Use of molecular and ultrastructural markers to evaluate stage conversion of Toxoplasma gondii in both the intermediate and definitive host

Toxoplasma gondii has a complex life cycle involving definite (cat) and intermediate (all warm blooded animals) hosts. This gives rise to four infectious forms each of which has a distinctive biological role. Two (tachyzoite and merozoite) are involved in propagation within a host and two (bradyzoite and sporozoite) are involved in transmission to new hosts. The various forms can be identified by their structure, host parasite relationship and distinctive developmental processes. In the present in vivo study, the various stages have been evaluated by electron microscopy and immunocytochemistry using a panel of molecular markers relating to surface and cytoplasmic molecules, metabolic iso-enzymes and secreted proteins that can differentiate between tachyzoite, bradyzoite and coccidian development. Tachyzoites were characterised as being positive for surface antigen 1, enolase isoenzyme 2, lactic dehydrogenase isoenzyme 1 and negative for bradyzoite antigen 1. In contrast, bradyzoites were negative for SAG1 but positive for BAG1, ENO1 and LDH2. When stage conversion was followed in brain lesion at 10 and 15 days post-infection, tachyzoites were predominant but a number of single intermediate organisms displaying tachyzoite and certain bradyzoite markers were observed. At later time points, small groups of organisms displaying only bradyzoite markers were also present. A number (9) of dense granule proteins (GRA1-8, NTPase) have also been identified in both tachyzoites and bradyzoites but there were differences in their location during parasite development. All the dense granule proteins extensively label the parasitophorous vacuole during tachyzoite development. In contrast the tissue cyst wall displays variable staining for the dense granule proteins, which also expresses an additional unique cyst wall protein. The molecular differences could be identified at the single cell stage consistent with conversion occurring at the time of entry into a new cell. These molecular differences were reflected in the structural differences in the parasitophorous vacuoles observed by electron microscopy. Stage conversion to enteric (coccidian) development was limited to the enterocytes of the cat small intestine. Although no specific markers were available, this form of development can be identified by the absence of specific tachyzoite (SAG1) and bradyzoite (BAG1) markers although the isoenzymes ENO2 and LHD1 were expressed. There was also a significant difference in the expression of the dense granule proteins. The coccidian stages and merozoites only expressed two (GRA7 and NTPase) of the nine dense granule proteins and this was reflected in significant differences in the structure of the parasitophorous vacuole. The coccidian stages also undergo conversion from asexual to sexual development. The mechanism controlling this process is unknown but does not involve any change in the host cell type or parasitophorous vacuole and may be pre-programmed, since the number of asexual cycles was self-limiting. In conclusion, it was possible using a combination of molecular markers to identify tachyzoite, bradyzoite and coccidian development in tissue sections.     

Identification of Neospora caninum proteins regulated during the differentiation process from tachyzoite to bradyzoite stage by DIGE

Identification of differentially expressed proteins during Neospora caninum tachyzoite–bradyzoite conversion processes may lead to a better knowledge of the pathogenic mechanisms developed by this important parasite of cattle. In the present work, a differential expression proteomic study of tachyzoite and bradyzoite stages was accomplished for the first time by applying DIGE technology coupled with MS analysis. Up to 72 differentially expressed spots were visualized (1.5‐fold in relative abundance, p<0.05, t‐test). A total of 53 spots were more abundant in bradyzoites and 19 spots in tachyzoites. MS analysis identified 26 proteins; 20 of them overexpressed in the bradyzoite stage and 6 in the tachyzoite stage. Among the novel proteins, enolase and glyceraldehyde‐3‐phosphate dehydrogenase (involved in glycolysis), HSP70 and HSP90 (related to stress response) as well as the dense granule protein GRA9, which showed higher abundance in the bradyzoite stage, might be highlighted. On the other hand, isocitrate dehydrogenase 2, involved in the Krebs cycle, was found to be more abundant in tachyzoites extract. Biological functions from most novel proteins were correlated with previously reported processes during the differentiation process in Toxoplasma gondii. Thus, DIGE technology arises as a suitable tool to study mechanisms involved in the N. caninum tachyzoite to bradyzoite conversion.     

The differential expression of multiple isoenzyme forms during stage conversion of Toxoplasma gondii: an adaptive developmental strategy.

