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.     

Disruption of the Toxoplasma gondii bradyzoite-specific gene BAG1 decreases in vivo cyst formation

The bradyzoite stage of the Apicomplexan protozoan parasite Toxoplasma gondii plays a critical role in maintenance of latent infection. We reported previously the cloning of a bradyzoite-specific gene BAG1/hsp30 (previously referred to as BAG5) encoding a cytoplasmic antigen related to small heat shock proteins. We have now disrupted BAG1 in the T. gondii PLK strain by homologous recombination. H7, a cloned null mutant, and Y8, a control positive for both cat and BAG1, were chosen for further characterization. Immunofluorescence and Western blot analysis of bradyzoites with BAG1 antisera demonstrated expression of BAG1 in the Y8 and the PLK strain but no expression in H7. All three strains expressed a 116 kDa bradyzoite cyst wall antigen, a 29 kDa matrix antigen and the 65 kDa matrix reactive antigen MAG1. Mice inoculated with H7 parasites formed significantly fewer cysts than those inoculated with the Y8 and the PLK strains. H7 parasites were complemented with BAG1 using phleomycin selection. Cyst formation in vivo for the BAG1-complemented H7 parasites was similar to wild-type parasites. We therefore conclude that BAG1 is not essential for cyst formation, but facilitates formation of cysts in vivo.     

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.     

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.     

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.     

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.     

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.     

Neospora caninum in vitro: evidence that the destiny of a parasitophorous vacuole depends on the phenotype of the progenitor zoite

We previously reported that Neospora caninum can be induced to express BAGI, a bradyzoite antigen, within 3 days of culture under stress conditions. The main goals of the present experiment were to increase the expression of BAGI in vitro (in part by extending cultures for 9 days), to observe parasitophorous vacuoles at various points of stage differentiation, and to test the ability of organisms produced in vitro to function like mature bradyzoites. Expression of BAG1 and of a tachyzoite antigen (NcSAGI) was monitored using a double-label immunofluorescence assay. For the purpose of this study, organisms expressing NcSAG1 were designated as tachyzoites, those expressing BAG1 were designated as bradyzoites, and those expressing both antigens were designated as intermediate zoites. The greatest percentage of intermediate zoites and bradyzoites (14%) occurred in bovine monocytes maintained for 9 days. These bradyzoites did not appear to be functionally mature; they did not induce patent infections in dogs. in contrast to bradyzoites that were produced in chronically infected mice. In vitro, large parasitophorous vacuoles contained either a pure population of tachyzoites or a mixture of tachyzoites and intermediate zoites, which is indicative of asynchronous stage conversion of organisms within a vacuole. Bradyzoites were first observed within small vacuoles on day 6. and bradyzoites never shared vacuoles with tachyzoites. This finding suggests that vacuoles containing bradyzoites may develop only if the cell is invaded by a zoite that has already begun bradyzoite differentiation. An alternative possibility is that cysts may develop if the establishing tachyzoite undergoes bradyzoite differentiation before multiplying. Cysts do not appear to arise from transformation of tachyzoites within large parasitophorous vacuoles.     

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.     

Microplate assay for screening Toxoplasma gondii bradyzoite differentiation with DUAL luciferase assay

Toxoplasma gondii can differentiate into tachyzoites or bradyzoites. To accelerate the investigation of bradyzoite differentiation mechanisms, we constructed a reporter parasite, PLK/DLUC_1C9, for a high-throughput assay. PLK/DLUC_1C9 expressed firefly luciferase under the bradyzoite-specific BAG1 promoter. Firefly luciferase activity was detected with a minimum of 10² parasites induced by pH 8.1. To normalize bradyzoite differentiation, PLK/DLUC_1C9 expressed Renilla luciferase under the parasite’s α-tubulin promoter. Renilla luciferase activity was detected with at least 10² parasites. By using PLK/DLUC_1C9 with this 96-well format screening system, we found that the protein kinase inhibitor analogs, bumped kinase inhibitors 1NM-PP1, 3MB-PP1, and 3BrB-PP1, had bradyzoite-inducing effects.     

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.     

Toxoplasma gondii: chimeric Dr fimbriae as a recombinant vaccine against toxoplasmosis

Toxoplasma gondii is responsible for fetopathy in farm animals and humans and severe disease in immunocompromised individuals (i.e. AIDS patients). Effective vaccines, inducing protective and long-lasting immunity to this global parasite, are still desired. In the work, we evaluated the immunogenic and immunoprotective activity of Escherichia coli chimeric Dr fimbriae bearing selected antigenic epitopes of three T. gondii antigens (SAG1, GRA1 and MAG1), in comparison with conventional recombinant antigens obtained in E. coli expression system. Our data demonstrate a very high protective efficacy of recombinant antigens supplemented with Freund's adjuvants, whereas chimeric Dr fimbriae as a vaccine proved non-protective. The recombinant antigen vaccine induced a strong specific antibody response and prevented the brain cysts formation by 89%. The results are promising and should be confirmed in further study on farm animals by use of less aggressive than Freund's adjuvant preparations.     

