Bradyzoite Stage Research Articles

Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of non-overlapping gene families to attach, invade, and replicate within feline enterocytes.

BackgroundThe apicomplexan parasite Toxoplasma gondii is cosmopolitan in nature, largely as a result of its highly flexible life cycle. Felids are its only definitive hosts and a wide range of mammals and birds serve as intermediate hosts. The latent bradyzoite stage is orally infectious in all warm-blooded vertebrates and establishes chronic, transmissible infections. When bradyzoites are ingested by felids, they transform into merozoites in enterocytes and expand asexually as part of their coccidian life cycle. In all other intermediate hosts, however, bradyzoites differentiate exclusively to tachyzoites, and disseminate extraintestinally to many cell types. Both merozoites and tachyzoites undergo rapid asexual population expansion, yet possess different effector fates with respect to the cells and tissues they develop in and the subsequent stages they differentiate into.ResultsTo determine whether merozoites utilize distinct suites of genes to attach, invade, and replicate within feline enterocytes, we performed comparative transcriptional profiling on purified tachyzoites and merozoites. We used high-throughput RNA-Seq to compare the merozoite and tachyzoite transcriptomes. 8323 genes were annotated with sequence reads across the two asexually replicating stages of the parasite life cycle. Metabolism was similar between the two replicating stages. However, significant stage-specific expression differences were measured, with 312 transcripts exclusive to merozoites versus 453 exclusive to tachyzoites. Genes coding for 177 predicted secreted proteins and 64 membrane- associated proteins were annotated as merozoite-specific. The vast majority of known dense-granule (GRA), microneme (MIC), and rhoptry (ROP) genes were not expressed in merozoites. In contrast, a large set of surface proteins (SRS) was expressed exclusively in merozoites.ConclusionsThe distinct expression profiles of merozoites and tachyzoites reveal significant additional complexity within the T. gondii life cycle, demonstrating that merozoites are distinct asexual dividing stages which are uniquely adapted to their niche and biological purpose.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1225-x) contains supplementary material, which is available to authorized users.

The immunomodulatory effects of rolipram abolish drug-resistant latent phase of Toxoplasma gondii infection in a murine model

BackgroundLatent toxoplasmosis always has the risk of reactivation leading to significant sequelae. The available medications, for chronic toxoplasmosis, are awfully limited by resistance of Toxoplasma cysts. Therefore, there is a growing necessity for novel therapeutic approaches. Agents increasing cAMP levels and downregulating proinflammatory cytokine could inhibit Toxoplasma conversion to the bradyzoite stage. This study explores a potential immunomodulatory effect of rolipram, a PDE4 inhibitor, on the course of experimental toxoplasmosis and links this role to deterrence of the resistant chronic phase of the disease. Materials and methodsMice infected with low pathogenic strain of Toxoplasma gondii were treated with rolipram for three weeks. The effect of rolipram was evaluated through tissue injury scoring, brain cyst count, specific IgG titers as well as TNF-α, IFN-γ and IL-12 assays. ResultsRolipram was partially able to prevent the progression to chronic toxoplasmosis. Toxoplasma brain cyst burden showed a 74% reduction while Toxoplasma-induced inflammatory foci per liver area and nucleated cells per inflammatory focus were significantly reduced: 57.14% and 61.3% respectively. Significant reduction of TNF-α (84.6%), IFN-γ (76.7%) and IL-12 (71%) levels was demonstrated along with significant inhibition of anti-Toxoplasma antibody response. ConclusionRolipram efficiently modulated the Toxoplasma-induced immunological changes with a consequent remission of chronic toxoplasmosis. This study is the first to report the utilization of PDE4 inhibitors as possible immune modulators of chronic phase of Toxoplasma infection.

