Bradyzoite Cysts Research Articles

A Toxoplasma gondii O-glycosyltransferase that modulates bradyzoite cyst wall rigidity is distinct from host homologues

Infection with the apicomplexan protozoan Toxoplasma gondii can be life-threatening in immunocompromised hosts. Transmission frequently occurs through the oral ingestion of T. gondii bradyzoite cysts, which transition to tachyzoites, disseminate, and then form cysts containing bradyzoites in the central nervous system, resulting in latent infection. Encapsulation of bradyzoites by a cyst wall is critical for immune evasion, survival, and transmission. O-glycosylation of the protein CST1 by the mucin-type O-glycosyltransferase T. gondii (Txg) GalNAc-T3 influences cyst wall rigidity and stability. Here, we report X-ray crystal structures of TxgGalNAc-T3, revealing multiple features that are strictly conserved among its apicomplexan homologues. This includes a unique 2nd metal that is coupled to substrate binding and enzymatic activity in vitro and cyst wall O-glycosylation in T. gondii. The study illustrates the divergence of pathogenic protozoan GalNAc-Ts from their host homologues and lays the groundwork for studying apicomplexan GalNAc-Ts as therapeutic targets in disease.

Lessons from a Multilaboratorial Task Force for Diagnosis of a Fatal Toxoplasmosis Outbreak in Captive Primates in Brazil.

Toxoplasmosis is an important zoonotic disease caused by the parasite Toxoplasma gondii and is especially fatal for neotropical primates. In Brazil, the Ministry of Health is responsible for national epizootic surveillance, but some diseases are still neglected. Here, we present an integrated investigation of an outbreak that occurred during the first year of the COVID-19 pandemic among eleven neotropical primates housed at a primatology center in Brazil. After presenting non-specific clinical signs, all animals died within four days. A wide range of pathogens were evaluated, and we successfully identified T. gondii as the causative agent within four days after necropsies. The liver was the most affected organ, presenting hemorrhage and hepatocellular necrosis. Tachyzoites and bradyzoite cysts were observed in histological examinations and immunohistochemistry in different organs; in addition, parasitic DNA was detected through PCR in blood samples from all specimens evaluated. A high prevalence of Escherichia coli was also observed, indicating sepsis. This case highlights some of the obstacles faced by the current Brazilian surveillance system. A diagnosis was obtained through the integrated action of researchers since investigation for toxoplasmosis is currently absent in national guidelines. An interdisciplinary investigation could be a possible model for future epizootic investigations in animals.

Association Between Ocular Trauma and Activation of Ocular Toxoplasmosis

ABSTRACT Purpose To evaluate the association between ocular trauma and activation of ocular toxoplasmosis. Methods Retrospective review of 686 patients with ocular toxoplasmosis and its association with trauma to the eye or the head within 1 week of activation. Results Ten patients with a history of trauma and activation of ocular toxoplasmosis were detected (10/686; 1.45%). Nine patients showed a primary focus of retinitis without a previous scar and one patient had a recurrent form of ocular toxoplasmosis. From these 10 patients, Toxoplasma IgG was positive in eight of them. The median age of the patients was 35.8 years-old (range 17 to 65). Conclusions These cases suggest that trauma can be associated with activation of retinal bradyzoite cysts in ocular toxoplasmosis.

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.

P18 (SRS35/TgSAG4) Plays a Role in the Invasion and Virulence of Toxoplasma gondii

Toxoplasmosis is a prevalent parasitic disease caused by Toxoplasma gondii (T. gondii). Under the control of the host immune system, T. gondii persists as latent bradyzoite cysts. Immunosuppression leads to their reactivation, a potentially life-threatening condition. Interferon-gamma (IFN-γ) controls the different stages of toxoplasmosis. Here, we addressed the role of the parasite surface antigen P18, belonging to the Surface-Antigen 1 (SAG-1) Related Sequence (SRS) family, in a cyst-forming strain. Deletion of P18 gene (KO P18) impaired the invasion of parasites in macrophages and IFN-γ-mediated activation of macrophages further reduced the invasion capacity of this KO, as compared to WT strain. Mice infected by KO P18, showed a marked decrease in virulence during acute toxoplasmosis. This was consequent to less parasitemia, accompanied by a substantial recruitment of dendritic cells, macrophages and natural killer cells (NK). Furthermore, KO P18 resulted in a higher number of bradyzoite cysts, and a stronger inflammatory response. A prolonged survival of mice was observed upon immunosuppression of KO P18 infected BALB/c mice or upon oral infection of Severe Combined Immunodeficiency (SCID) mice, with intact macrophages and natural killer (NK) cells. In stark contrast, oral infection of NSG (NOD/Shi-scid/IL-2Rγnull) mice, defective in macrophages and NK cells, with KO P18, was as lethal as that of the control strain showing that the conversion from bradyzoites to tachyzoites is intact and, suggesting a role of P18 in the response to host IFN-γ. Collectively, these data demonstrate a role for P18 surface antigen in the invasion of macrophages and in the virulence of the parasite, during acute and chronic toxoplasmosis.

