Structural analysis of glycosyl-phosphatidylinositol membrane anchor of the Toxoplasma gondii tachyzoite surface glycoprotein gp23

In this study we describe the biochemical features of the Toxoplasma gondii tachyzoite surface glycoprotein, gp23, demonstrating that it is attached to the parasite membrane by a glycosyl-phosphatidyl inositol anchor. Gp23 was metabolically labeled with tritiated palmitate, myristate, ethanolamine, inositol, glucosamine, mannose and galactose, as expected for a GPI-anchor structure. Gp23 was released from the surface of living parasites after treatment with phosphatidyl inositol-specific phospholipase C (PI-PLC) and the resulting water-soluble protein was immunoprecipitated with a monoclonal antibody specific for gp23. The GPIcore glycan was generated after aqueous-HF dephosphorylation followed by nitrous acid deamination and its carbohydrate structure was analyzed using selective exo- and endoglycosidase treatments. Finally, the phosphatidylinositol moiety of gp23 was characterized using PI-PLC and phospholipase A₂ (PLA₂) digestions. Our cumulative data suggest that gp23 of T gondii tachyzoites contains a modified GPI-backbone similar to the mammalian Thy-1 anchor, consisting of a conserved core structure (ethanolaminePO₄-6-Manαl-2-Manαl-6-Manαl-4-GIcNαl-6-PI) bearing β-linked N-acetylgalactosamine residue(s).     

Comparison of the major antigens of Neospora caninum and Toxoplasma gondii

The Apicomplexa are a diverse group of parasitic protozoa with very ancient phylogenetic roots. Consistent with their phylogeny, the extant species share conserved proteins and traits that were found in their apicomplexan progenitor, but at the same time they have diverged to occupy different biological niches (e.g. host-range and cell type). Characterisation of gene and protein diversity is important for distinguishing between related parasites, for determining their phylogeny, and for providing insight into factors that determine host restriction, cell preference, and virulence. The value of molecular characterisations and comparisons between species is well illustrated by the close phylogenetic relationship between Neospora caninum and Toxoplasma gondii. These two organisms have nearly identical morphology and can cause similar pathology and disease. Consequently, N. caninum has often been incorrectly identified as T. gondii, thus demonstrating the need for studies addressing the molecular and antigenic composition of Neospora. In this review, we describe the major antigenic proteins that have been characterised in N. caninum. These show homology to T. gondii proteins, yet possess unique antigenic characteristics that distinguish them from their homologues and enable their use for specific serological diagnoses and parasite identification.     

Structural analysis of glycosyl-phosphatidylinositol antigens of Leishmania major

Three glycosyl-phosphatidylinositol glycolipids recognized by antibodies from patients with cutaneous leishmaniasis were extracted from Leishmania major promastigotes by hexane:isopropanol and then purified by thin layer chromatography and LH-20 gel chromatography. Structural analysis was carried out using chemical analyses, fast atom bombardment mass spectrometry, and 1H NMR. The major structures deduced can be summarized as follows: (formula: see text) where n = 0, 1, 2; R1 = (CH2)23-CH3; R2 = (CH2)14-CH3 or (CH2)16-CH3. Alkyl-acyl substitutions in the glycerol backbone showed considerable heterogeneity. These three glycolipids belong to a relatively new class of compounds and may represent sequential steps in the biosynthesis of glycosyl-phosphatidylinositols which anchor proteins or other glycoconjugates to Leishmania cell membranes.     

Evidence for Heterotrimeric GTP-Binding Proteins in Toxoplasma gondii

Toxoplasma gondii , an intracellular protozoan parasite, resides within a host-derived vacuole that is rapidly modified by a parasite-secreted membranous tubular network. In this study we investigated the involvement of heterotrimeric G proteins in the secretory pathway of T. gondii. Aluminum fluoride (AIF_n), a specific activator of heterotrimeric G proteins, induced secretion from isolated tachyzoites of T. gondii in vitro, as seen by light optics and electron microscopy. In Western blot analyses, antibodies to G protein α subunits reacted with 39–42 kDa proteins from T. gondii isolates. Antibodies to G_oα and G_sα coupled to the fluorescent probe fluorescein isothiocyanate localized to the paranuclear region of T. gondii. G_i3α immunoprobes were confined to the cytoplasmic matrix of T. gondii and also labeled the parasitophorous vesicle. Fluorescein isothiocyanate-conjugated GA/1, an antipeptide antisera directed toward the GTP binding site common to G protein α subunits, was confined to the lateral cytoplasmic domain of the parasites where secretion is most prominent. In time-sequence studies using the GA/1 probe, the immunoreactive material shifted position daring invasion of target cell to areas of active secretion.     

