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.     

Comparative analysis of stress agents in a simplified in vitro system of Neospora caninum bradyzoite production

Neospora caninum is an apicomplexan parasite identified as a major cause of abortion in cattle and neurological disease in various animal species. It is closely related to Toxoplasma gondii, sharing the ability to persist indefinitely in latent stage within the host as a tissue cyst containing slow-dividing bradyzoites. In this study, we compared different stress methods to induce in vitro bradyzoite conversion, using MARC-145 cells infected with Nc-Liverpool isolate. The tachyzoite-to-bradyzoite conversion rate was monitored at days 3, 5, and 7 after stress in a double-immunofluorescence assay using a monoclonal antibody against the tachyzoite antigen SAG1 (alphaSAG1) and a rabbit serum directed to the intracytoplasmic bradyzoite antigen BAG1 (alphaBAG1). Seven days of treatment with 70 microM sodium nitroprusside offered the highest bradyzoite transformation rate and the best yield of total parasitophorous vacuoles observed. In the present work, we introduce an alternative, simplified, and more advantageous method for bradyzoite production of N. caninum, using a reliable cell culture system easy to handle and with promising capacity of parasite purification.     

Primary culture of skeletal muscle cells as a model for studies of Toxoplasma gondii cystogenesis

Toxoplasma gondii is a protozoan pathogen of birds and mammals, including humans. The infective stage, the bradyzoite, lives within cysts, which occur predominantly in cells of the central nervous system and skeletal and cardiac muscles, characterizing the chronic phase of toxoplasmosis. In the present study, we employed for the first time primary mouse culture of skeletal muscle cells (SkMC) infected with bradyzoites, as a cellular model for cystogenesis. The interconversion of bradyzoite and tachyzoite was analyzed by immunofluorescence using 2 stage-specific antibodies, i.e., anti-bradyzoite (anti-BAG1) and anti-tachyzoite (anti-SAG1). After 24 hr of interaction only bradyzoites were multiplying, as revealed by anti-BAG1 incubation; interconversion to tachyzoites was not observed. After 48 hr of infection, 2 types of vacuoles were seen, i.e., BAG1+ and SAG1+, indicating the presence of bradyzoites as well as their interconversion to tachyzoites. After 96 hr of infection, BAG1+ vacuoles presented a higher number of parasites when compared to 48 hr, indicating multiplication of bradyzoites without interconversion. Using ultrastructural analysis, bradyzoites were found to adhere to the cell membranes via both the apical and posterior regions or were associated with SkMC membrane expansions. During bradyzoite invasion of SkMC, migration of the rough endoplasmic reticulum (RER) profiles to the parasite invasion site was observed. Later, RER profiles were localized between the mitochondria and parasitophorous vacuole membrane (PVM) that contained the parasite. After 31 days of parasite-host cell infection, RER profiles and mitochondria were not observed in association with the cyst wall. Alterations of the PVM, including increased thickness and electrondensity gain on its inner membrane face, were observed 48 hr after infection. Cystogenesis was complete 96 hr after infection, resulting in the formation of the cyst wall, which displayed numerous membrane invaginations. In addition, an electron-dense granular region enriched with vesicles and tubules was present, as well as numerous intracystic bradyzoites. These results show that the in vitro T. gondii model and SkMC are potential tools for both the study of cystogenesis using molecular approaches and the drug screening action on tissue cysts and bradyzoites.     

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.     

Autofluorescence of Toxoplasma gondii and Neospora caninum cysts in vitro

Autofluorescence of Toxoplasma gondii and Neospora caninum was studied by fluorescence microscopy during their differentiation from tachyzoites to bradyzoites in vitro using Vero as host cells. Stage conversion into bradyzoites and cysts was confirmed by immunofluorescent microscopy and Western blot analysis using SAG1- and BAG1-specific antibody, respectively. From day 4 postinfection (PI), pale blue autofluorescence of the bradyzoites and tissue cysts was observed with UV light at 330-385 nm, which coincided with the onset of cyst development. This autofluorescence under UV light of bradyzoites and tissue cysts increased in intensity from days 8 to 10 PI. In contrast to the autofluorescence shown by bradyzoites and cysts, tachyzoites and parasitophorous vacuoles containing tachyzoites never autofluoresced at any time examined. Autofluorescence of the cystic stages was of sufficient intensity and duration to allow the detection of cysts and bradyzoites of T. gondii and N. caninum. In this study, we describe for the first time the autofluorescence properties of in vitro-induced bradyzoites and cysts of T. gondii and N. caninum.     