The apicomplexan parasite Toxoplasma gondii has the ability to switch between a rapidly replicating tachyzoite and a slowly dividing encysted bradyzoite within its intermediate hosts such as humans or other warm-blooded vertebrates. It is likely that in vivo, the tachyzoites differentiate into encysted bradyzoites in response to the immune system attack during disease progression. As part of a developmental strategy and, in order to survive within infected hosts, T. gondii tachyzoites undergo profound metabolic and morphological changes by differentiating into encysted bradyzoites. Bradyzoites are characterised by their resistance to both the immune system and chemotherapy. The stimulus that triggers Toxoplasma encystation and the molecular mechanisms triggering the switch from tachyzoite to bradyzoite remain unknown. It is very important to elucidate these mechanisms since bradyzoites within tissue cysts are not only the source of infection transmitted from domestic animals to humans, but can also be converted into tachyzoites that are the cause of fatal toxoplasmic encephalitis in acquired immunodeficiency syndrome patients. In this review, I focus on recent efforts towards the characterisation of genes that encode several stage-specific isoenzymes. The picture emerging from these studies is that stage-specific expression of isoenyzmes having different biochemical properties accompanies the interconversion of tachyzoite into bradyzoite, and vice versa. It can be hypothesised that the difference found between these enzymatic activities may be instrumental in maintaining some major parasitic metabolisms such as glycolysis in pace with the stage-specific requirements of carbohydrate or polysaccharide biosynthesis.     

Host cell binding of GRA10, a novel, constitutively secreted dense granular protein from Toxoplasma gondii

Monoclonal antibodies (mAbs) against Toxoplasma gondii, Tg378 and Tg556 clones, are specifically observed to localize to the dense granules of tachyzoites by immunofluorescence microscopy. mAb Tg556 is directed against GRA3, a previously described 30kDa dense granular protein. mAb Tg378 is directed against a novel 36kDa dense granular protein, which we refer to as GRA10. These are major proteins in the excretory/secretory proteins from T. gondii before the parasite's entry into host cells, and they are released into the parasitophorous vacuole (PV) during or shortly after invasion to be associated with the PV membrane. GRA10 binds to the membrane of the host cells regardless of its anchorage-dependence or -independence. The cDNA sequence encoding GRA10 was determined by screening a T. gondii cDNA expression library with mAb Tg378. The deduced amino acid sequence of GRA10 consists of a polypeptide of 364 amino acids, and it has no significant homology to any other known proteins. The sequence contains amino terminal signal peptides and two potential transmembrane domains in the middle of sequence that are not near the carboxy terminus. GRA10 has a RGD motif between the two potential transmembrane domains.     

Genomic data reveal Toxoplasma gondii differentiation mutants are also impaired with respect to switching into a novel extracellular tachyzoite state

Toxoplasma gondii pathogenesis includes the invasion of host cells by extracellular parasites, replication of intracellular tachyzoites, and differentiation to a latent bradyzoite stage. We present the analysis of seven novel T. gondii insertional mutants that do not undergo normal differentiation to bradyzoites. Microarray quantification of the variation in genome-wide RNA levels for each parasite line and times after induction allowed us to describe states in the normal differentiation process, to analyze mutant lines in the context of these states, and to identify genes that may have roles in initiating the transition from tachyzoite to bradyzoite. Gene expression patterns in wild-type parasites undergoing differentiation suggest a novel extracellular state within the tachyzoite stage. All mutant lines exhibit aberrant regulation of bradyzoite gene expression and notably some of the mutant lines appear to exhibit high proportions of the intracellular tachyzoite state regardless of whether they are intracellular or extracellular. In addition to the genes identified by the insertional mutagenesis screen, mixture model analysis allowed us to identify a small number of genes, in mutants, for which expression patterns could not be accounted for using the three parasite states--genes that may play a mechanistic role in switching from the tachyzoite to bradyzoite stage.     

