INTRODUCTION

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Toxoplasma gondii is an obligate intracellular apicomplexan parasite responsible for encephalitis in immunocompromised individuals and birth defects in children infected in utero. Although some individuals present with toxoplasmosis during acute infection, most clinically apparent disease results from reactivation of dormant bradyzoites and their conversion to tachyzoites. Unchecked multiplication of the rapidly growing tachyzoite is thought to be responsible for disease, and control of tachyzoites by the immune system results in their conversion to latent bradyzoite forms. Thus, elucidation of the signaling pathways responsible for tachyzoite-bradyzoite interconversion is critical for understanding pathogenesis of toxoplasmosis.

Recent studies by several investigators have established that a variety of stress conditions including pH shock, heat shock, mitochondrial inhibitors, chemical stress, and nitric oxide induce bradyzoite formation (2, 3, 21, 23). Induction of a variety of heat shock proteins (HSPs) including HSP70 is associated with bradyzoite transition (19, 25), and knockout of a bradyzoite-specific small HSP gene, BAG1, results in reduced numbers of bradyzoites in mouse brains (29). These data collectively suggest that the transition from tachyzoite to bradyzoite is a stress-induced differentiation response. Because of the remarkable conservation of cyclic nucleotide signaling pathways in the stress response in a wide variety of organisms including other eukaryotic pathogens, we examined the role of cyclic nucleotide signaling in bradyzoite differentiation in T. gondii.

Our data suggest that both cyclic GMP (cGMP) and cyclic AMP (cAMP) can induce bradyzoite formation. These effects could be due to an increase in host or parasite cyclic nucleotides. PLK, a T. gondii strain able to differentiate in vitro, exhibited a rise in cAMP in response to bradyzoite-inducing conditions, but elevation of cAMP under the same conditions was not evident in RH, a strain that does not differentiate well. These data suggest that cAMP elevation within the parasite may be important for bradyzoite differentiation.

	MATERIALS AND METHODS

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Parasite and tissue culture. ME49, PLK (a clonal derivative of ME49 [10]), and RH were the three T. gondii strains used. PLK parasites had been previously passaged though mice and were known to generate cysts efficiently in vivo (29). Parasites were maintained by serial passage in confluent monolayers of human foreskin fibroblasts (HFF) grown in Dulbecco's modified Eagle's medium (Gibco BRL, Grand Island, N.Y.) supplemented with 10% fetal bovine serum (Gibco BRL) and 1% penicillin-streptomycin (Gibco BRL).

Materials. Forskolin, 1-methyl-3-isobutylxanthine (IBMX), 8-(4-chlorophenylthio)-cAMP (CPT-cAMP), 8-(4-chlorophenylthio)-cGMP (CPT-cGMP), and sodium nitroprusside (SNP) were obtained from Sigma (St. Louis, Mo.). LY83583 (6-anilino-5,8-quinolinequinone) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1- one (ODQ) were obtained from Calbiochem (La Jolla, Calif.). Forskolin, CPT-cAMP, CPT-cGMP, and IBMX were dissolved in dimethyl sulfoxide; SNP was dissolved in Dulbecco's modified Eagle's medium.

In vitro bradyzoite assay. T. gondii in vitro differentiation assays were performed using an indirect immunofluorescence assay as described by Weiss et al. (23). Approximately 4,000 parasites were inoculated with the agent to be tested onto confluent HFF monolayers growing in four-chambered coverglass slides or Permanox slides (Lab-Tek; Fisher Scientific, Pittsburgh, Pa.). Each condition to be analyzed was set up in duplicate for each experiment. Cultures were grown for 2 or 3 days. (Cultures were monitored daily and fixed when tachyzoite vacuoles were on the verge of lysing [generally 2 days for RH and 3 days for PLK].) Fixation of cultures at 1 day was not routinely performed because staining was frequently weak or uneven and SNP-induced vacuoles were often too small to count reliably.

Cyclic nucleotide measurements. Freshly lysed RH, PLK, and ME49 strains of T. gondii were purified from host cells by serial passage through 20-, 23-, 25-, and 27-gauge needles and filtration through a 3.0-µm-pore-size Nuclepore filter. Parasites were washed and resuspended in complete medium supplemented with the agents to be tested. Incubations were performed at 37°C in a CO₂ incubator to maintain normal medium pH. At 15, 30, and 60 min, cells were placed in 20 mM phosphate buffer (pH 7.0) containing 20 mM EDTA and 1 mM IBMX. The cells were then boiled for 7 min and quick cooled on ice. The extract was briefly centrifuged, and then the supernatant was stored at 20°C. Cyclic nucleotide levels were measured in duplicate using the cGMP and cAMP enzyme immunoassay (EIA) kits from Stratagene (La Jolla, Calif.) following the manufacturer's instructions. cGMP samples were acetylated prior to testing, and cAMP levels were measured with nonacetylated samples. Absolute levels of cyclic nucleotide were calculated by comparison to a standard curve generated during each experiment.