Murine immune responses to recombinant Toxoplasma gondii antigens

Recombinant proteins of the RH strain of Toxoplasma gondii were produced by expression in Escherichia coli as glutathione S-transferase (GST) fusion proteins. Enzyme-linked immunosorbent assays were established using 2 of these fusion proteins termed H4/GST and H11/GST. The assays were able to detect antibodies in the sera of mice orally infected with either the cyst or oocyst stage of a pork isolate of T. gondii. In addition, the sera from mice infected with 1 of 4 different T. gondii isolates were investigated for their binding to these fusion proteins. Antibodies in the sera of all mice bound to H11/GST, but not all sera recognized H4/GST. Delayed-type hypersensitivity (DTH) responses to the fusion proteins were found when the mice were sensitized intradermally with H4/GST and H11/GST and challenged with the homologous fusion protein. However, no DTH response was recorded when mice were challenged with homologous fusion proteins after infection with T. gondii, or after immunization with a sonicate of the RH strain of the parasite. In addition, cellular responses were not stimulated against either of the fusion proteins in in vitro assays. These 2 fusion proteins were recognized by anti-T. gondii antibodies in experimental murine infections, and they are therefore potential candidates as antigens in assays for the diagnosis of human toxoplasmosis.     

Evidence for de novo sphingolipid biosynthesis in Toxoplasma gondii

Glycolipids are important components of cellular membranes involved in various biological functions. In this report, we describe the identification of the de novo synthesis of glycosphingolipids by Toxoplasma gondii tachyzoites. Parasite-specific glycolipids were identified by metabolic labelling of parasites with tritiated serine and galactose. These glycolipids were characterised as sphingolipids based on the labelling protocol and their insensitivity towards alkaline treatment. Synthesis of parasite glycosphingolipids were inhibited by threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol and L-cycloserine, two well-established inhibitors of de novo sphingolipid biosynthesis. The identified glycolipids were insensitive towards treatment with endoglycoceramidase II indicating that they might belong to globo-type glycosphingolipids. Taken together, we provide evidence for the first time that T. gondii is capable of synthesising glycosphingolipids de novo.     

Technique for the detection of Toxoplasma gondii antigens in mouse urine

A simple and rapid staphylococcal coagglutination test for the detection of Toxoplasma gondii antigens in mice urine is described. A suspension of protein-A containing Staphylococcus aureus coated with rabbit hyperimmune serum was used as reagent. The sensitivity of the antigen assay was found to be at least 118 ng of the antigen protein per ml. No coagglutination was observed when the reagent was challenged against antigenic solutions of other parasites. The suitability of the method for detecting antigens of T. gondii in urine samples was studied by experimental toxoplasma infection in mice. Before the staphylococcal test, the urine samples were double serially diluted in 0.1 M PBS. From the second day on all samples from infected mice were positive at 1/16 dilution. At this dilution, all samples from non infected mice were negative or did not produce coagglutination. This method might be used in the rapid etiological diagnosis also in human cases of acute toxoplasmosis.     

Quantitative immunolocalization of four immunodominant antigens of Toxoplasma gondii

Ultrastructural localization of four immunodominant antigens of Toxoplasma gondii was investigated quantitatively on thin sections and replicas by an immunogold technique using four monoclonal antibodies (Mab). On immunoblot Mab IV47, GII9, II38 and IE10 identified proteins of 28, 30, 45 and 66-70 kDa, respectively. Use of digital image analyzer and a semi-automatic procedure developed by us, the patterns of label distribution were compared in three cell structures: cell surface, submembrane area and rhoptries. On the whole cell surface, protein P28 and P30 were 2.5 and 4 times more abundant than P66-70 respectively. The protein P28 was essentially concentrated in the submembrane area with a labeling of 195.4 +/- 46.7 gold particles/microns 2 that follows a decreasing gradient from this area to the cell centre. In the rhoptries, all four antigens were detected, P45 and P66-70 being major with a labeling of 97.1 +/- 31.1 gold particles/microns 2 and 155.1 +/- 39.3 gold particles/microns 2 respectively. The results support the hypothesis that rhoptries are the essential site for antigen storage.     