Real-Time RT-PCR on SAG1 and BAG1 Gene Expression during Stage Conversion in Immunosuppressed Mice Infected with Toxoplasma gondii Tehran Strain

Toxoplasmic encephalitis is caused by reactivation of bradyzoites to rapidly dividing tachyzoites of the apicomplexan parasite Toxoplasma gondii in immunocompromised hosts. Diagnosis of this life-threatening disease is problematic, because it is difficult to discriminate between these 2 stages. Toxoplasma PCR assays using gDNA as a template have been unable to discriminate between an increase or decrease in SAG1 and BAG1 expression between the active tachyzoite stage and the latent bradyzoite stage. In the present study, real-time RT-PCR assay was used to detect the expression of bradyzoite (BAG1)- and tachyzoite-specific genes (SAG1) during bradyzoite/tachyzoite stage conversion in mice infected with T. gondii Tehran strain after dexamethasone sodium phosphate (DXM) administration. The conversion reaction was observed in the lungs and brain tissues of experimental mice, indicated by SAG1 expression at day 6 after DXM administration, and continued until day 14. Bradyzoites were also detected in both organs throughout the study; however, it decreased at day 14 significantly. It is suggested that during the reactivation period, bradyzoites not only escape from the cysts and reinvade neighboring cells as tachyzoites, but also converted to new bradyzoites. In summary, the real-time RT-PCR assay provided a reliable, fast, and quantitative way of detecting T. gondii reactivation in an animal model. Thus, this method may be useful for diagnosing stage conversion in clinical specimens of immunocompromised patients (HIV or transplant patients) for early identification of tachyzoite-bradyzoite stage conversion.     

Silencing of GRA10 protein expression inhibits Toxoplasma gondii intracellular growth and development

Toxoplasma gondii dense granule proteins (GRAs) are secreted abundantly in both the tachyzoite and bradyzoite stages of the parasite and are known to localize to various compartments of the parasitophorous vacuole (PV) that interfaces with the host cell milieu. Thus, GRAs may play significant roles in the biogenesis of the PV that is important for survival of intracellular T. gondii. GRA10 is a dense granule protein whose role in T. gondii has not yet been characterized. Therefore, in this study, we endeavored to determine the role of GRA10 in the growth and survival of intracellular T. gondii by using phosphorodiamidate morpholino oligomers (PPMOs) antisense knockdown approach to disrupt the translation of GRA10 mRNA in the parasites. We expressed and purified a truncated recombinant GRA10 protein to generate anti-GRA10 polyclonal antibodies that we used to characterize GRA10 in T. gondii. We found that GRA10 is a soluble, dense granule-associated protein that is secreted into the parasite cytosol and the parasitophorous vacuole milieu. Using in vitro cultures, we found that knockdown of GRA10 results in severe inhibition of T. gondii growth in human fibroblasts and in ovine monocytic cells. Together, our findings define GRA10 as a dense granule protein that plays a significant role in the growth and propagation of intracellular T. gondii in human fibroblasts and in ovine monocytic cells.     

Toxoplasma gondii in primary rat CNS cells: differential contribution of neurons, astrocytes, and microglial cells for the intracerebral development and stage differentiation.

The central nervous system (CNS) of the intermediate host plays a central role in the lifelong persistence of Toxoplasma gondii as well as in the pathogenesis of congenital toxoplasmosis and reactivated infection in immunocompromised patients. In order to analyze the parasite-host interaction within the CNS, the host cell invasion, the intracellular replication, and the stage conversion from tachyzoites to bradyzoites was investigated in mixed cultures of dissociated CNS cells from cortices of Wistar rat embryos. Two days post infection (p.i.) with T. gondii tachyzoites, intracellular parasites were detected within neurons, astrocytes, and microglial cells as assessed by double immunofluorescence and confocal microscopy. Quantitative analyses revealed that approximately 10% of neurons and astrocytes were infected with T. gondii, while 30% of the microglial cells harbored intracellular parasites. However, the replication of T. gondii within microglial cells was considerably diminished, since 93% of the parasitophorous vacuoles (PV) contained only one to two parasites which often appeared degenerated. This toxoplasmacidal activity was not abrogated after treatment with NO synthase inhibitors or neutralization of IFN-gamma production. In contrast, 30% of the PV in neurons and astrocytes harbored clearly proliferating parasites with at least four to eight parasites per vacuole. Four days p.i. with tachyzoites of T. gondii, bradyzoites were detected within neurons, astrocytes, and microglial cells of untreated cell cultures. However, the majority of bradyzoite-containing vacuoles were located in neurons. Spontaneous differentiation to the bradyzoite stage was not inhibited after addition of NO synthase inhibitors or neutralization of IFN-gamma. In conclusion, our results indicate that intracerebral replication of T. gondii as well as spontaneous conversion from the tachyzoite to the bradyzoite stage is sustained predominantly by neurons and astrocytes, whereas microglial cells may effectively inhibit parasitic growth within the CNS.     