	RESULTS

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In vitro bradyzoite induction assay. DBA lectin reactivity has been reported to be a sensitive early marker of bradyzoite differentiation similar to BAG1 and p36/BSR4 (4). The lectin recognizes carbohydrate modifications of a 116-kDa cyst wall glycoprotein previously identified with bradyzoite-specific monoclonal antibody 73.18 (24, 28). We adapted DBA to the in vitro differentiation assay. In our hands, DBA gave results similar to those obtained with BAG1 immunostaining (23).

Induction of bradyzoite formation by cyclic nucleotides. Initially, the membrane-permeable nonhydrolyzable forms of cAMP and cGMP, CPT-cAMP and CPT-cGMP, were compared to control medium for the ability to induce bradyzoite conversion in culture. Parasites were infected onto HFF in medium supplemented with the agent to be tested and then grown for 3 days or until tachyzoites had begun to lyse. Each condition was tested in duplicate. Bradyzoite-positive vacuoles were counted for each experiment and compared to the control cultures. Cultures with SNP, which induces bradyzoite formation, were analyzed in parallel. Results from a representative experiment are shown in Table 1.

Effects of guanylate cyclase inhibitors. Nitric oxide exerts some of its effects on cells by sequestering the heme group in soluble guanylate cyclase, leading to cyclase activation and increased levels of cGMP. The downstream effects of cGMP can be mediated by activation of cGMP-dependent kinase and either activation or inhibition of phosphodiesterase activity. Therefore, we tested the effects of the soluble guanylate cyclase inhibitors LY83583 (1) and ODQ (7, 18) on bradyzoite differentiation.

Cyclic nucleotide measurements. Cyclic nucleotide levels were measured in PLK and RH strain extracellular parasites as well as host cells by using cAMP and cGMP EIA assays. Samples were tested at 15, 30, and 60 min. For all conditions tested, absolute cGMP levels were lower than cAMP levels, and acetylated cGMP samples were used for detection. Acetylation increases the sensitivity of the assay at least 10-fold (Stratagene cAMP and cGMP EIA user manual). Host cells (HFF) exhibited cAMP elevation in response to 100 µM SNP, 100 µM forskolin, and pH 8.1 (Fig. 1). In HFF, SNP consistently led to an elevation of cGMP, but consistent results were not seen in response to other bradyzoite-inducing conditions (data not shown).

View larger version (15K): [in this window] [in a new window]   FIG. 1.   cAMP levels in HFF after exposure to various extracellular stimuli. Monolayers of host HFF were incubated in media with the indicated conditions. At the indicated times, samples were harvested and processed for cAMP measurements using a Stratagene EIA kit. Cyclic nucleotide concentration was determined by comparison to a standard curve. Numbers are plotted as fold change in concentration compared to untreated control cells. Shown are fold change in HFF cAMP levels in response to 100 µM SNP (circles), pH 8.1 (triangles), and 100 µM forskolin (squares) compared to untreated control cells (diamonds). Results of a representative experiment are shown.

View larger version (15K): [in this window] [in a new window]   FIG. 2.   cAMP levels in T. gondii PLK tachyzoites in response to various stimuli. Extracellular PLK parasites were incubated in media with the indicated conditions. At the indicated times, samples were harvested and processed for cAMP measurements using a Stratagene EIA kit. cAMP concentration was determined by comparison to a standard curve generated for each experiment. Numbers are plotted as fold change in concentration compared to untreated control cells. (A) cAMP levels in PLK parasites exposed to 100 µM SNP (circles), pH 8.1 (triangles), and 100 µM forskolin (squares) compared to untreated control cells (diamonds). Results of a representative experiment are shown. (B) cAMP levels in PLK parasites treated with 100 µM SNP (circles), 250 µM CPT-cGMP (triangles), or 500 µM IBMX (squares) compared to untreated control cells (diamonds). Results of representative experiment are shown.

	DISCUSSION

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Studies from a number of laboratories suggest that bradyzoite formation is a response of parasites to environmental stress. Although changes in the parasite's environment frequently reflect changes in the host cell, there need not be changes in the host cell for bradyzoite induction to occur. Exposure of extracellular parasites to stress-inducing bradyzoite conditions is sufficient to induce bradyzoite differentiation whether pH induction or SNP is used (25). These data suggest that parasites sense and respond to changes in their extracellular environment by differentiating into bradyzoites and that continuous induction is not necessary. Similarly, Yahiaoui et al. (27) found that parasites incubated in media extracellularly for 12 h efficiently differentiated into bradyzoites once inoculated onto HFF grown in normal media.

Our studies indicate that both cGMP and cAMP can mediate bradyzoite differentiation, and data from other systems suggest that these signaling pathways are likely to interact. It remains to be determined whether these effects are extracellular (i.e., host mediated), intracellular (i.e., within the parasite), or, more likely, due to stimulation of both host and parasite signaling pathways. It is likely that bradyzoite differentiation involves the interaction of multiple host and parasite signaling pathways including both cGMP and cAMP pathways (Fig. 3).

