acute murine infection, Δ mag1 parasites displayed significantly reduced virulence and dissemination. In the chronic stage of infection, Δ mag1 parasites generated almost no brain cysts. To identify the mechanism behind the attenuated pathology seen with Δ mag1 parasites, various immune responses were screened in vitro using bone marrow-derived macrophages (BMDM). Infection of BMDM with Δ mag1 parasites induced a significant increase in interleukin 1β (IL-1β) secretion, which is a hallmark of inflammasome activation. Heterologous complementation of MAG1 in BMDM cells prevented this Δ mag1 parasite-induced IL-1β release, indicating that secreted MAG1 in host cytosol dampens inflammasome activation. Furthermore, knocking out GRA15 (an inducer of IL-1β release) in Δ mag1 parasites completely inhibited all IL-1β release by host cells following infection. These data suggest that MAG1 has a role as an immunomodulatory molecule and that by suppressing inflammasome activation, it would favor survival of the parasite and the establishment of latent infection. IMPORTANCE Toxoplasma gondii is an Apicomplexan that infects a third of humans, causing encephalitis in AIDS patients and intellectual disabilities in congenitally infected patients. We determined that one of the cyst matrix proteins, MAG1, which had been thought to be an innate structural protein, can be secreted into the host cell and suppress the host immune reaction. This particular immune reaction is initiated by another parasite-secreted protein, GRA15. The intricate balance of inflammasome activation by GRA15 and suppression by MAG1 protects mice from acute death while enabling parasites to disseminate and establish chronic cysts. Our finding contributes to our understanding of how parasites persist in the host and how T. gondii modulates the host immune system., (Copyright © 2021 Tomita et al.)

Previous studies showed that three clonal strain types (I, II, and III) of Toxoplasma gondii can be distinguished using serotyping based on a series of polymorphic proteins. However, to establish a systematic serotyping method with higher resolution even being equal to that of genotyping, more specific peptide markers are needed. The objective of the present study was to determine the possibility of the polymorphic dense granule protein 15 (GRA15) for diagnosis and serotyping of T. gondii infection. Three different T. gondii GT1 strain GRA15 gene fragments encoding a 584-residue peptide, a 199-residue peptide and a 84-residue peptide were amplified, expressed and purified, respectively. Anti-T. gondii GT1 strain antibodies, anti-T. gondii RH strain antibodies and anti-T. gondii PRU strain antibodies were used for immunoblotting analysis of the three peptides. Western blotting analysis showed that the 584-residue peptide of GT1 strain GRA15 was a potential candidate for serological diagnosis of T. gondii infection. RH strain from GT1 strain could be distinguished by serotyping based on the GRA15 199 or GRA15 84 , and T. gondii GT1 strain could be distinguished from PRU strain by using serotyping based on the GRA15 84 . These findings reveal, for the first time, a novel potential role of GRA15-derived peptides in diagnosis and serological differentiation of T. gondii infection., Competing Interests: Declaration of competing interest All authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Intracellular pathogens must egress from the host cell to continue their infectious cycle. Apicomplexans are a phylum of intracellular protozoans that have evolved members of the membrane attack complex and perforin (MACPF) family of pore forming proteins to disrupt cellular membranes for traversing cells during tissue migration or egress from a replicative vacuole following intracellular reproduction. Previous work showed that the apicomplexan Toxoplasma gondii secretes a perforin-like protein (TgPLP1) that contains a C-terminal Domain (CTD) which is necessary for efficient parasite egress. However, the structural basis for CTD membrane binding and egress competency remained unknown. Here, we present evidence that TgPLP1 CTD prefers binding lipids that are abundant in the inner leaflet of the lipid bilayer. Additionally, solving the high-resolution crystal structure of the TgPLP1 APCβ domain within the CTD reveals an unusual double-layered β-prism fold that resembles only one other protein of known structure. Three direct repeat sequences comprise subdomains, with each constituting a wall of the β-prism fold. One subdomain features a protruding hydrophobic loop with an exposed tryptophan at its tip. Spectrophotometric measurements of intrinsic tryptophan fluorescence are consistent with insertion of the hydrophobic loop into a target membrane. Using CRISPR/Cas9 gene editing we show that parasite strains bearing mutations in the hydrophobic loop, including alanine substitution of the tip tryptophan, are equally deficient in egress as a strain lacking TgPLP1 altogether. Taken together our findings suggest a crucial role for the hydrophobic loop in anchoring TgPLP1 to the membrane to support its cytolytic activity and egress function., Competing Interests: The authors have declared that no competing interests exist.

