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.     

Primary brain cell infection by Toxoplasma gondii reveals the extent and dynamics of parasite differentiation and its impact on neuron biology

Toxoplasma gondii is a eukaryotic parasite that forms latent cysts in the brain of immunocompetent individuals. The latent parasite infection of the immune-privileged central nervous system is linked to most complications. With no drug currently available to eliminate the latent cysts in the brain of infected hosts, the consequences of neurons'' long-term infection are unknown. It has long been known that T. gondii specifically differentiates into a latent form (bradyzoite) in neurons, but how the infected neuron responds to the infection remains to be elucidated. We have established a new in vitro model resulting in the production of mature bradyzoite cysts in brain cells. Using dual, host and parasite RNA-seq, we characterized the dynamics of differentiation of the parasite, revealing the involvement of key pathways in this process. Moreover, we identified how the infected brain cells responded to the parasite infection revealing the drastic changes that take place. We showed that neuronal-specific pathways are strongly affected, with synapse signalling being particularly affected, especially glutamatergic synapse signalling. The establishment of this new in vitro model allows investigating both the dynamics of parasite differentiation and the specific response of neurons to long-term infection by this parasite.     

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.     

Releasing latent Toxoplasma gondii cysts from host cells to the extracellular environment induces excystation

Toxoplasma gondii, an obligate intracellular protozoan parasite, infects a wide variety of mammals and birds. Although T. gondii infects the brain and muscles in its latent cyst form containing bradyzoite stage parasites during chronic infection, when a chronically infected host becomes immunodeficient or is preyed upon by a predator, the latent cyst undergoes excystation. However, it is not yet known how T. gondii recognises the triggers of excystation in the microenvironment surrounding the cyst. In this study, we incubated T. gondii cysts from host cells in several solutions containing a variety of ionic compositions. Excystation occurred in a solution with an ionic composition which mimicked that of the extracellular environment. However, excystation did not occur in a solution that mimicked the intracellular environment. We also found that the specific Na⁺/K⁺ ratio and the presence of Ca²⁺, mimicking the extracellular environment, are required to trigger excystation. To examine whether the stage conversion of bradyzoite to tachyzoite occurs prior to egress, we constructed a gene-modified T. gondii strain expressing a green fluorescent protein specifically in the tachyzoite stage. During the process of cyst reactivation of this strain, green fluorescence was detected prior to excystation. This suggests that stage conversion from bradyzoite to tachyzoite occurs prior to cyst disruption. These results indicate that T. gondii bradyzoites monitor the ionic composition of their surroundings to recognise their expulsion from host cells, to effectively time their excystation and stage conversion.     

Toxoplasma gondii: DNA vaccination with bradyzoite antigens induces protective immunity in mice against oral infection with parasite cysts

Infection of the host by Toxoplasma gondii leads to an acute systemic dissemination of tachyzoites, followed by a chronic phase, in which bradyzoites, enclosed in cysts, persist in the brain, the heart, and other tissues. Among putative vaccine candidates, the bradyzoite antigens BAG1 and MAG1 look promising since they are preferentially expressed during the chronic stage of the parasite. This work focused on studying the immunogenicity of bradyzoite antigens in a mouse model of chronic toxoplasmosis. A mixture of plasmids directing the cytoplasmic expression of MAG1 and BAG1 in mammalian cells was used to immunize mice. We show here that immunized mice developed, preferentially, specific anti-MAG1 and anti-BAG1 IgG2a subclass antibodies, indicating a shift towards a Th1-like response after DNA immunization. We then demonstrated that DNA immunization followed by challenge infection elicited effective protection in mice, suggesting that bradyzoite antigens should be considered in the design of vaccines against toxoplasmosis.     

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.     

Exogenous nitric oxide triggers Neospora caninum tachyzoite-to-bradyzoite stage conversion in murine epidermal keratinocyte cell cultures

Neospora caninum, like Toxoplasma gondii, undergoes stage conversion in chronically infected animals, and forms tissue cysts which contain the slowly proliferating bradyzoite stage. These tissue cysts are delineated by a cyst wall, protect the parasite from physiological and immunological reactions on part of the host, and bradyzoites remain viable within an infected host for many years. However, unlike T. gondii, N. caninum bradyzoites have been difficult to obtain using in vitro culture techniques, and current protocols, based on those developed for T. gondii, have been shown to be not very efficient in promoting tachyzoite-to-bradyzoite stage conversion. We report here an alternative in vitro culture method to obtain stage conversion of N. caninum from the proliferative to the cystic stage by using the Nc-Liverpool isolate, murine epidermal keratinocytes as host cells, and continuous treatment of infected cultures with 70 microM sodium nitroprusside for up to 8 days. This treatment significantly reduced parasite proliferation as assessed by Neospora-specific quantitative real-time PCR. The expression of bradyzoite markers was analysed by immunofluorescence following 4 and 8 days of in vitro culture using antibodies directed against bradyzoite antigen 1, the mAbCC2, and the lectin Dolichos biflorus agglutinin. Expression of the tachyzoite-specific immunodominant antigen NcSAG1 and the tachyzoite antigen NcMIC1 was also assessed. Transmission electron microscopy revealed that the majority of parasitophorous vacuoles were in the process of forming a distinct cyst wall through accumulation of granular material at the periphery of the vacuole, and parasites exhibited the typical features of bradyzoites. These findings demonstrate the usefulness of this culture technique as a promising way to study tachyzoite-to-bradyzoite stage conversion in N. caninum in vitro.     