Developmental differentiation between tachyzoites and bradyzoites of Toxoplasma gondii

An important event in the pathogenesis of toxoplasmosis is the interconversion between the bradyzoite and the tachyzoite stage of Toxoplasma gondii within the intermediate host. The factors that influence either cyst formation (bradyzoites) or reactivation (tachyzoites) are unknown. Uwe Gross, Wolfgang Bohne, Martine Soête and Jean Fran?ois Dubremetz here describe current knowledge about the mechanisms that might lead to the induction of stage differentiation of this protozoan parasite.     

Neospora caninum: identification of 19-, 38-, and 40-kDa surface antigens and a 33-kDa dense granule antigen using monoclonal antibodies.

Neospora caninum, a coccidian parasite closely related to Toxoplasma gondii, can infect a broad host range and is regarded as an important cause of bovine abortion worldwide. In the present study, four antigens of N. caninum were partially characterized using monoclonal antibodies. Immunofluorescence of viable tachyzoites as well as the immunoprecipitation of antigens extracted from tachyzoites previously labeled by surface biotinylation revealed that three of these antigens with apparent molecular weights of 40, 38, and 19 kDa are located in the outer surface membrane of this parasite stage. Further evidence for the surface localization of the 38-kDa antigen was obtained by immunoelectron microscopy. In addition to the surface molecules, an antigen located in dense granules and in the tubular network of the parasitophorous vacuole was detected by another monoclonal antibody. When tachyzoite antigens separated under nonreducing conditions were probed on Western blots, this antibody reacted mainly with a 33-kDa antigen. Immunohistochemical analysis of infected tissue sections indicated that the 33-kDa dense granule antigen is present in both tachyzoites and bradyzoites, while the 38-kDa surface antigen from tachyzoites seems to be absent in bradyzoites.     

Dynamics of Toxoplasma gondii differentiation

Parasite differentiation is commonly associated with transitions between complex life cycle stages and with long-term persistence in the host, and it is therefore critical for pathogenesis. In the protozoan parasite Toxoplasma gondii, interconversion between rapidly growing tachyzoites and latent encysted bradyzoites is accompanied by numerous morphological and metabolic adaptations. In order to explore early cell biological events associated with this differentiation process, we have exploited fluorescent reporter proteins targeted to various subcellular locations. Combining these markers with efficient in vitro differentiation and time-lapse video microscopy provides a dynamic view of bradyzoite development in living cultures, demonstrating subcellular reorganization, maintenance of the mitochondrion, and missegregation of the apicoplast. Bradyzoites divide asynchronously, using both endodyogeny and endopolygeny, and are highly motile both within and between host cells. Cysts are able to proliferate without passing through an intermediate tachyzoite stage, via both the migration of free bradyzoites and the fission of bradyzoite cysts, suggesting a mechanism for dissemination during chronic infection.     

Transmembrane insertion of the Toxoplasma gondii GRA5 protein occurs after soluble secretion into the host cell

The intracellular parasite Toxoplasma gondii resides within a specialized compartment, the parasitophorous vacuole (PV), that resists fusion with host cell endocytic and lysosomal compartments. The PV is extensively modified by secretion of parasite proteins, including the dense granule protein GRA5 that is specifically targeted to the delimiting membrane of the PV (PVM). We show here that GRA5 is present both in a soluble form and in hydrophobic aggregates. GRA5 is secreted as a soluble form into the PV after which it becomes stably associated with the PVM. Topological studies demonstrated that GRA5 was inserted into the PVM as a transmembrane protein with its N-terminal domain extending into the cytoplasm and its C terminus in the vacuole lumen. Deletion of 8 of the 18 hydrophobic amino acids of the single predicted transmembrane domain resulted in the failure of GRA5 to associate with the PVM; yet it remained correctly packaged in the dense granules and was secreted as a soluble protein into the PV. Collectively, these studies demonstrate that the secretory pathway in Toxoplasma is unusual in two regards; it allows soluble export of proteins containing typical transmembrane domains and provides a mechanism for their insertion into a host cell membrane after secretion from the parasite.     