Discovery and Optimization of Triazine Nitrile Inhibitors of Toxoplasma gondii Cathepsin L for the Potential Treatment of Chronic Toxoplasmosis in the CNS.

With roughly 2 billion people infected, the neurotropic protozoan Toxoplasma gondii remains one of the most pervasive and infectious parasites. Toxoplasma infection is the second leading cause of death due to foodborne illness in the United States, causes severe disease in immunocompromised patients, and is correlated with several cognitive and neurological disorders. Currently, no therapies exist that are capable of eliminating the persistent infection in the central nervous system (CNS). In this study we report the identification of triazine nitrile inhibitors of Toxoplasma cathepsin L (TgCPL) from a high throughput screen and their subsequent optimization. Through rational design, we improved inhibitor potency to as low as 5 nM, identified pharmacophore features that can be exploited for isoform selectivity (up to 7-fold for TgCPL versus human isoform), and improved metabolic stability (t1/2 > 60 min in mouse liver microsomes) guided by a metabolite ID study. We demonstrated that this class of compounds is capable of crossing the blood-brain barrier in mice (1:1 brain/plasma at 2 h). Importantly, we also show for the first time that treatment of T. gondii bradyzoite cysts in vitro with triazine nitrile inhibitors reduces parasite viability with efficacy equivalent to a TgCPL genetic knockout.

Experimental Toxoplasmosis in Pigeons (Columba livia)

The purpose of the present study was to establish experimental model of toxoplasmosis in pigeons, to investigate pathogenesis and compare tissue lesions by clinical, histopathological, serological and bioassay techniques. Total of 60 unknown aged pigeons (Columba livia), 21 males and 39 females were used. They were divided into groups as oral (Group I and II) and parenteral (Group III, IV, V and VI) infection groups (Table 1). While some pigeons in Group IV showed acute infection signs such as anorexia, weight loss, pale cockscomb, bend of head and neck and partial paralyze; chronic infection signs such as anorexia, weakness, weight loss, diarrhea, difficulties in breathing, and conjunctivitis were seen in Group IV, V, and VI. In necropsy, the pigeons in Group IV had hyperemia and focal hemorrhages in the meninges and brain; the pigeons in Groups V and VI had yellowish color of the liver, the pigeons in Group V had the pale chest muscles, pericardial thickening and opaqueness. There were no macroscopic findings in pigeons in Group I and III. Histopathological examination revealed nonsuppurative meningoencephalitis and tachyzoites and bradyzoite cysts formation of T. gondii in brain tissue, lymphoid cell infiltration and necrotic focal hepatitis and nephritis in Group IV. While pigeons in Group V had nonsuppurative focal myositis, myocarditis, hepatitis, gastritis, enteritis, pneumonitis, and necrotic pancreatitis, one of them had toxoplasma bradyzoite cyst in the sinusoid in the liver. In group VI, nonsuppurative focal hepatitis, myocarditis, nephritis and necrotic pancreatitis were detected in pigeons. Bioassay tests were performed with tissue samples taken from seropositive pigeons and parasitic tachyzoites were isolated from the peritoneal fluid of the mice. Seropositivity in the oral and parenteral groups was determined by Sabin-Feldman Dye Test (SFDT) and Indirect Hemagglutination Assay (IHA). As a result; in similar studies that will be performed investigating pathogenesis of Toxoplasmosis and subclinical cases that may be overlooked, serologic tests and bioassay applications should be used together for the diagnosis of toxoplasmosis.