Locus expansion and diversification is pervasive in apicomplexan genomes and is predominantly found in loci encoding secreted proteins that interact with factors outside of the parasite. Key for understanding the impact of each of these loci on the host requires identification and functional characterization of their protein products, but these repetitive loci often are refractory to genome assembly. In this review we focus on Toxoplasma gondii and its nearest relatives to highlight the known impact of duplicated and diversified loci on our understanding of the host-pathogen molecular arms race. We describe current tools used for the identification and characterization of these loci, and review the most recent examples of how gene-expansion driven diversification can lead to novel gene functions., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective To investigate the effect of rhoptry protein 38 (ROP38) from Toxoplasma gondii (T. gondii) on the maturation of dendritic cells (DCs) by Toll-like receptor 4 (TLR4) induction in vitro. Methods The total RNA from T. gondii RH strain was extracted by guanidine thiocyanate method, and then cDNA was synthesized with reverse transcription reaction. After ROP38 gene was amplified by PCR, the recombinant pGEX-4T-ROP38 was constructed and expressed under IPTG induction. The recombinant ROP38 protein was detected by SDS-PAGE and Western blot analysis. The secondary structure and antigenicity of the ROP38 were predicted through DNAStar8.0 and ProtScale. In vitro, DCs were isolated and cultured for 6 days, then reacted with ROP38 antigen for 2 hours. The CD11c was detected by flow cytometry, and the data were analyzed with ANOVA by SPSS 23.0 software. Results The amplified gene was about 516 bp as expected. The sequence analysis showed that its homology was 99% compared with the reported sequence (XM_002366710.2) from GenBank. It was found that the relative molecular mass (M r ) of recombinant ROP38 was 45 kD. The prediction of structure indicated that there were 5 α-helices, 5 β-sheets, 5 hydrophilic regions and 8 potential epitopes in ROP38 protein. In vitro, the expression of CD11c on DCs was significantly up-regulated after stimulated with ROP38, and the expression level of CD11c in ROP38 infection group was significantly higher than that of the other groups. Conclusion ROP38 promotes the maturation of DCs mediated by TLR4.