A single mutation in the gatekeeper residue in TgMAPKL-1 restores the inhibitory effect of a bumped kinase inhibitor on the cell cycle

Toxoplasma gondii is the causative pathogen for Toxoplasmosis. Bumped kinase inhibitor 1NM-PP1 inhibits the growth of T. gondii by targeting TgCDPK1. However, we recently reported that resistance to 1NM-PP1 can be acquired via a mutation in T. gondii mitogen-activated protein kinase like 1 (TgMAPKL-1). Further characterization of how this TgMAPKL-1 mutation restores the inhibitory effect of 1NM-PP1 would shed further light on the function of TgMAPKL-1 in the parasite life cycle. Therefore, we made parasite clones with TgMAPKL-1 mutated at the gatekeeper residue Ser 191, which is critical for 1NM-PP1 susceptibility. Host cell lysis of RH/ku80-/HA-TgMAPKL-1S191A was completely inhibited at 250 nM 1NM-PP1, whereas that of RH/ku80-/HA-TgMAPKL-1S191Y was not. By comparing 1NM-PP1-sensitive (RH/ku80-/HA-TgMAPKL-1S191A) and -resistant (RH/ku80-/HA-TgMAPKL-1S191Y) clones, we observed that inhibition of TgMAPKL-1 blocked cell cycle progression after DNA duplication. Morphological analysis revealed that TgMAPKL-1 inhibition caused enlarged parasite cells with many daughter cell scaffolds and imcomplete cytokinesis. We conclude that the mutation in TgMAPKL-1 restored the cell cycle-arresting effect of 1NM-PP1 on T. gondii endodyogeny. Given that endodyogeny is the primary mechanism of cell division for both the tachyzoite and bradyzoite stages of this parasite, TgMAPKL-1 may be a promising target for drug development. Exploration of the signals that regulate TgMAPKL-1 will provide further insights into the unique mode of T. gondii cell division.

Structural and Functional Divergence of the Aldolase Fold in Toxoplasma gondii

Parasites of the phylum Apicomplexa are highly successful pathogens of humans and animals worldwide. As obligate intracellular parasites, they have significant energy requirements for invasion and gliding motility that are supplied by various metabolic pathways. Aldolases have emerged as key enzymes involved in these pathways, and all apicomplexans express one or both of fructose 1,6-bisphosphate (F16BP) aldolase and 2-deoxyribose 5-phosphate (dR5P) aldolase (DERA). Intriguingly, Toxoplasma gondii, a highly successful apicomplexan parasite, expresses F16BP aldolase (TgALD1), d5RP aldolase (TgDERA), and a divergent dR5P aldolase-like protein (TgDPA) exclusively in the latent bradyzoite stage. While the importance of TgALD1 in glycolysis is well established and TgDERA is also likely to be involved in parasite metabolism, the detailed function of TgDPA remains elusive. To gain mechanistic insight into the function of different T. gondii aldolases, we first determined the crystal structures of TgALD1 and TgDPA. Structural analysis revealed that both aldolases adopt a TIM barrel fold accessorized with divergent secondary structure elements. Structural comparison of TgALD1 and TgDPA with members of their respective enzyme families revealed that, while the active-site residues are conserved in TgALD1, key catalytic residues are absent in TgDPA. Consistent with this observation, biochemical assays showed that, while TgALD1 was active on F16BP, TgDPA was inactive on dR5P. Intriguingly, both aldolases are competent to bind polymerized actin in vitro. Altogether, structural and biochemical analyses of T. gondii aldolase and aldolase-like proteins reveal diverse functionalization of the classic TIM barrel aldolase fold.

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 merozoite gene expression analysis with comparison to the life cycle discloses a unique expression state during enteric development.