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 transcription of bradyzoite genes in Toxoplasma gondii is controlled by autonomous promoter elements

Experimental evidence suggests that apicomplexan parasites possess bipartite promoters with basal and regulated cis -elements similar to other eukaryotes. Using a dual luciferase model adapted for recombinational cloning and use in Toxoplasma gondii , we show that genomic regions flanking 16 parasite genes, which encompass examples of constitutive and tachyzoite- and bradyzoite-specific genes, are able to reproduce the appropriate developmental stage expression in a transient luciferase assay. Mapping of cis -acting elements in several bradyzoite promoters led to the identification of short sequence spans that are involved in control of bradyzoite gene expression in multiple strains and under different bradyzoite induction conditions. Promoters that regulate the heat shock protein BAG1 and a novel bradyzoite-specific NTPase during bradyzoite development were fine mapped to a 6–8 bp resolution and these minimal cis -elements were capable of converting a constitutive promoter to one that is induced by bradyzoite conditions. Gel-shift experiments show that mapped cis -elements are bound by parasite protein factors with the appropriate functional sequence specificity. These studies are the first to identify the minimal sequence elements that are required and sufficient for bradyzoite gene expression and to show that bradyzoite promoters are maintained in a 'poised' chromatin state throughout the intermediate host life cycle in low passage strains. Together, these data demonstrate that conventional eukaryotic promoter mechanisms work with epigenetic processes to regulate developmental gene expression during tissue cyst formation.     

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.     

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 review: Competence, compromise, and concomitance-reaction of the host cell to Toxoplasma gondii infection and development

Toxoplasma gondii is an important zoonotic parasite with a worldwide distribution. It infects about one-third of the world's population, causing serious illness in immunosuppressed individuals, fetuses, and infants. Toxoplasma gondii biology within the host cell includes several important phases: (1) active invasion and establishment of a nonfusogenic parasitophorous vacuole in the host cell, (2) extensive modification of the parasitophorous vacuolar membrane for nutrient acquisition, (3) intracellular proliferation by endodyogeny, (4) egress and invasion of new host cells, and (5) stage conversion from tachyzoite to bradyzoite and establishment of chronic infection. During these processes, T. gondii regulates the host cell by modulating morphological, physiological, immunological, genetic, and cellular biological aspects of the host cell. Overall, the infection/development predispositions of T. gondii -host cell interactions overtakes the infection resistance aspects. Upon invasion and development, host cells are modulated to keep a delicate balance between facilitating and eliminating the infection.     

Cyclic nucleotide kinases and tachyzoite-bradyzoite transition in Toxoplasma gondii

The ability of Toxoplasma gondii to cycle between the tachyzoite and bradyzoite life stages in intermediate hosts is key to parasite survival and the pathogenesis of toxoplasmosis. Studies from a number of laboratories indicate that differentiation in T. gondii is a stress-induced phenomenon. The signalling pathways or molecular mechanisms that control formation of the latent bradyzoite stage are unknown and specific effectors of differentiation have not been identified. We engineered a reporter parasite to facilitate simultaneous comparison of differentiation and replication after various treatments. Chloramphenicol acetyltransferase (CAT), expressed constitutively from the alpha-tubulin promoter (TUB1), was used to quantitate parasite number. beta-galactosidase (beta-GAL), expressed from a bradyzoite specific promoter (BAG1), was used as a measure of bradyzoite gene expression. Sodium nitroprusside, a well-known inducer of bradyzoite differentiation, reduced reporter parasite replication and caused bradyzoite differentiation. Stress-induced differentiation in many other pathogens is regulated by cyclic nucleotide kinases. Specific inhibitors of the cAMP dependent protein kinase and apicomplexan cGMP dependent protein kinase inhibited replication and induced differentiation. The beta-GAL/CAT reporter parasite provides a method to quantify and compare agents that cause differentiation in T. gondii.     

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.     

A Hammondia-like parasite from the European fox (Vulpes vulpes) forms biologically viable tissue cysts in cell culture

Tissue cysts of parasites of the genus Hammondia are rarely described in naturally or experimentally infected intermediate hosts. However, ultrastructural examinations on tissue cyst stages of Hammondia sp. are needed, e.g. to compare these stages with those of Neospora caninum and other related parasites. We describe a cell culture system employed to examine the in vitro development of tissue cysts of a Hammondia sp.-like parasite (isolate FOX 2000/1) which uses the European fox as a definitive host. Cells of a diploid finite cell line from embryonal bovine heart (KH-R; CCLV, RIE 090) were infected by inoculation of sporozoites und cultivated for up to 3 months. Transmission electron microscopic examination of 17 day old cell culture material revealed the presence of cyst walls. Infected cell cultures cultivated for 2 months were used to feed a fox. Six to 13 days post infection the fox shed large numbers (n=1.2 x 10(7)) of Hammondia-sp. like oocysts which could not be distinguished from those used to infect the cell culture as determined by DNA sequencing of the internal transcribed spacer 1 and the D2/D3 domain of the large subunit ribosomal DNA. To find out the proportion of parasitophorous vacuoles that had developed into tissue cysts, the expression of bradyzoite markers was examined by probing infected cell cultures with mouse polyclonal antibodies against Toxoplasma gondii bradyzoite antigen 1 (anti-BAG1) and rat monoclonal antibodies against a cyst wall protein (mAbCC2). Nineteen and 90 days post infection all parasitophorous vacuoles in the cell cultures were positive with anti-BAG1 and mAbCC2. This shows that biologically viable (i.e. infectious) tissue cysts of a fox-derived Hammondia sp. isolate (FOX 2000/1) can be efficiently produced in this cell culture system. Since in vitro cystogenesis of dog-derived Hammondia heydorni has not been observed yet, in vitro cyst formation might be one trait to separate fox-derived Hammondia sp. from H. heydorni on a species level.