SAG1-related sequence 3 (SRS3) is one of the major Toxoplasma gondii tachyzoite surface antigens and has been shown to be potentially useful for the detection of toxoplasmosis. This protein is highly conformational due to the presence of six disulfide bonds. To achieve solubility and antigenicity, SRS3 depends on proper disulfide bond formation. The aim of this study was to over-express the SRS3 protein with correct folding for use in serodiagnosis of the disease. To achieve this, a truncated SRS3 fusion protein (rtSRS3) was produced, containing six histidyl residues at both terminals and purified by immobilized metal affinity chromatography. The refolding process was performed through three methods, namely dialysis in the presence of chemical additives along with reduced/oxidized glutathione and drop-wise dilution methods with reduced/oxidized glutathione or reduced DTT/oxidized glutathione. Ellman's assay and ELISA showed that the protein folding obtained by the dialysis method was the most favorable, probably due to the correct folding. Subsequently, serum samples from individuals with chronic infection (n = 76), probable acute infection (n = 14), and healthy controls (n = 81) were used to determine the usefulness of the refolded rtSRS3 for Toxoplasma serodiagnosis. The results of the developed IgG-ELISA showed a diagnostic specificity of 91% and a sensitivity of 82.89% and 100% for chronic and acute serum samples, respectively. In conclusion, correctly folded rtSRS3 has the potential to be used as a soluble antigen for the detection of human toxoplasmosis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Protein antigens comprising peptide motifs with high binding affinity to major histocompatibility complex class I (MHC-I) molecules are expected to induce a stronger cytotoxic T-lymphocyte response and thus provide better protection against infection with microorganisms where cytotoxic T-cells are the main effector arm of the immune system., Methods: Data on cyst formation and survival were extracted from past studies on the DNA immunization of mice with plasmids coding for Toxoplasma gondii antigens. From in silico analyses of the vaccine antigens, the correlation was tested between the predicted affinity for MHC-I molecules of the vaccine peptides and the survival of immunized mice after challenge with T. gondii. ELISPOT analysis was used for the experimental testing of peptide immunogenicity., Results: Predictions for the Db MHC-I molecule produced a strong, negative correlation between survival and the dissociation constant of vaccine-derived peptides. The in silico analyses of nine T. gondii antigens identified peptides with a predicted dissociation constant in the interval from 10nM to 40μM. ELISPOT assays with splenocytes from T. gondii-infected mice further supported the importance of the peptide affinity for MHC-I., Conclusions: In silico analysis clearly helped the search for protective vaccine antigens. The ELISPOT analysis confirmed that the predicted T-cell epitopes were immunogenic by their ability to release interferon gamma in spleen cells., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Toxoplasmosis is a zoonosis distributed all over the world, which the etiologic agent is an intracellular protozoan parasite, Toxoplasma gondii. This disease may cause abortions and severe diseases in many warm-blood hosts, including humans, particularly the immunocompromised patients. The parasite specialized secretory organelles, as micronemes, rhoptries and dense granules, are critical for the successful parasitism. The dense granule protein 2 (GRA2) is a parasite immunogenic protein secreted during infections and previous studies have been shown that this parasite component is crucial for the formation of intravacuolar membranous nanotubular network (MNN), as well as for secretion into the vacuole and spatial organization of the parasites within the vacuole. In the present study, we produced a monoclonal antibody to GRA2 (C3C5 mAb, isotype IgG2b), mapped the immunodominant epitope of the protein by phage display and built GRA2 synthetic epitopes to evaluate their ability to protect mice in a model of experimental infection. Our results showed that synthetic peptides for B- and T-cell epitopes are able to improve survival of immunized animals. In contrast with non-immunized animals, the immunized mice with both B- and T-cell epitopes had a better balance of cytokines and demonstrated higher levels of IL-10, IL-4 and IL-17 production, though similar levels of TNF-α and IL-6 were observed. The immunization with both B- and T-cell epitopes resulted in survival rate higher than 85% of the challenged mice. Overall, these results demonstrate that immunization with synthetic epitopes for both B- and T-cells from GRA2 protein can be more effective to protect against infection by T. gondii.