BackgroundConsiderable work has been carried out to understand the biology of tachyzoites and bradyzoites of Toxoplasma gondii in large part due to in vitro culture methods for these stages. However, culturing methods for stages that normally develop in the gut of the definitive felid host, including the merozoite and sexual stages, have not been developed hindering the ability to study a large portion of the parasite’s life cycle. Here, we begin to unravel the molecular aspects of enteric stages by providing new data on merozoite stage gene expression.ResultsTo profile gene expression differences in enteric stages we harvested merozoites from the intestine of infected cats and hybridized mRNA to the Affymetrix Toxoplasma GeneChip. We analyzed the merozoite data in context of the life cycle by comparing it to previously published data for the oocyst, tachyzoite, and bradyzoite stages. Principal component analysis highlighted the unique profile of merozoites, placing them approximately half-way on a continuum between the tachyzoite/bradyzoite and oocyst samples. Prior studies have shown that antibodies to surface antigen one (SAG1) and many dense granule proteins do not label merozoites: our microarray data confirms that these genes were not expressed at this stage. Also, the expression for many rhoptry and microneme proteins was drastically reduced while the expression for many surface antigens was increased at the merozoite stage. Gene Ontology and KEGG analysis revealed that genes involved in transcription/translation and many metabolic pathways were upregulated at the merozoite stage, highlighting unique growth requirements of this stage. To functionally test these predictions, we demonstrated that an upstream promoter region of a merozoite specific gene was sufficient to control expression in merozoites in vivo.ConclusionsMerozoites are the first developmental stage in the coccidian cycle that takes place within the gut of the definitive host. The data presented here describe the global gene expression profile of the merozoite stage and the creation of transgenic parasite strains that show stage-specific expression of reporter genes in the cat intestine. These data and reagents will be useful in unlocking how the parasite senses and responds to the felid gut environment to initiate enteric development.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-350) contains supplementary material, which is available to authorized users.

A Toxoplasma Patatin-Like Protein Changes Localization and Alters the Cytokine Response during Toxoplasmic Encephalitis

Toxoplasma gondii is an obligate intracellular parasite that forms a lifelong infection within the central nervous system of its host. The T. gondii genome encodes six members of the patatin-like phospholipase family; related proteins are associated with host-microbe interactions in bacteria. T. gondii patatin-like protein 1 (TgPL1) was previously determined to be necessary for parasites to suppress nitric oxide and prevent degradation in activated macrophages. Here, we show that in the rapidly replicating tachyzoite stage, TgPL1 is localized within vesicles inside the parasite that are distinct from the dense granules; however, in the encysted bradyzoite stage, TgPL1 localizes to the parasitophorous vacuole (PV) and cyst wall. While we had not previously seen a defect of the TgPL1 deletion mutant (ΔTgPL1) during acute and early chronic infection, the localization change of TgPL1 in bradyzoites caused us to reevaluate the ΔTgPL1 mutant during late chronic infection and in a toxoplasmic encephalitis (TE) mouse model. Mice infected with ΔTgPL1 are more resistant to TE and have fewer inflammatory lesions than mice infected with the wild type and ΔTgPL1 genetically complemented with TgPL1. This increased resistance to TE could result from several contributing factors. First, we found that ΔTgPL1 bradyzoites did not convert back to tachyzoites readily in tissue culture. Second, a subset of cytokine levels were higher in ΔTgPL1-infected mice, including gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and monocyte chemotactic protein 1 (MCP-1). These studies suggest that TgPL1 plays a role in the maintenance of chronic T. gondii infection.

DNA vaccine encoding the Toxoplasma gondii bradyzoite-specific surface antigens SAG2CDX protect BALB/c mice against type II parasite infection

The surface antigens SAG2C, SAG2D, and SAG2X, which expressed specifically on bradyzoite stage of Toxoplasma gondii, have been demonstrated to be important for persistence of cyst in the brain. In this study, DNA vaccines expressing SAG2C, SAG2D, and SAG2X of T. gondii were constructed and their protective efficacy were evaluated in BALB/c mice. Mice vaccinated with pVAX1-SAG2C (pSAG2C), pVAX1-2D (pSAG2D) or pVAX1-2X (pSAG2C) showed higher levels of serum IgG antibodies and lymphocyte proliferation response compared to PBS and pVAX1 treated mice (p<0.05). The immune response was characterized by a strong Th1 response and increased cytokine production of IL-2 and IFN-γ. Vaccinated mice displayed significant protection against the challenge with the cyst of T. gondii genotype II strain of PRU (cyst-forming in mouse). A significant reduction in the brain cyst burden was detected in the mice immunized with pSAG2C (72%), pSAG2D (23%), pSAG2X (69%) alone and even more reduction rate, 77%, was achieved in the combination group compared to PBS treated mice. The results implied that immunization with DNA vaccines expressing SAG2C, SAG2D, and SAG2X, and, in particular, a combination of all three DNA plasmids, could effectively protect the mice against T. gondii chronic infection.