Molecular weight analysis of soluble antigens from Toxoplasma gondii

Ultrasonicated Toxoplasma gondii (RH strain) tachyzoites were fractionated into a water-soluble and a deoxycholate-soluble fraction. Polyclonal immune mouse serum was prepared by challenging chronically-infected mice with viable RH strain tachyzoites. The parasite fractions were labelled with 125I, and the radio-labelled antigens were precipitated by the immune mouse serum or a monoclonal anti-Toxoplasma antibody (FMC 20), that reacts only in the indirect hemagglutination antibody test. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the immunoprecipitates showed that the water-soluble fraction contained 10 antigenic polypeptides, and the deoxycholate-soluble fraction contained seven antigenic peptides. The FMC 20 reacted against a 98,000-dalton antigen that was present in the water-soluble fraction only.     

Stage-specific expression of surface antigens by Toxoplasma gondii as a mechanism to facilitate parasite persistence

Toxoplasma persists in the face of a functional immune system. This success critically depends on the ability of parasites to activate a strong adaptive immune response during acute infection with tachyzoites that eliminates most of the parasites and to undergo stage conversion to bradyzoites that encyst and persist predominantly in the brain. A dramatic change in antigenic composition occurs during stage conversion, such that tachyzoites and bradyzoites express closely related but antigenically distinct sets of surface Ags belonging to the surface Ag 1 (SAG1)-related sequence (SRS) family. To test the contribution of this antigenic switch to parasite persistence, we engineered parasites to constitutively express the normally bradyzoite-specific SRS9 (SRS9(c)) mutants and tachyzoite-specific SAG1 (SAG1(c)) mutants. SRS9(c) but not wild-type parasites elicited a SRS9-specific immune response marked by IFN-gamma production, suggesting that stage-specificity of SRS Ags determines their immunogenicity in infection. The induction of a SRS9-specific immune response correlated with a continual decrease in the number of SRS9(c) cysts persisting in the brain. In contrast, SAG1(c) mutants produced reduced brain cyst loads early in chronic infection, but these substantially increased over time accompanying a hyperproduction of IFN-gamma, TNF-alpha, and IL-10, and severe encephalitis. We conclude that stage-specific expression of SRS Ags is among the key mechanisms by which optimal parasite persistency is established and maintained.     

Strain and stage specific variation in Toxoplasma gondii antigens

The antigenic profile of virulent (RH, ENT, Martin) and avirulent (RRA, DEG, ME49) Toxoplasma strains was compared directly by western blotting using a panel of immune mouse sera. Dominant antigens of approximate MR 30-33, 21 and 25 x 10(3) were common to tachyzoites of all strains, however, there were significant quantitative and qualitative differences in the antigen profiles, indicating a moderate degree of strain specific polymorphism in tachyzoite antigens. We found no specific association between antigenic variation and strain virulence. Comparison of tachyzoite and bradyzoite antigens from homologous strains (RRA, DEG, ME49) confirmed the existence of stage specific antigens and demonstrated a conserved antigen profile among bradyzoites.     

Use of recombinant antigens for detection of Toxoplasma gondii infection in swine

Five recombinant Toxoplasma gondii antigens, designated B427, C51, C55, V22, and MBP30 were assessed for their potential use in an enzyme-linked immunoassay (EIA) for detection of T. gondii infection in swine. The antigens were evaluated with sera from young pigs that had been fed 1-10,000 T. gondii oocysts of the VEG or GT-1 strains. Results were compared with an EIA using a native T. gondii antigen extract. All 5 recombinant antigens, as well as native antigen, detected antibody responses as soon as 3 wk after infection in pigs inoculated with 1 or 10 oocysts of the VEG strain. This antibody response persisted, at varying levels, for 14 wk when the experiment was terminated. All antigens also detected antibody responses in pigs 4 wk after inoculation with 10,000 oocysts of the GT-1 strain. The antibody response recognized by native antigen remained high through 51 wk after inoculation. However, there was considerable animal-to-animal variation in responses to the individual recombinant antigens. Only antigens C51 and MBP30 consistently detected a positive antibody response over the entire 51-wk course of the experiment. These results suggest that these antigens might be useful for the serological detection of T. gondii infection in pigs.     