Immunoproteomic technology offers an exceptional tool to fill the blanks that still exist in diagnosis of Toxoplasma gondii infection despite its annotated sequence. The pitfalls of serological assays and current immunoproteomic approaches are accentuated, and new approaches are presented to improve the signal and to eliminate the noise produced by blocking-specific background. This review also highlights examples where immunoproteomic studies have contributed to broaden our understanding of toxoplasmosis diagnosis. Further promising solutions, which immunoproteomic technology can grant for toxoplasmosis diagnosis are part of an intense discussion., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Polymorphic rhoptry-secreted kinases (ROPs) are essential virulence factors of Toxoplasma gondii. In particular, the pseudokinase ROP5 is the major determinant of acute virulence in mice, but the underlying mechanisms are unclear. We developed a tandem affinity protein tagging and purification approach in T. gondii and used it to show that ROP5 complexes with the active kinases ROP18 and ROP17. Biochemical analyses indicate that ROP18 and ROP17 have evolved to target adjacent and essential threonine residues in switch region I of immunity-related guanosine triphosphatases (GTPases) (IRGs), a family of host defense molecules that function to control intracellular pathogens. The combined activities of ROP17 and ROP18 contribute to avoidance of IRG recruitment to the intracellular T. gondii-containing vacuole, thus protecting the parasite from clearance in interferon-activated macrophages. These studies reveal an intricate, multilayered parasite survival strategy involving pseudokinases that regulate multiple active kinase complexes to synergistically thwart innate immunity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Microtubules are versatile biopolymers that support numerous vital cellular functions in eukaryotes. The specific properties of microtubules are dependent on distinct microtubule-associated proteins, as the tubulin subunits and microtubule structure are exceptionally conserved. Highly specialized microtubule-containing assemblies are often found in protists, which are rich sources for novel microtubule-associated proteins. A protozoan parasite, Toxoplasma gondii, possesses several distinct tubulin-containing structures, including 22 microtubules closely associated with the cortical membrane. Early ultrastructural studies have shown that the cortical microtubules are heavily decorated with associating proteins. However, little is known about the identities of these proteins. Here, we report the discovery of a novel protein, TrxL1 (for Thioredoxin-Like protein 1), and an associating complex that coats the cortical microtubules. TrxL1 contains a thioredoxin-like fold. To visualize its localization in live parasites by fluorescence, we replaced the endogenous TrxL1 gene with an mEmeraldFP-TrxL1 fusion gene. Structured illumination-based superresolution imaging of this parasite line produced a detailed view of the microtubule cytoskeleton. Despite its stable association with the cortical microtubules in the parasite, TrxL1 does not seem to bind to microtubules directly. Coimmunoprecipitation experiments showed that TrxL1 associates with a protein complex containing SPM1, a previously reported microtubule-associated protein in T. gondii. We also found that SPM1 recruits TrxL1 to the cortical microtubules. Besides SPM1, several other novel proteins are found in the TrxL1-containing complex, including TrxL2, a close homolog of TrxL1. Thus, our results reveal for the first time a microtubule-associated complex in T. gondii.

Macrophage migration inhibitory factor (MIF) is a proinflammatory molecule in mammals that, unusually for a cytokine, exhibits tautomerase and oxidoreductase enzymatic activities. Homologues of this well conserved protein are found within diverse phyla including a number of parasitic organisms. Herein, we produced recombinant histidine-tagged Toxoplasma gondii MIF (TgMIF), a 12-kDa protein that lacks oxidoreductase activity but exhibits tautomerase activity with a specific activity of 19.3 μmol/min/mg that cannot be inhibited by the human MIF inhibitor ISO-1. The crystal structure of the TgMIF homotrimer has been determined to 1.82 Å, and although it has close structural homology with mammalian MIFs, it has critical differences in the tautomerase active site that account for the different inhibitor sensitivity. We also demonstrate that TgMIF can elicit IL-8 production from human peripheral blood mononuclear cells while also activating ERK MAPK pathways in murine bone marrow-derived macrophages. TgMIF may therefore play an immunomodulatory role during T. gondii infection in mammals.

Toxoplasma gondii is a protozoan parasite capable of infecting humans and animals. Surface antigen glycoproteins, SAG2C, -2D, -2X, and -2Y, are expressed on the surface of bradyzoites. These antigens have been shown to protect bradyzoites against immune responses during chronic infections. We studied structures of SAG2C, -2D, -2X, and -2Y proteins using bioinformatics methods. The protein sequence alignment was performed by T-Coffee method. Secondary structural and functional domains were predicted using software PSIPRED v3.0 and SMART software, and 3D models of proteins were constructed and compared using the I-TASSER server, VMD, and SWISS-spdbv. Our results showed that SAG2C, -2D, -2X, and -2Y are highly homologous proteins. They share the same conserved peptides and HLA-I restricted epitopes. The similarity in structure and domains indicated putative common functions that might stimulate similar immune response in hosts. The conserved peptides and HLA-restricted epitopes could provide important insights on vaccine study and the diagnosis of this disease.