Glycolysis is important for optimal asexual growth and formation of mature tissue cysts by Toxoplasma gondii

Toxoplasma gondii can grow and replicate using either glucose or glutamine as the major carbon source. Here, we have studied the essentiality of glycolysis in the tachyzoite and bradyzoite stages of T. gondii, using transgenic parasites that lack a functional hexokinase gene (Δhk) in RH (Type-1) and Prugniaud (Type-II) strain parasites. Tachyzoite stage Δhk parasites exhibit a fitness defect similar to that reported previously for the major glucose transporter mutant, and remain virulent in mice. However, although Prugniaud strain Δhk tachyzoites were capable of transforming into bradyzoites in vitro, they were severely compromised in their ability to make mature bradyzoite cysts in the brain tissue of mice. Isotopic labelling studies reveal that glucose-deprived tacyzoites utilise glutamine to replenish glycolytic and pentose phosphate pathway intermediates via gluconeogenesis. Interestingly, while glutamine-deprived intracellular Δhk tachyzoites continued to replicate, extracellular parasites were unable to efficiently invade host cells. Further, studies on mutant tachyzoites lacking a functional phosphoenolpyruvate carboxykinase (Δpepck1) revealed that glutaminolysis is the sole source of gluconeogenic flux in glucose-deprived parasites. In addition, glutaminolysis is essential for sustaining oxidative phosphorylation in Δhk parasites, while wild type (wt) and Δpepck1 parasites can obtain ATP from either glycolysis or oxidative phosphorylation. This study provides insights into the role of nutrient metabolism during asexual propagation and development of T. gondii, and validates the versatile nature of central carbon and energy metabolism in this parasite.

Global Analysis of Palmitoylated Proteins in Toxoplasma gondii

Post-translational modifications (PTMs) such as palmitoylation are critical for the lytic cycle of the protozoan parasite Toxoplasma gondii. While palmitoylation is involved in invasion, motility, and cell morphology, the proteins that utilize this PTM remain largely unknown. Using a chemical proteomic approach, we report a comprehensive analysis of palmitoylated proteins in T. gondii, identifying a total of 282 proteins, including cytosolic, membrane-associated, and transmembrane proteins. From this large set of palmitoylated targets, we validate palmitoylation of proteins involved in motility (myosin light chain 1, myosin A), cell morphology (PhIL1), and host cell invasion (apical membrane antigen 1, AMA1). Further studies reveal that blocking AMA1 palmitoylation enhances the release of AMA1 and other invasion-related proteins from apical secretory organelles, suggesting a previously unrecognized role for AMA1. These findings suggest that palmitoylation is ubiquitous throughout the T. gondii proteome and reveal insights into the biology of this important human pathogen.

The Toxoplasma nuclear factor TgAP2XI‐4 controls bradyzoite gene expression and cyst formation

Toxoplasma gondii undergoes many phenotypic changes during its life cycle. The recent identification of AP2 transcription factors in T. gondii has provided a platform for studying the mechanisms controlling gene expression. In the present study, we report that a recombinant protein encompassing the TgAP2XI-4 AP2 domain was able to specifically bind to a DNA motif using gel retardation assays. TgAP2XI-4 protein is localized in the parasite nucleus throughout the tachyzoite life cycle in vitro, with peak expression occurring after cytokinesis. We found that the TgAP2XI-4 transcript level was higher in bradyzoite cysts isolated from brains of chronically infected mice than in the rapidly replicating tachyzoites. A knockout of the TgAP2XI-4 gene in both T. gondii virulent type I and avirulent type II strains reveals its role in modulating expression and promoter activity of genes involved in stage conversion of the rapidly replicating tachyzoites to the dormant cyst forming bradyzoites. Furthermore, mice infected with the type II KO mutants show a drastically reduced brain cyst burden. Thus, our results validate TgAP2XI-4 as a novel nuclear factor that regulates bradyzoite gene expression during parasite differentiation and cyst formation.

Mechanisms of Toxoplasma gondii persistence and latency.

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes opportunistic disease, particularly in immunocompromised individuals. Central to its transmission and pathogenesis is the ability of the proliferative stage (tachyzoite) to convert into latent tissue cysts (bradyzoites). Encystment allows Toxoplasma to persist in the host and affords the parasite a unique opportunity to spread to new hosts without proceeding through its sexual stage, which is restricted to felids. Bradyzoite tissue cysts can cause reactivated toxoplasmosis if host immunity becomes impaired. A greater understanding of the molecular mechanisms orchestrating bradyzoite development is needed to better manage the disease. Here, we will review key studies that have contributed to our knowledge about this persistent form of the parasite and how to study it, with a focus on how cellular stress can signal for the reprogramming of gene expression needed during bradyzoite development.

Toxoplasmaon the Brain: Understanding Host-Pathogen Interactions in Chronic CNS Infection

Toxoplasma gondii is a prevalent obligate intracellular parasite which chronically infects more than a third of the world's population. Key to parasite prevalence is its ability to form chronic and nonimmunogenic bradyzoite cysts, which typically form in the brain and muscle cells of infected mammals, including humans. While acute clinical infection typically involves neurological and/or ocular damage, chronic infection has been more recently linked to behavioral changes. Establishment and maintenance of chronic infection involves a balance between the host immunity and parasite evasion of the immune response. Here, we outline the known cellular interplay between Toxoplasma gondii and cells of the central nervous system and review the reported effects of Toxoplasma gondii on behavior and neurological disease. Finally, we review new technologies which will allow us to more fully understand host-pathogen interactions.