Surface antigens of Toxoplasma gondii: variations on a theme

Toxoplasma gondii is an obligate intracellular protozoan parasite with an exceptionally broad host range. Recently, it has become apparent that the number of surface antigens (SAGs) it expresses may rival the number of genera it can infect. Most of these antigens belong to the developmentally regulated and distantly related SAG1 or SAG2 families. The genes encoding the surface antigens are distributed throughout the T. gondii genome, with remarkably little polymorphism observed at each locus. Results from a number of studies have suggested that the surface antigens play an important role in the biology of the parasite. For example, SAG3 null mutants generated by targeted disruption provide convincing evidence that this surface antigen, at least, functions during parasite attachment. Analyses of a SAG1 knockout in rodents, however, indicate that this surface antigen may play a crucial role in immune modulation or virulence attenuation. The current understanding of the SAG1 and SAG2 families will be discussed here.     

The major surface antigen, P30, of Toxoplasma gondii is anchored by a glycolipid

P30, the major surface antigen of the parasitic protozoan Toxoplasma gondii, can be specifically labeled with [3H]palmitic acid and with myo-[2-3H]inositol. The fatty acid label can be released by treatment of P30 with phosphatidylinositol-specific phospholipase C (PI-PLC). Such treatment exposes an immunological "cross-reacting determinant" first described on Trypanosoma brucei variant surface glycoprotein. PI-PLC cleavage of intact parasites metabolically labeled with [35S]methionine results in the release of intact P30 polypeptide in a form which migrates faster in polyacrylamide gel electrophoresis. These results argue that P30 is anchored by a glycolipid. Results from thin layer chromatography analysis of purified [3H] palmitate-labeled P30 treated with PI-PLC, together with susceptibility to mild alkali hydrolysis and to cleavage with phospholipase A2, suggest that the glycolipid anchor of T. gondii P30 includes a 1,2-diacylglycerol moiety.     

Cellular receptor for urokinase plasminogen activator. Carboxyl-terminal processing and membrane anchoring by glycosyl-phosphatidylinositol.

The cellular receptor for human urokinase-type plasminogen activator (u-PAR) is shown by several independent criteria to be a true member of a family of integral membrane proteins, anchored to the plasma membrane exclusively by a COOH-terminal glycosyl-phosphatidylinositol moiety. 1) Amino acid analysis of u-PAR after micropurification by affinity chromatography and N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]glycine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of 2-3 mol of ethanolamine/mol protein. 2) Membrane-bound u-PAR is efficiently released from the surface of human U937 cells by trace amounts of purified bacterial phosphatidylinositol-specific phospholipase C. This soluble form of u-PAR retains the binding specificity toward both u-PA and its amino-terminal fragment holding the receptor-binding domain. 3) Treatment of purified u-PAR with phosphatidylinositol-specific phospholipase C or mild alkali completely alters the hydrophobic properties of the receptor as judged by temperature-induced detergent-phase separation and charge-shift electrophoresis. 4) Biosynthetic labeling of u-PAR was obtained with [3H]ethanolamine and myo-[3H]inositol. 5) Finally, comparison of amino acid compositions derived from cDNA sequence and amino acid analysis shows that a polypeptide of medium hydrophobicity is excised from the COOH terminus of the nascent u-PAR. A similar proteolytic processing has been reported for other proteins that are linked to the plasma membrane by a glycosyl-phosphatidylinositol membrane anchor.     