Genetic Manipulation in &lt;em&gt;&amp;Delta;ku80&lt;/em&gt; Strains for Functional Genomic Analysis of &lt;em&gt;Toxoplasma gondii&lt;/em&gt;

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The Δku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The Δku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.Here, we report methods for using type I and type II Δku80Δhxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in Δku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Optogenetic Modulation of an Adenylate Cyclase in Toxoplasma gondii Demonstrates a Requirement of the Parasite cAMP for Host-Cell Invasion and Stage Differentiation

cAMP research in intracellular parasites remains underappreciated, and it requires a specific method for cyclic nucleotide regulation. Optogenetic induction of cAMP in T. gondii affects host-cell invasion, stage-specific expression, and parasite differentiation. The underlying method allows a versatile control of parasite cAMP. Optogenetic parasite strains offer valuable tools for dissecting cAMP-mediated processes. The method is applicable to other gene-tractable intertwined systems. Successful infection and transmission of the obligate intracellular parasite Toxoplasma gondii depends on its ability to switch between fast-replicating tachyzoite (acute) and quiescent bradyzoite (chronic) stages. Induction of cAMP in the parasitized host cells has been proposed to influence parasite differentiation. It is not known whether the parasite or host cAMP is required to drive this phenomenon. Other putative roles of cAMP for the parasite biology also remain to be identified. Unequivocal research on cAMP-mediated signaling in such intertwined systems also requires a method for an efficient and spatial control of the cAMP pool in the pathogen or in the enclosing host cell. We have resolved these critical concerns by expressing a photoactivated adenylate cyclase that allows light-sensitive control of the parasite or host-cell cAMP. Using this method, we reveal multiple roles of the parasite-derived cAMP in host-cell invasion, stage-specific expression, and asexual differentiation. An optogenetic method provides many desired advantages such as: (i) rapid, transient, and efficient cAMP induction in extracellular/intracellular and acute/chronic stages; (ii) circumvention of the difficulties often faced in cultures, i.e. poor diffusion, premature degradation, steady activation, and/or pleiotropic effects of cAMP agonists and antagonists; (iii) genetically encoded enzyme expression, thus inheritable to the cell progeny; and (iv) conditional and spatiotemporal control of cAMP levels. Importantly, a successful optogenetic application in Toxoplasma also illustrates its wider utility to study cAMP-mediated signaling in other genetically amenable two-organism systems such as in symbiotic and pathogen-host models.

Identification ofBesnoitia besnoitiproteins that showed differences in abundance between tachyzoite and bradyzoite stages by difference gel electrophoresis

Bovine besnoitiosis is a chronic and debilitating disease, caused by the apicomplexan parasite Besnoitia besnoiti. Infection of cattle by B. besnoiti is governed by the tachyzoite stage, which is related to acute infection, and the bradyzoite stage gathered into macroscopic cysts located in subcutaneous tissue in the skin, mucosal membranes and sclera conjunctiva and related to persistence and chronic infection. However, the entire life cycle of this parasite and the molecular mechanisms underlying tachyzoite-to-bradyzoite conversion remain unknown. In this context, a different antigenic pattern has been observed between tachyzoite and bradyzoite extracts. Thus, to identify stage-specific proteins, a difference gel electrophoresis (DIGE) approach was used on tachyzoite and bradyzoite extracts followed by mass spectrometry (MS) analysis. A total of 130 and 132 spots were differentially expressed in bradyzoites and tachyzoites, respectively (average ratio ± 1.5, P<0.05 in t-test). Furthermore, 25 differentially expressed spots were selected and analysed by MALDI-TOF/MS. As a result, 5 up-regulated bradyzoite proteins (GAPDH, ENO1, LDH, SOD and RNA polymerase) and 5 up-regulated tachyzoite proteins (ENO2; LDH; ATP synthase; HSP70 and PDI) were identified. The present results set the basis for the identification of new proteins as drug targets. Moreover, the role of these proteins in tachyzoite-to-bradyzoite conversion and the role of the host cell environment should be a subject of further research.