Methods to produce and safely work with large numbers of Toxoplasma gondii oocysts and bradyzoite cysts

Two major obstacles to conducting studies with Toxoplasma gondii oocysts are the difficulty in reliably producing large numbers of this life stage and safety concerns because the oocyst is the most environmentally resistant stage of this zoonotic organism. Oocyst production requires oral infection of the definitive feline host with adequate numbers of T. gondii organisms to obtain unsporulated oocysts that are shed in the feces for 3–10 days after infection. Since the most successful and common mode of experimental infection of kittens with T. gondii is by ingestion of bradyzoite tissue cysts, the first step in successful oocyst production is to ensure a high bradyzoite tissue cyst burden in the brains of mice that can be used for the oral inoculum. We compared two methods for producing bradyzoite brain cysts in mice, by infecting them either orally or subcutaneously with oocysts. In both cases, oocysts derived from a low passage T. gondii Type II strain (M4) were used to infect eight-ten week-old Swiss Webster mice. First the number of bradyzoite cysts that were purified from infected mouse brains was compared. Then to evaluate the effect of the route of oocyst inoculation on tissue cyst distribution in mice, a second group of mice was infected with oocysts by one of each route and tissues were examined by histology. In separate experiments, brains from infected mice were used to infect kittens for oocyst production. Greater than 1.3 billion oocysts were isolated from the feces of two infected kittens in the first production and greater than 1.8 billion oocysts from three kittens in the second production. Our results demonstrate that oral delivery of oocysts to mice results in both higher cyst loads in the brain and greater cyst burdens in other tissues examined as compared to those of mice that received the same number of oocysts subcutaneously. The ultimate goal in producing large numbers of oocysts in kittens is to generate adequate amounts of starting material for oocyst studies. Given the potential risks of working with live oocysts in the laboratory, we also tested a method of oocyst inactivation by freeze–thaw treatment. This procedure proved to completely inactivate oocysts without evidence of significant alteration of the oocyst molecular integrity.

Toxoplasma gondii Lysine Acetyltransferase GCN5-A Functions in the Cellular Response to Alkaline Stress and Expression of Cyst Genes

Parasitic protozoa such as the apicomplexan Toxoplasma gondii progress through their life cycle in response to stimuli in the environment or host organism. Very little is known about how proliferating tachyzoites reprogram their expressed genome in response to stresses that prompt development into latent bradyzoite cysts. We have previously linked histone acetylation with the expression of stage-specific genes, but the factors involved remain to be determined. We sought to determine if GCN5, which operates as a transcriptional co-activator by virtue of its histone acetyltransferase (HAT) activity, contributed to stress-induced changes in gene expression in Toxoplasma. In contrast to other lower eukaryotes, Toxoplasma has duplicated its GCN5 lysine acetyltransferase (KAT). Disruption of the gene encoding for TgGCN5-A in type I RH strain did not produce a severe phenotype under normal culture conditions, but here we show that the TgGCN5-A null mutant is deficient in responding to alkaline pH, a common stress used to induce bradyzoite differentiation in vitro. We performed a genome-wide analysis of the Toxoplasma transcriptional response to alkaline pH stress, finding that parasites deleted for TgGCN5-A fail to up-regulate 74% of the stress response genes that are induced 2-fold or more in wild-type. Using chromatin immunoprecipitation, we verify an enrichment of TgGCN5-A at the upstream regions of genes activated by alkaline pH exposure. The TgGCN5-A knockout is also incapable of up-regulating key marker genes expressed during development of the latent cyst form, and is impaired in its ability to recover from alkaline stress. Complementation of the TgGCN5-A knockout restores the expression of these stress-induced genes and reverses the stress recovery defect. These results establish TgGCN5-A as a major contributor to the alkaline stress response in RH strain Toxoplasma.