Toxoplasma gondii glycosylphosphatidylinositols (GPIs) bind to galectin-3 to induce TNF-α production in macrophages via Toll-like receptors 2 and 4. Here we show that T. gondii GPIs stimulate human macrophages to synthesize matrix metalloproteinase-9 in a TNF-α-dependent pathway and degrade extracellular galectin-3., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

By the separation of Toxoplasma lysate using two-dimensional gel electrophoresis and its analysis with human serum samples and mass spectrometry, the subtilisin-like protein (SUB1) was identified to be a potential marker for acute toxoplasmosis. Following expression of the C-terminal domain of SUB1 in Escherichia coli, it was tested in a line blot assay using a total of 80 human serum samples. Two computer programs based on different evaluation strategies were used for judgment of the line blot results: (i) a time-dependent method with a predefined cutoff value and (ii) a fixed-time-point method with a calculated cutoff. Thereby, SUB1 was proven to be rather reactive with specific immunoglobulin A (IgA), IgM, and IgG of patients with an acute infection. This finding makes this antigen an attractive candidate for improving diagnosis of toxoplasmosis and demonstrates that not only the selection of respective antigens but also the evaluation method chosen are important for the evaluation of new diagnostic markers.

Recombinant Toxoplasma gondii small heat shock protein HSP20, surface antigen SAG1 and dense granule GRA7 were analyzed by IgG-ELISA with serum samples of Toxoplasma infected humans grouped as I (IgG+, IgM+), II (IgG+, IgM-) and III (IgG-, IgM-). rHSP20 reacted against 80% and 62.5% of serum samples from groups I and II, respectively. rSAG1 was recognized by 85% of the samples from group I and 70.8% from group II, whereas rGRA7 was recognized by 85% and 66.6% of the serum samples from groups I and II, respectively. When a combination of two or three recombinant antigens was used, the sensitivity values improved to 85-95% for group I and 87.5-91.7% for group II. All combinations tested produced similar reactivity profiles. None of the recombinant proteins reacted against group III serum samples. In conclusion, we demonstrated that T. gondii HSP20 elicits an important B-cell response during human infection, and could be suitable for the development of serodiagnosis tools., (Copyright 2010 Elsevier Inc. All rights reserved.)

How eukaryotic pathogens export and sort membrane-bound proteins destined for host-cell compartments is still poorly understood. The dense granules of the intracellular protozoan Toxoplasma gondii constitute an unusual secretory pathway that allows soluble export of the GRA proteins which become membrane-associated within the parasite replicative vacuole. This process relies on both the segregation of the proteins routed to the dense granules from those destined to the parasite plasma membrane and on the sorting of the secreted GRA proteins to their proper final membranous system. Here, we provide evidence that the soluble trafficking of GRA6 to the dense granules relies on the N-terminal domain of the protein, which is sufficient to prevent GRA6 targeting to the parasite plasma membrane. We also show that the GRA6 N-terminal domain, possibly by interacting with negatively charged lipids, is fundamental for proper GRA6 association with the vacuolar membranous network of nanotubes. These results support our emerging model: sorting of transmembrane GRA proteins to the host cell vacuole is mainly driven by the dual role of their N-terminal hydrophilic domain and is compartmentally regulated., (Copyright (c) 2010. Published by Elsevier Ltd.)

In this study, human sera reactivity against nine peptides derived from the Toxoplasma gondii P30 protein was assessed by ELISA in patients with different clinical forms of toxoplasmosis. Same as has been reported in mice, sera from congenital, ocular and chronic asymptomatic toxoplasmosis patients recognized more strongly peptides from the protein's carboxy-terminus, being peptide 2017 (amino acids 301-320) the one most strongly recognized by sera from patients with ocular toxoplasmosis. Serum samples collected from 13 patients without ocular infection, 13 with inactive chorioretinal scars, 6 with active ocular infection and 10 seronegative individuals were then screened for anti-2017 IgG. Peptide 2017 was recognized by all patients' samples but not by sera from T. gondii-seronegative individuals. No statistically significant differences were found between the absorbance levels of groups with and without lesions or with active or inactive ocular lesions, as determined by ANOVA.