Tachyzoite-specific isoform of Toxoplasma gondii lactate dehydrogenase is the target antigen of a murine CD4(+) T-cell clone

In two-dimensionally separated Toxoplasma gondii lysate, mouse Th1 clone 3Tx15 detects two proteins of apparent molecular weight 40000 and pI of 5.8 and 5.9. Microsequencing of peptide fragments from tryptic digestion of one of these proteins yielded partial sequences of T. gondii lactate dehydrogenase (LDH)1. As shown by Western blot, toxoplasmic LDH co-migrates in two-dimensional gel electrophoresis with both T-cell antigenic proteins. With synthetic peptides spanning the complete primary structure of T. gondii LDH1, the T-cell epitope was mapped to a nine amino acid partial sequence which exhibits a motif for binding to I-E(k), the class II restriction element of antigen recognition by clone 3Tx15. From the two known isoforms of T. gondii LDH, clone 3Tx15 specifically recognises tachyzoite LDH1, but not bradyzoite LDH2, as shown with the corresponding epitope peptides and recombinant proteins. Antigen-presenting cells infected with live bradyzoites stimulate 3Tx15 T cells, while killed bradyzoites provide no antigenic stimulus. This finding implies that a transformation into the tachyzoite stage occurs in cells challenged with bradyzoites. Although LDH1 represents one major constituent of the tachyzoite proteome, the protein does not seem to be immunogenic in T. gondii infection of mice. This is evident from the lack of serum anti-LDH immunoreactivity and the failure of adoptively transferred 3Tx15 T cells to protect against lethal challenge. In conclusion, a T-cell-stimulatory Toxoplasma antigen is identified by means of a novel, high-resolution T-cell blot technique, the clones antigenic fine specificity allowing detection of parasite-stage conversion.     

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.     

Toxoplasma gondii: enzyme-linked immunosorbent assay using different fragments of recombinant microneme protein 1 (MIC1) for detection of immunoglobulin G antibodies

Three different fragments of microneme 1 protein termed, r-MIC1ex2, r-MIC1ex34 and r-MIC1 of Toxoplasma gondii, were expressed in Escherichia coli as fusion proteins containing six histidyl residues at N- and C-terminal. After purification by metal affinity chromatography, these recombinant proteins were tested for their usefulness as antigens in an enzyme-linked immunosorbent assay for the detection of immunoglobulin G. Ninety-eight sera from patients with different stages of invasion and 24 sera from seronegative patients were examined. There was no significant difference observed in the antigenicity for human serum samples from patients with acute toxoplasmosis between three recombinant types of MIC1 antigen (96.1% for r-MIC1ex2 antigen and 100% for both r-MIC1ex34 and r-MIC1 proteins). Sera from chronic infections (with low titers of IgG antibody) showed significant lower sensitivity, especially for r-MIC1ex34 and r-MIC1 antigens (75%, 52.7% and 36.1% for r-MIC1ex2, r-MIC1ex34 and r-MIC1, respectively). These results indicate that the strongest antigenic region of the MIC1 is encoding by the second exon of mic1 gene. When r-MIC1ex2 (N-terminal fragment of protein) was combined with MAG1 (matrix antigen) and MIC3 (microneme 3 protein), the sensitivity increased to 88.9%. This result was comparable to an ELISA using a Toxoplasma lysate antigen (TLA) and two combinations of recombinant antigens: M1 (GRA1+GRA7+SAG1) and M2 (P35+SAG2+GRA6) with the sensitivity for serum samples tested 94.4%, 88.9% and 94.4%, respectively.     

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.     

Evaluation of Recombinant SAG1, SAG2, and SAG3 Antigens for Serodiagnosis of Toxoplasmosis

Serologic tests are widely accepted for diagnosing Toxoplasma gondii but purification and standardization of antigen needs to be improved. Recently, surface tachyzoite and bradyzoite antigens have become more attractive for this purpose. In this study, diagnostic usefulness of 3 recombinant antigens (SAG1, SAG2, and SAG3) were evaluated, and their efficacy was compared with the available commercial ELISA. The recombinant plasmids were transformed to JM109 strain of Escherichia coli, and the recombinants were expressed and purified. Recombinant SAG1, SAG2, and SAG3 antigens were evaluated using different groups of sera in an ELISA system, and the results were compared to those of a commercial IgG and IgM ELISA kit. The sensitivity and specificity of recombinant surface antigens for detection of anti-Toxoplasma IgG in comparison with commercially available ELISA were as follows: SAG1 (93.6% and 92.9%), SAG2 (100.0% and 89.4%), and SAG3 (95.4% and 91.2%), respectively. A high degree of agreement (96.9%) was observed between recombinant SAG2 and commercial ELISA in terms of detecting IgG anti-Toxoplasma antibodies. P22 had the best performance in detecting anti-Toxoplasma IgM in comparison with the other 2 recombinant antigens. Recombinant SAG1, SAG2, and SAG3 could all be used for diagnosis of IgG-specific antibodies against T. gondii.