ApiAP2 transcription factor restricts development of the Toxoplasma tissue cyst

Cellular differentiation leading to formation of the bradyzoite tissue cyst stage is the underlying cause of chronic toxoplasmosis. Consequently, mechanisms responsible for controlling development in the Toxoplasma intermediate life cycle have long been sought. Here, we identified 15 Toxoplasma mRNAs induced in early bradyzoite development that encode proteins with apicomplexan AP2 (ApiAP2) DNA binding domains. Of these 15 mRNAs, the AP2IX-9 mRNA demonstrated the largest expression increase during alkaline-induced differentiation. At the protein level, we found that AP2IX-9 was restricted to the early bradyzoite nucleus and is repressed in tachyzoites and in mature bradyzoites from 30-d infected animals. Conditional overexpression of AP2IX-9 significantly reduced tissue cyst formation and conferred alkaline pH-resistant growth, whereas disruption of the AP2IX-9 gene increased tissue cyst formation, indicating AP2IX-9 operates as a repressor of bradyzoite development. Consistent with a role as a repressor, AP2IX-9 specifically inhibited the expression of bradyzoite mRNAs, including the canonical bradyzoite marker, bradyzoite antigen 1 (BAG1). Using protein binding microarrays, we established the AP2 domain of AP2IX-9 binds a CAGTGT DNA sequence motif and is capable of binding cis-regulatory elements controlling the BAG1 and bradyzoite-specific nucleoside triphosphatase (B-NTPase) promoters. The effect of AP2IX-9 on BAG1 expression was direct because this factor inhibits expression of a firefly luciferase reporter under the control of the BAG1 promoter in vivo, and epitope-tagged AP2IX-9 can be immunoprecipitated with the BAG1 promoter in parasite chromatin. Altogether, these results indicate AP2IX-9 restricts Toxoplasma commitment to develop the mature bradyzoite tissue cyst.

Evidence of tRNA cleavage in apicomplexan parasites: Half-tRNAs as new potential regulatory molecules of Toxoplasma gondii and Plasmodium berghei

Several lines of evidence demonstrated that organisms ranging from bacteria to higher animals possess a regulated endonucleolytic cleavage pathway producing half-tRNA fragments. In the present study, we investigated the occurrence of this phenomenon in two distantly related apicomplexan parasites, Toxoplasma gondii, the agent of toxoplasmosis, and the rodent malaria parasite Plasmodium berghei. A low-scale molecular characterization of the small RNA fraction of T. gondii revealed the endonucleolytic processing of 10 distinct tRNA species, with cleavage in the anticodon loop and upstream of the 3′-terminal CCA sequence yielding 5′- or 3′-end half-tRNAs. T. gondii and P. berghei exhibited variable rates of tRNA cleavage upon egress from host cells and in response to stage differentiation, amino acid starvation and heat-shock. Moreover, avirulent isolates of T. gondii and attenuated P. berghei parasites showed a higher rate of tRNA cleavage than virulent strains. Interestingly, half-tRNA production was significantly higher in the metabolically quiescent bradyzoite and sporozoite stages of T. gondii, compared to the fast-growing tachyzoite. Collectively, our findings shed light for the first time on the occurrence of tRNA cleavage in apicomplexan parasites and suggest a relationship between half-tRNA production and growth rate in this important group of organisms.

Determination of stage interconversion in vitro and in vivo by construction of transgenic Toxoplasma gondii that stably express stage-specific fluorescent proteins

Detection of Toxoplasma gondii conversion from the tachyzoite stage to the bradyzoite stage in living brain tissue is difficult because the parasites are small and conversion and reactivation of the parasites are transient events. To better understand the mechanisms of T. gondii stage conversion between tachyzoites and bradyzoites, and to recognize stage conversion in an intermediate host, we constructed a transgenic cyst-forming strain (PLK) of T. gondii. The parasites stably expressed enhanced green fluorescence protein (EGFP) in the tachyzoite stage and red fluorescence protein (RFP) in the bradyzoite stage, under the control of the SAG1 and BAG1 promoters, respectively. The resulting transgenic parasite was designated as PLK/Bi. The PLK/Bi zoites expressed only green fluorescence in the tachyzoite stage and only red fluorescence in the bradyzoite stage in vitro and in vivo. Fluorescence analyses showed that recombinant GFP and RFP were located to the intracellular vacuolar spaces. In addition, an analysis of growth and culture conditions of transgenic T. gondii was performed in vitro and the virulence was evaluated in vivo. Our data suggested that the stage-specific fluorescence expression by PLK/Bi may be rationally designed for in vitro and in vivo studies on stage conversion and reactivation of T. gondii.