<em>Toxoplasma gondii</em> Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFNγ). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFNγ and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFNγ and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

<em>Toxoplasma gondii</em> Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFNgamma). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFNgamma and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFNgamma and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

Toxoplasma gondii: determinants of tachyzoite to bradyzoite conversion

Apicomplexa are primarily obligate intracellular protozoa that have evolved complex developmental stages important for pathogenesis and transmission. Toxoplasma gondii, responsible for the disease toxoplasmosis, has the broadest host range of the Apicomplexa as it infects virtually any warm-blooded vertebrate host. Key to T. gondii's pathogenesis is its ability to differentiate from a rapidly replicating tachyzoite stage during acute infection to a relatively non-immunogenic, dormant bradyzoite stage contained in tissue cysts. These bradyzoite cysts can reconvert back to tachyzoites years later causing serious pathology and death if a person becomes immune-compromised. Like the sexual stage sporozoites, bradyzoites are also orally infectious and a major contributor to transmission. Because of the critical role of stage conversion to pathogenesis and transmission, a major research focus is aimed at identifying molecular mediators and pathways that regulate differentiation. Tachyzoite to bradyzoite development can occur spontaneously in vitro and be induced in response to exogenous stress including but not limited to host immunity. The purpose of this review is to explore the potential contributors to stage differentiation in infection and how a determination is made by the parasite to differentiate from tachyzoites to bradyzoites.

Persistent toxoplasma bradyzoite cysts in the brain: incidental finding in an immunocompetent patient without evidence of a toxoplasmosis

We report on a 72-year-old patient in whom autopsy demonstrated incidentally intracerebral Toxoplasma gondii cysts, locally restricted in the occipital lobe, in association with only a few CD4+ and CD8+ T cells and a mild microglial activation. The patient was HIV-negative. Serologically, there was no evidence for an active inflammation (Toxoplasma gondii specific antibody IgG-positive, IgM-negative). This unusual observation may indicate that in patients with sepsis, who may yield to a state of immunodysbalance, a focal reactivation of parasites may ensue in the absence of conditions predisposing for opportunistic infection.

Pyogranulomatous cystitis associated with Toxoplasma gondii infection in a cat after renal transplantation

An 8-year-old spayed female domestic shorthair cat was evaluated for azotemia and a suspected mass in the urinary bladder 6 weeks after receiving a renal transplant. Ultrasonography revealed a mass at the ureteroneocystostomy site, and the mass was excised. Both the donor and recipient cats were seronegative for Toxoplasma gondii-specific IgG antibodies prior to transplantation. Histologic evaluation of the mass revealed lesions indicative of extensive necrotizing pyogranulomatous cystitis with numerous intralesional T gondii tachyzoites and bradyzoite cysts. Treatment with clindamycin was initiated; however, the cat's clinical condition continued to decline, and it was euthanized 9 days after the mass was excised. Necropsy revealed T gondii cysts within the renal allograft and the transplanted ureter, with no evidence of systemic spread of organisms. Toxoplasmosis should be considered as a differential diagnosis for azotemia in feline renal transplant recipients regardless of the results of assays for T gondii antibodies in the serum of donors or recipients. This report illustrated the need for improved screening of donor and recipient cats and the importance of minimizing exposure to potential sources of T gondii after transplantation.

Bradyzoite-Specific Surface Antigen SRS9 Plays a Role in Maintaining Toxoplasma gondii Persistence in the Brain and in Host Control of Parasite Replication in the Intestine

Toxoplasma gondii is a ubiquitous parasite that persists for the life of a healthy mammalian host. A latent, chronic infection can reactivate upon immunosuppression and cause life-threatening diseases, such as encephalitis. A key to the pathogenesis is the parasite's interconversion between the tachyzoite (in acute infection) and bradyzoite (in chronic infection) stages. This developmental switch is marked by differential expression of numerous, closely related surface proteins belonging to the SRS (SAG1-related sequence) superfamily. To probe the functions of bradyzoite-specific SRSs, we created a bioluminescent strain lacking the expression of SRS9, one of the most abundant SRSs of the bradyzoite stage. Imaging of mice intraperitoneally infected with tachyzoites revealed that during an acute infection, wild-type and Deltasrs9 strains replicated at similar rates, disseminated systemically following similar kinetics, and initially yielded similar brain cyst numbers. However, during a chronic infection, Deltasrs9 cyst loads substantially decreased compared to those of the wild type, suggesting that SRS9 plays a role in maintaining parasite persistence in the brain. In oral infection with bradyzoite cysts, the Deltasrs9 strain showed oral infectivity and dissemination patterns indistinguishable from those of the wild type. When chronically infected mice were treated with the immunosuppressant dexamethasone, however, the Deltasrs9 strain reactivated in the intestinal tissue after only 8 to 9 days, versus 2 weeks for the wild-type strain. Thus, SRS9 appears to play an important role in both persistence in the brain and reactivation in the intestine. Possible mechanisms for this are discussed.