View larger version (20K): [in this window] [in a new window]   FIG. 3.   A working model for stress-induced bradyzoite differentiation. Stress conditions, including pH shock and exposure to nitric oxide, result in elevation of cAMP and cGMP (in response to nitric oxide) within host cells. Host cell stress and cAMP response genes are induced. Stress also induces formation of cAMP within the parasite by adenylate cyclase, which may be coupled to a receptor that senses an extracellular signaling molecule or nutrient. cAMP activates PKA and leads to transcription of bradyzoite-specific genes. Prolonged cAMP also activates pathways inhibitory to bradyzoite differentiation. TPK3, a GSK3 family member, may also interact with the PKA signaling pathway as seen in other stress-induced differentiation models such as Dictyostelium. PVM, parasitophorous vacuole membrane.

HFF generated a significant cAMP elevation after exposure to conditions associated with bradyzoite induction. It is likely that host cell environments including cAMP elevations contribute to the bradyzoite differentiation process. It is not known if T. gondii has a receptor or sensor for cyclic nucleotides in its extracellular environment. In Dictyostelium, cAMP secreted into the environment binds to cAMP receptors to regulate the differentiation program of cells within the fruiting body (20). A similar pathway may also be important in T. gondii, but elevation in host cell cAMP does not appear to be sufficient for differentiation given that T. gondii RH differentiated much less efficiently than T. gondii PLK or ME49.

The role of cGMP in T. gondii bradyzoite differentiation remains to be clarified. CPT-cGMP consistently induced PLK tachyzoites to differentiate into bradyzoites. CPT-cGMP also elevated cAMP levels in parasites, suggesting that cGMP effects may be due to regulation of cAMP levels. We addressed the role of cGMP in bradyzoite differentiation by using inhibitors of guanylate cyclase. While these experiments are consistent with a role for cGMP in nitric oxide-mediated bradyzoite induction, the effects of these inhibitors was complex and effects on other signaling molecules cannot be ruled out.

A cAMP response within the parasite may be associated with differentiation. We could not generate a significant cAMP response in RH parasites, but our RH isolate did not differentiate well under any conditions tested. In contrast, PLK parasites had a significant elevation in intracellular cAMP levels in conditions known to induce bradyzoite differentiation and were able to differentiate efficiently. Because the cyclic nucleotide measurements were performed on a population of cells, we were unable to determine if the small subpopulation of RH parasites differentiating to bradyzoites had changes in cyclic nucleotide levels.

It seems likely that cAMP has both stimulatory and antagonistic effects on bradyzoite differentiation. While both forskolin and IBMX elevated cAMP in parasites, only forskolin induced bradyzoite formation. Since IBMX, which inhibits cAMP degradation, blocked bradyzoite induction by forskolin, the degree of cAMP elevation or kinetics of cAMP elevation may be critical. It may be that sustained elevation of cAMP leads to activation of pathways that are inhibitory to bradyzoite differentiation. In support of this hypothesis, CPT-cAMP, which is not degraded by phosphodiesterases, was also inhibitory for bradyzoite formation. Signaling molecules such as the mitogen-activated protein kinase KSS1 (6, 13) and cAMP-dependent kinases (14, 17) have been described to have both positive and negative regulatory effects in stress-induced pseudohyphal differentiation in Saccharomyces cerevisiae.

Studies of many pathogens have correlated cAMP signaling with differentiation. cAMP signaling has been implicated in Plasmodium falciparum differentiation to gametocytes (11), but as with T. gondii, cAMP effects have not been observed in all strains (5, 9). cAMP signaling has also been found to be critical for virulence in a number of fungal pathogens, including Cryptococcus neoformans and Candida albicans (12).

Our data would predict that T. gondii protein kinase A (PKA) plays a significant role in the conversion of tachyzoites to bradyzoite forms. In P. falciparum, a correlation between PKA levels and ability to transform into gametocytes has been described (16). PKA could have stimulatory or inhibitory effects on bradyzoite gene transcription or effects on other kinases important in cell fate decisions and stress-induced differentiation (8) such as the T. gondii GSK3/shaggy homologue TPK3 (15, 26) (Fig. 3). Recent data for S. cerevisiae reveal that one of the three PKA catalytic subunits mediates stress-induced differentiation (14, 17), and studies of Dictyostelium have suggested that cAMP is not required for differentiation if sufficient levels of PKA activity are present (22).

Studies in other organisms suggest that the signaling pathways utilized by eukaryotic pathogens in response to stress are phylogenetically conserved and significantly affect virulence and pathogenicity. Further studies of T. gondii will be needed to clarify the complex interactions of cGMP and cAMP signaling molecules with upstream sensors and downstream effector molecules.