The goal of the present study is to develop an enzyme-linked immunosorbent assay (ELISA) using a truncated surface antigen 1 (SAG1) gene of Toxoplasma gondii for the diagnosis of human toxoplasmosis. The truncated SAG1 gene was highly expressed in Escherichia coli. An ELISA kit based on the purified recombinant truncated SAG1 (rtSAG1) was developed, which was used to detect antibodies against T. gondii in human sera. The results showed that the infection of T. gondii could be detected sensitively and specifically by this serologic method. The positive concordance between rtSAG1-ELISA and Western blot, the gold standard, was 93.9% (31/33). However, the positive concordance between the commercial available ELISA Kit 1 (Haitai, Zhuhai, China) and ELISA Kit 2 (DiaSorin ETI-TOXOK-M reverse Plus, Italy) with Western blot was 79.5% (31/39) and 91.2% (31/34), respectively. Comparatively, the positive concordance of ELISA Kit 1 and 2 with Western blot was lower than rtSAG1-ELISA, in particular, the ELISA Kit 1 (P < 0.01), which indicated that the rtSAG1 protein could be used as the diagnostic antigen for human toxoplasmosis.

Toxoplasma gondii is a haploid protozoan parasite infecting about one in seven people in the United States. Key to the worldwide prevalence of T. gondii is its ability to establish a lifelong, chronic infection by evading the immune system, and central to this is the developmental switch between the two asexual forms, tachyzoites and bradyzoites. A library of mutants defective in tachyzoite-to-bradyzoite differentiation (Tbd(-)) was created through insertional mutagenesis. This library contains mutants that, compared to the wild type, are between 20% and 74% as efficient at stage conversion. Two mutants, TBD5 and TBD8, with disruptions in a gene encoding a putative pseudouridine synthase, PUS1, were identified. The disruption in TBD8 is in the 5' end of the PUS1 gene and appears to produce a null allele with a 50% defect in differentiation. This is about the same switch efficiency as obtained with an engineered pus1 deletion mutant (Deltapus1). The insertion in TBD5 is within the PUS1 coding region, and this appears to result in a more extreme phenotype of only approximately 10% switch efficiency. Complementation of TBD8 with the genomic PUS1 allele restored wild-type differentiation efficiency. Infection of mice with pus1 mutant strains results in increased mortality during the acute phase and higher cyst burdens during the chronic infection, demonstrating an aberrant differentiation phenotype in vivo due to PUS1 disruption. Our results suggest a surprising and important role for RNA modification in this biological process.

ATP-binding cassette (ABC) transporters represent an important family of membrane proteins involved in drug resistance and other biological activities. The present study reports on the characterization of a P-glycoprotein (Pgp), TgABC.B1, in the protozoan parasite Toxoplasma gondii. The protein encoded by the TgABC.B1 gene displays the typical (TMD-NBD)2 structural organization of the "full" ABC transporter and shows significant identity and similarity with two apicomplexan Pgps; Pgh1 in Plasmodium falciparum and CpABC3 in Cryptosporidium parvum. The TgABC.B1 gene is a single copy gene transcribed into a full-length mRNA of 4.3kb and expressed as a protein of approximately 150kDa, which cellular localization revealed a membrane-associated labelling in tachyzoites. The TgABC.B1 gene is constitutively expressed in the three major T. gondii genotypes but demonstrated a higher expression in virulent type I, at both transcriptional and translational levels. Further characterization of this Pgp-like protein will increase our knowledge of the membrane transport system in this parasite and could result in the identification of a new therapeutic target in Toxoplasma.

Interleukin-12 (IL-12) is important in inducing Th1 responses during infection with microbial pathogens such as the protozoan Toxoplasma gondii. In this issue of Cell Host & Microbe, Plattner and colleagues describe an engineered Toxoplasma strain that lacks profilin, an actin-binding molecule previously implicated in Toll-like receptor-11-dependent IL-12 induction and now shown to be important in parasite motility and host cell invasion.