Toxoplasma gondii: characterization and localization of antigens secreted from tachyzoites

Since we had previously demonstrated the protective role played by Toxoplasma excreted-secreted antigens, the aim of the present work was to produce monoclonal antibodies directed against these antigens in order to determine if their localization in the parasite is compatible with a mechanism of excretion or secretion. Western immunoblotting analysis revealed three monoclonal antibodies (TG17-179, TG17-43, and TG17-113) raised against excreted-secreted antigens of 28.5, 27, and 21 kDa, respectively. The TG17-179 which reacts with antigens isolated by Concanavalin A affinity chromatography is directed against a glycosylated 28.5-kDa component. Colloidal immunogold labeling showed the ultrastructural localization of the 21-, 27-, and 28.5-kDa antigens in the matrix of the dense granules of tachyzoites and associated with the microvilli network of the parasitophorous vacuole, after host cell invasion. These observations suggest the following mechanism of Toxoplasma secretion: secreted antigens are first stored in tachyzoite-dense granules and are then released inside the parasitophorous vacuole. Among the secretory molecules characterized here, the native 27-kDa antigen recognized by TG17-43 is a calcium-binding protein found to be intermixed with the 21- and 28.5-kDa antigens inside the dense granules and hence could play a role in the packaging of secretory products. In addition, the 21- and 28.5-kDa antigens were also located beneath the parasite plasma-lemma. This particular location could reflect a transient step characteristic of T. gondii secretion.     

Toxoplasma gondii: immunocytochemistry of four immunodominant antigens with monoclonal antibodies

Four monoclonal antibodies in which diagnostic usefulness has been observed, concerning congenital, acquired, and reactivated toxoplasmosis, were raised against Toxoplasma gondii tachyzo?tes in order to localize immunodominant antigens. On immunoblots, it appears that McAb IV47, McAB GII9, McAb II38, and McAb IE10 identify families of proteins with estimated molecular weights of 28-30 kDa, 30 kDa, 45-50 kDa, and 66-70 kDa, respectively. By immunogold preembedding techniques one can observe an homogeneous labeling of the outer pellicle of the tachyzo?tes with the McAb GII9 and IV47 and a light labeling with the McAb II38 and IE10. The three-dimensional observation of cell surface antigens is performed by applying a modified metal extraction replica method, i.e., A plasma polymerization method of glow discharge by Tanaka (1979). By immunogold preembedding techniques [with saponin permeabilization (0.1%)], and by immunogold postembedding techniques, a labeling of the rhoptries is observed with McAb GII9 and McAb IV47 but essentially all label is found with McAb II38 and IE10. With McAb GII9 a uniform labeling is observed on the cell surface. By immunoenzymatic techniques (peroxidase) a cell surface labeling is observed with the four McAb. Intracellular Toxoplasma, the outer pellicle, and the vesicles of the network (elaborated by Toxoplasma in parasitophorous vacuole) are also labeled with McAb IE10. These results indicate that McAb GII9 recognizes antigens of the antigen family (P 30) located on the cell surface and in the rhoptries. The antigen recognized by McAb IV47 is essentially located on and beneath the Toxoplasma cell surface membrane, and McAb II38 and IE10 identify preferentially rhoptry proteins.     

Development of an immunochromatographic strip for the rapid detection of Toxoplasma gondii circulating antigens

We developed a rapid immunochromatographic strip (ICS) procedure that can detect circulating antigens in the blood of animals during the acute stage of toxoplasmosis. The aim of this study was to evaluate this test using sera from field samples and from experimentally infected animals. The sensitivity and specificity of the ICS were compared with those of an enzyme-linked immunosorbent assay (ELISA). Both assays detected circulating antigens in the sera of animals experimentally infected with the Gansu Jingtai strain of Toxoplasma gondii, and the agreement between the two assays was 100%. In the infected animals, circulating antigens could be detected as early as the second day post-infection (PI) and in all animals by the fourth day. In the 381 field serum samples, the positive rates of the ICS and ELISA were 5.2% and 5.8%, respectively. In addition, there was no cross-reactivity of the antigens with Neospora caninum. The results presented here suggest that the ICS is a feasible, convenient, rapid and effective method to detect infection by T. gondii. This test could be a powerful supplement to the current diagnostic methods. Taken together, the results of this study encourage further research toward the production of commercial diagnostic tests for detecting T. gondii in animals.