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.     

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.     

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.     

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.     

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.     

The expression and distribution of dense granule proteins in the enteric (Coccidian) forms of Toxoplasma gondii in the small intestine of the cat

The expression and distribution of dense granule proteins in the enteric (coccidian) forms of Toxoplasma gondii in the small intestine of the cat. Experimental Parasitology 91, 203-211. The expression and location of the dense granule proteins (GRA1-6 and NTPase) in the merozoite and during asexual and sexual development of Toxoplasma gondii in the small intestine of the cat (definitive host) was examined by immuno-light and electron microscopy. This was compared with that of tachyzoites and bradyzoites present in the intermediate host. It was found that the merozoite contained the characteristic apical organelles plus a few large dense granules. By immunocytochemistry, dense granules in merozoites were negative for GRA proteins 1 to 6 in contrast to both tachyzoites and bradyzoites in which dense granules were positive for all six proteins. The GRA proteins were associated with the parasitophorous vacuole (PV) during tachyzoite and bradyzoite development but were absent from the PV of the enteric stages. However, the merozoite dense granules were positive for NTPase, which was similar to the tachyzoite while this antigen was down regulated in the bradyzoite. The apparent release of the NTPases into the PV formed by merozoites was also similar to that described for the tachyzoite, possibly reflecting the relative metabolic activity of the various stages. This study shows that the majority of GRA proteins have a similar stage-specific expression, which is independent of NTPases expression. These observations are consistent with T. gondii having a different host parasite relationship in the enteric forms, which does not involve the GRA proteins 1-6.     

Toxoplasma gondii: Simple duplex RT-PCR assay for detecting SAG1 and BAG1 genes during stage conversion in immunosuppressed mice

Toxoplasmic encephalitis (TE) is caused by reactivation of dormant bradyzoites into rapidly dividing tachyzoites of the apicomplexan parasite Toxoplasma gondii in immune-compromised hosts. Diagnosis of this life-threatening disease is complicated, since it is difficult to distinguish between these two stages. It is, therefore, mainly based on a test positive for T. gondii antibodies, and specific clinical symptoms. We developed a duplex RT-PCR to detect the expression of bradyzoite (BAG1) and tachyzoite (SAG1) specific genes simultaneously during tachyzoite/bradyzoite stage conversion. The conversion reaction was observed in many organs of experimental mice, indicated by tachyzoites in the cerebrum, cerebellum, heart and lung, beginning in week 1 after the suppression period, and continuing until the end. Bradyzoites were also detected in nearly all organs throughout the study, suggesting that during the reactivation period, bradyzoites not only escape from cysts and reinvade neighboring cells as tachyzoites, but are also driven into developing new bradyzoites. The results of our study show that duplex RT-PCR is an easy, rapid, sensitive, and reproducible method, which is particularly valuable when numerous samples must be analyzed. This technique may usefully serve as an alternate tool for diagnosing TE in severely immunocompromised patients.     

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.     

A Cell Culture System for Study of the Development of Toxoplasma gondii Bradyzoites

ABSTRACT. Toxoplasma gondii is a ubiquitous apicomplexan parasite and a major opportunistic pathogen under AIDS-induced conditions, where it causes encephalitis when the bradyzoite (cyst) stage is reactivated. A bradyzoite-specific Mab, 74.1.8, reacting with a 28 kDa antigen, was used to study bradyzoite development in vitro by immuno-electron microscopy and immunofluorescence in human fibroblasts infected with ME49 strain T. gondii . Bradyzoites were detected in tissue culture within 3 days of infection. Free floating cyst-like structures were also identified. Western blotting demonstrated the expression of bradyzoite antigens in these free-floating cysts as well as in the monolayer. Bradyzoite development was increased by using media adjusted to pH 6.8 or 8.2. The addition of γ-interferon at day 3 of culture while decreasing the total number of cysts formed prevented tachyzoite overgrowth and enabled study of in vitro bradyzoites for up to 25 days. The addition of IL-6 increased the number of cysts released into the medium and increased the number of cysts formed at pH 7.2. Confirmation of bradyzoite development in vitro was provided by electron microscopy. It is possible that the induction of an acute phase response in the host cell may be important for bradyzoite differentiation. This system should allow further studies on the effect of various agents on the development of bradyzoites.     

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.