The apical complex of Toxoplasma gondii is widely believed to serve essential functions in both invasion of its host cells (including human cells), and in replication of the parasite. The understanding of apical complex function, the basis for its novel structure, and the mechanism for its motility are greatly impeded by lack of knowledge of its molecular composition. We have partially purified the conoid/apical complex, identified approximately 200 proteins that represent 70% of its cytoskeletal protein components, characterized seven novel proteins, and determined the sequence of recruitment of five of these proteins into the cytoskeleton during cell division. Our results provide new markers for the different subcompartments within the apical complex, and revealed previously unknown cellular compartments, which facilitate our understanding of how the invasion machinery is built. Surprisingly, the extreme apical and extreme basal structures of this highly polarized cell originate in the same location and at the same time very early during parasite replication.

Development of effective therapies for intracellular eukaryotic pathogens is a serious challenge, given the protected location of these pathogens and the similarity of their biology to that of the host. Identifying cellular processes that are unique to the parasite is therefore a crucial step towards defining appropriate drug targets. In the case of the apicomplexan parasite Toxoplasma gondii, the need to find alternative treatments is imperative because of the poor tolerability and frequent side-effects associated with existing therapeutic strategies. The discovery that the parasite uses lipid synthetic pathways which are different from, or absent in, the mammalian host is now driving a renewed interest in T. gondii lipid biology. Recent achievements in this field are promising and suggest that the elucidation of lipid pathways will provide new opportunities for designing potent antiparasitic strategies.

Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems to both humans and livestock and of great economic impact worldwide. Oligodeoxynucleotides (ODN) which contain immunostimulatory CG motifs (CpG ODN) can promote Th1 responses, an adjuvant activity that is desirable for vaccination against intracellular pathogens. We investigated the feasibility of using CpG as an adjuvant combined with Toxoplasma lysate antigen (TLA) as a vaccine against toxoplasmosis. Genetically susceptible C57BL/6 mice were vaccinated with TLA with or without CpG ODN as an adjuvant and then challenged with 85 cysts of the moderately virulent RRA (Beverley) strain of T. gondii. Prior to challenge infection, immunization with TLA plus CpG ODN directed cellular and humoral immunity toward a Th1 pattern, characterized by enhanced INF gamma production by splenic cells in response to TLA, and enhanced production of toxoplasma-specific IgG and IgG (2a) antibodies. Consequently, CpG/TLA-treated mice showed prolonged survival and 64% reduction in brain parasite burden compared to non-CpG/TLA treated group. Our results suggest that CpG ODN would provide a stable and effective adjuvant for use in vaccination against toxoplasmosis.

The article presents data concerning methods of proteomics and main achievements of studies on Toxoplasma gondii proteom.

This study was carried out to investigate the immune response against 97 kDa (p97) molecular marker of Toxoplasma gondii that has been characterized as a cytosolic protein and a component of the excreted-secreted antigens from this parasite. A total of 60 serum samples from patients were analyzed by enzime-linked immunosorbent assay and Western blot for toxoplasmosis. These samples were organized in three groups, based on clinical symptoms and results of serological tests. Group I: 20 samples reactive to IgG and IgM (acute phase); group II: 20 non-reactive samples (control group); and group III: 20 samples reactive only to IgG (chronic phase). Western blot was performed with total antigenic extracts or with excreted and secreted antigen from T. gondii to identify the fraction correspondent to p97. It was observed that this cytosolic component from T. gondii stimulates the immunologic system to produce both IgM and IgG antibodies in the beginning of the acute infection and IgG throughout the chronic stage of the asymptomatic toxoplasmosis.

Excreted secreted antigens of the protozoan parasite Toxoplasma gondii play a key role in stimulating the host immune system during acute and chronic infection. With the aim of identifying the immunodominant epitopes of T. gondii antigens involved in the human B-cell response against the parasite, we employed a novel immunological approach. A library of cDNA fragments from T. gondii tachyzoites was displayed as fusion proteins to the amino-terminus of lambda bacteriophage capsid protein D. The lambda D-tachyzoite library was then affinity-selected by using a panel of sera of pregnant women, all infected with the parasite. Some of the clones identified through this procedure matched the sequence of the dense granule GRA1 protein (p24), allowing us to identify its antigenic regions. In particular, the analysis of human antibody response against the recombinant GRA1 antigen fragments revealed the existence of an immunodominant epitope (epi-24 peptide).