Bradyzoite Pseudokinase 1 Is Crucial for Efficient Oral Infectivity of the Toxoplasma gondii Tissue Cyst

The tissue cyst formed by the bradyzoite stage of Toxoplasma gondii is essential for persistent infection of the host and oral transmission. Bradyzoite pseudokinase 1 (BPK1) is a component of the cyst wall, but nothing has previously been known about its function. Here, we show that immunoprecipitation of BPK1 from in vitro bradyzoite cultures, 4 days postinfection, identifies at least four associating proteins: MAG1, MCP4, GRA8, and GRA9. To determine the role of BPK1, a strain of Toxoplasma was generated with the bpk1 locus deleted. This BPK1 knockout strain (Δbpk1) was investigated in vitro and in vivo. No defect was found in terms of in vitro cyst formation and no difference in pathogenesis or cyst burden 4 weeks postinfection (wpi) was detected after intraperitoneal (i.p.) infection with Δbpk1 tachyzoites, although the Δbpk1 cysts were significantly smaller than parental or BPK1-complemented strains at 8 wpi. Pepsin-acid treatment of 4 wpi in vivo cysts revealed that Δbpk1 parasites are significantly more sensitive to this treatment than the parental and complemented strains. Consistent with this, 4 wpi Δbpk1 cysts showed reduced ability to cause oral infection compared to the parental and complemented strains. Together, these data reveal that BPK1 plays a crucial role in the in vivo development and infectivity of Toxoplasma cysts.

Spontaneous cystogenesis in vitro of a Brazilian strain of Toxoplasma gondii

Conversion of Toxoplasma gondii tachyzoites to the bradyzoite stage and tissue cyst formation in the life cycle of the parasite have crucial roles in the establishment of chronic toxoplasmosis. In this work we investigated the in vitro cystogenesis and behavior of the EGS strain, isolated from human amniotic fluid. We observed that tachyzoites of the EGS strain converted to intracellular cysts spontaneously in LLC-MK2 epithelial cells, HSFS fibroblasts and C6 glial cell lineage. The peak of conversion occurred in the LLC-MK2 cells after 4days of infection, when 72.3±15.9 of the infected cells contained DBA positive cysts. Using specific markers against bradyzoite, tachyzoite and cyst wall components, we confirmed stage conversion and distinguished immature from mature cysts. It was also observed that the deposition of cyst wall components occurred before the total conversion of parasites. Transmission electron microscopy confirmed the fully conversion of parasites presenting the typical characteristics of bradyzoites as the posterior position of the nucleus and the presence of amylopectin granules. A thick cyst wall was also detected. Besides, the scanning microscopy revealed that the intracyst matrix tubules were shorter than those from the parasitophorous vacuole intravacuolar network and were immersed in a granular electron dense material. The EGS strain spontaneously forms high burden of cysts in cell culture without artificial stress conditions, and constitutes a useful tool to study this stage of the T. gondii life cycle.