An unexpected response to vaccination with a purified major membrane tachyzoite antigen (P30) of Toxoplasma gondii

A purified Toxoplasma gondii tachyzoite membrane protein (P30) and a monoclonal antibody directed against this antigen were used to immunize mice. The P30 protein has an apparent m.w. of 30,000 and is the major radioiodinated tachyzoite membrane antigen identified by human and mouse antitoxoplasma antisera. Polyclonal and monoclonal antibody to the P30 antigen are parasiticidal in the presence of human serum. A series of mice were immunized with affinity column-purified P30 protein. This produced a dose-dependent, antigen-specific IgG and IgM response. The mice were challenged with the less virulent C strain tachyzoite. Immunized mice showed a statistically significant increase in mortality over nonimmunized control mice. In addition, vaccinated mice had an increased number of intracerebral tissue cysts when compared with the control group. Similar results were obtained with passive transfer immunization by using monoclonal antibody directed against the P30 antigen. Immunofluorescence assay of brain tissue cyst bradyzoites revealed a total absence of P30 antigen. Bradyzoites were also deficient in another major tachyzoite antigen of approximate m.w. 22,000 (P22). Mouse antibradyzoite serum absorbed with tachyzoites recognized bradyzoites but failed to identify tachyzoites. This suggests that there are stage-specific bradyzoite antigens of Toxoplasma gondii.     

Proteomic Analysis of Toxoplasma gondii KI-1 Tachyzoites

We studied on the proteomic characteristics of Toxoplasma gondii KI-1 tachyzoites which were originally isolated from a Korean patient, and compared with those of the well-known virulent RH strain using 2-dimensional electrophoresis (2-DE), mass spectrometry, and quantitative real-time PCR. Two-dimensional separation of the total proteins isolated from KI-1 tachyzoites revealed up to 150 spots, of which 121 were consistent with those of RH tachyzoites. Of the remaining 29 spots, 14 showed greater than 5-fold difference in density between the KI-1 and RH tachyzoites at a pH of 5.0-8.0. Among the 14 spots, 5 from the KI-1 isolate and 7 from the RH strain were identified using MALDI-TOF mass spectrometry and database searches. The spots from the KI-1 tachyzoites were dense granule proteins (GRA 2, 3, 6, and 7), hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGRPTase), and uracil phosphoribosyltransferase (UPRTase). The spots from the RH strain were surface antigen 1 (SAG 1), L-lactate dehydrogenase (LDH), actin, chorismate synthase, peroximal catalase, hexokinase, bifunctional dihydrofolate reductase-thymidylate synthase (DHTR-TS), and nucleoside-triphosphatases (NTPases). Quantitative real-time PCR supported our mass spectrometric results by showing the elevated expression of the genes encoding GRA 2, 3, and 6 and UPRTase in the KI-1 tachyzoites and those encoding GRA 7, SAG 1, NTPase, and chorismate synthase in the RH tachyzoites. These observations demonstrate that the protein compositions of KI-1 and RH tachyzoites are similar but differential protein expression is involved in virulence.     

Identification and characterization of differentiation mutants in the protozoan parasite Toxoplasma gondii

Two forms of the protozoan parasite Toxoplasma gondii are associated with intermediate hosts such as humans: rapidly growing tachyzoites are responsible for acute illness, whereas slowly dividing encysted bradyzoites can remain latent within the tissues for the life of the host. In order to identify genetic factors associated with parasite differentiation, we have used a strong bradyzoite-specific promoter (identified by promoter trapping) to drive the expression of T. gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) in stable transgenic parasites, providing a stage-specific positive/negative selectable marker. Insertional mutagenesis has been carried out on this parental line, followed by bradyzoite induction in vitro and selection in 6-thioxanthine to identify misregulation mutants. Two different mutants fail to induce the HXGPRT gene efficiently during bradyzoite differentiation. These mutants are also defective in other aspects of differentiation: they replicate well under bradyzoite growth conditions, lysing the host cell monolayer as effectively as tachyzoites. Expression of the major bradyzoite antigen BAG1 is reduced, and staining with Dolichos biflorus lectin shows reduced cyst wall formation. Microarray hybridizations show that these mutants behave more like tachyzoites at a global level, even under bradyzoite differentiation conditions.