Redefining Chronic Toxoplasmosis—A T Cell Exhaustion Perspective

Toxoplasmosis caused by Toxoplasma gondii (T. gondii), an obligate intercellular protozoan, is considered to be a leading cause of foodborne mortality in the United States (www.cdc.gov). Even in the post-HAART era, fatal toxoplasmic encephalitis (TE) due to reactivation of chronic Toxoplasma infection remains a major problem in Toxoplasma-seropositive AIDS patients in developing countries [1]. In warm-blooded intermediate hosts (including humans), the parasite undergoes stage conversion between the rapidly proliferating tachyzoite, which is considered to be responsible for acute toxoplasmosis, and the relatively quiescent, slowly replicating, encysted bradyzoite that can persist for life. However, in the immunocompromised such as AIDS patients, the parasite converts from a bradyzoite to a tachyzoite stage, leading to TE [2]. Similarly, repeated reactivations can also occur in congenitally infected individuals [3]. Tachyzoite– bradyzoite interconversion is believed to play a central role not only in establishing the chronic infection but also in disease recrudescence [2]. However, factors responsible for the reactivation of chronic infection in vivo remain poorly understood [2,4]. Studies in murine models of chronic toxoplasmosis have demonstrated that CD8 T cells are pivotal for long-term protection [3]. Paradoxically, despite a robust CD8 T cell response during the acute phase of infection, long-term immunity against this pathogen is compromised in susceptible mouse strains, leading to reactivation and host mortality. Differential susceptibility to T. gondii reactivation in AIDS patients was also noted in a study conducted during the pre-HAART era, which reported that only 30% of AIDS patients with low CD4 T cell count and Toxoplasma seropositivity, who were not on effective prophylaxis, developed reactivated toxoplasmosis [5]. Why does a modest subset of this high-risk group develop TE? Considering that memory CD8 T cells can persist for a lifetime and can mediate protective recall responses upon antigen reencounter in other infectious diseases [6], it remains to be addressed whether this differential outcome is a consequence of potential attrition of T. gondii–specific memory CD8 T cells due to genetic polymorphisms or other microenvironment-associated factors. Recent studies from our group, which utilized a susceptible mouse model (C57BL/6), have demonstrated that CD8 T cells during the later phase of chronic toxoplasmosis exhibit progressive attrition of functionality, increased apoptosis, and poor recall response along with elevated expression of PD-1, an inhibitory receptor-a phenomenon referred to as CD8 exhaustion [7]. Concomitant with graded CD8 exhaustion, parasites undergo reactivation resulting in the mortality of the infected host (Figure 1). While the paradigm of CD8 exhaustion has been extensively explored in chronic viral models, it is just beginning to unfold in parasitic infections. Unlike chronic viral models of CD8 exhaustion, which are characterized by persistent high viremia, the T. gondii model represents a unique situation where, despite initial control of parasitemia, CD8 T cells eventually become exhausted [7,8]. Considering that current drugs against T. gondii are toxic and inefficacious against the encysted bradyzoite stage of the parasite [2,3], a thorough understanding of T cell exhaustion during chronic toxoplasmosis is critical for the development of improved immunotherapeutics against this pathogen. Significantly, our laboratory has demonstrated that a blockade of PD-1 interaction with its receptor PDL1, via anti-PD-L1 antibody treatment of chronically infected animals, not only reinvigorates CD8 response and controls parasite reactivation but also prevents host mortality [7].

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.

Cactin is essential for G1 progression in Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular protozoan parasite whose rapid lytic replication cycles define its pathogenicity. We identified a temperature-sensitive growth mutant, FV-P6, which irreversibly arrests before the middle of the G1 stage of the tachyzoite cell cycle. This arrest is caused by a point mutation in a gene conserved across eukaryotes, Cactin, whose product localizes to the nucleus. To elucidate the role of TgCactin we performed genome-wide expression profiling. Besides the expected G1 expression profile, many genes associated with the extracellular state as well as with the bradyzoite cyst stage were identified. Consistent with these profiles were the expression of AP2 transcription factors typically associated with extracellular and bradyzoite stage parasites. This suggests a role for TgCactin in control of gene expression. As TgCactin does not contain any functionally defined domains we reasoned TgCactin exerts its function through interactions with other proteins. In support of this model we demonstrated that TgCactin is present in a protein complex and can oligomerize. Taken together, these results suggest that TgCactin acts as a pivotal protein potentially regulating gene expression at several transition points in parasite development.

A mathematical model for within-host &lt;em&gt;Toxoplasma gondii&lt;/em&gt; invasion dynamics

Toxoplasma gondii (T. gondii) is a protozoan parasite that infects a wide range of intermediate hosts, including all mammals and birds. Up to 20% of the human population in the US and 30% in the world are chronically infected. This paper presents a mathematical model to describe intra-host dynamics of T. gondii infection. The model considers the invasion process, egress kinetics, interconversion between fast-replicating tachyzoite stage and slowly replicating bradyzoite stage, as well as the host's immune response. Analytical and numerical studies of the model can help to understand the influences of various parameters to the transient and steady-state dynamics of the disease infection.