Host metabolism regulates growth and differentiation of Toxoplasma gondii

International Journal for Parasitology 42 (2012) 947–959

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Host metabolism regulates growth and differentiation of Toxoplasma gondii Dina R. Weilhammer a,⇑, Anthony T. Iavarone b, Eric N. Villegas a,1, George A. Brooks c, Anthony P. Sinai d, William C. Sha a a

Immunology and Pathogenesis Division, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720, USA c Exercise Physiology Laboratory, Department of Integrative Biology, University of California, Berkeley, CA 94720, USA d Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, Lexington, KY, USA b

a r t i c l e

i n f o

Article history: Received 12 April 2012 Received in revised form 28 July 2012 Accepted 30 July 2012 Available online 24 August 2012 Keywords: Toxoplasma gondii Bradyzoite differentiation Metabolism Glycolysis Akt

a b s t r a c t A critical step in the pathogenesis of Toxoplasma gondii is conversion from the fast-replicating tachyzoite form experienced during acute infection to the slow-replicating bradyzoite form that establishes longlived tissue cysts during chronic infection. Bradyzoite cyst development exhibits a clear tissue tropism in vivo, yet conditions of the host cell environment that influence this tropism remain unclear. Using an in vitro assay of bradyzoite conversion, we have found that cell types differ dramatically in the ability to facilitate differentiation of tachyzoites into bradyzoites. Characterization of cell types that were either resistant or permissive for conversion revealed that resistant cell lines release low molecular weight metabolites that could support tachyzoite growth under metabolic stress conditions and thereby inhibit bradyzoite formation in permissive cells. Biochemical analysis revealed that the glycolytic metabolite lactate is an inhibitory component of supernatants from resistant cells. Furthermore, upregulation of glycolysis in permissive cells through the addition of glucose or by overexpression of the host kinase, Akt, was sufficient to convert cells from a permissive to a resistant phenotype. These results suggest that the metabolic state of the host cell may play a role in determining the predilection of the parasite to switch from the tachyzoite to bradyzoite form. Ó 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Toxoplasma gondii is an obligate intracellular protozoan parasite of the phylum Apicomplexa. One of the most widespread parasites in nature, it can infect a wide range of host species, including approximately one-third of the world’s human population (Tenter et al., 2000). The asexual life cycle of T. gondii occurs within all intermediate hosts and involves conversion between two distinct life forms: tachyzoites and bradyzoites. Tachyzoites are the fastreplicating form responsible for flu-like symptoms experienced during acute infection. During the course of infection, tachyzoites differentiate into bradyzoites, which are the slow-replicating form responsible for establishment of long-lived tissue cysts that persist during chronic infection. While tachyzoites can infect and replicate within virtually any nucleated cell, bradyzoite cysts are typically found within neural and muscle tissue (Black and Boothroyd, 2000; Coppens and Joiner, 2001; Ferreira da Silva et al., 2009a). ⇑ Corresponding author. Address: Biosciences and Biotechnology Division, Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA 94550, USA. Tel.: +1 925 422 2977; fax: +1 925 422 2282. E-mail address: [email protected] (D.R. Weilhammer). 1 Present address: National Exposure Research Laboratory, US Environmental Protection Agency, Cincinnati, OH 45268, USA.

Vertical transmission of tachyzoites from mother fetus, as well as reactivation of bradyzoite cysts in immunocompromised patients, can result in severe neural pathology (Coppens and Joiner, 2001; Latkany, 2007). The host factors that regulate the stage conversion of parasites from tachyzoites into bradyzoites are just beginning to be unraveled (Ferreira da Silva et al., 2008; Skariah et al., 2010). A wide variety of general stress conditions, such as change in pH (Soete et al., 1994; Weiss et al., 1995), heat shock (Soete et al., 1994), and mitochondrial inhibitors (Bohne et al., 1994; Tomavo and Boothroyd, 1995), have also been shown to induce tachyzoite to bradyzoite conversion in vitro; therefore, bradyzoite differentiation has been viewed primarily as a stress response (Ferreira da Silva et al., 2008). Thus, it has been postulated that stress exerted by release of inflammatory mediators during the host immune response may also be critical in triggering tachyzoites to convert to bradyzoites in vivo (Gross et al., 1996). IFN-c is a major mediator of resistance to parasite infection in vivo (Suzuki et al., 1988; Scharton-Kersten et al., 1996), and treatment with the cytokine consistently results in slowed parasite growth in vitro. Its impact on parasite conversion is less consistent however, and differential effects that have been reported may be due to cell-type differences (Jones et al., 1986; Bohne et al., 1993, 1994; Weiss et al., 1995). The presence of nitric oxide also results in slowed parasite replication

0020-7519/$36.00 Ó 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijpara.2012.07.011

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and bradyzoite antigen expression in vitro (Bohne et al., 1994). However, the in vivo significance of this result is unclear, as bradyzoites still develop normally in inducible nitric oxide synthase knockout mice (Scharton-Kersten et al., 1997). Conversely, it has also been postulated that the absence of an effective immune response within immune privileged sites, particularly neural tissue, could allow increased tachyzoite replication that ultimately contributes to enhanced parasite persistence in immune privileged tissues (Jones et al., 2006). While the extent of the contribution remains unclear, there is evidence that the host cell environment plays an important role in the regulation of bradyzoite conversion. The in vivo tropism of bradyzoite development in brain and muscle tissue is well established (Coppens and Joiner, 2001), and parasites have been shown to spontaneously convert to bradyzoites in vitro within astrocyte, neural and muscle cell types (Lüder et al., 1999; Ferreira da Silva et al., 2009a,b), thus suggesting that molecular characteristics of certain host cell environments are more conducive to bradyzoite development. The most direct line of evidence implicating host cell contribution to conversion is the demonstration that bradyzoite conversion can be induced in vitro by the expression of the human cell division autoantigen 1 gene (CDA-1, Radke et al., 2006), which triggers growth arrest of both parasites and host cells, and results in robust induction of bradyzoite conversion through an unknown mechanism. In order to further investigate the effects of host cell environment on conversion, we used CO2 restriction to trigger conversion (Dzierszinski et al., 2004), together with a flow cytometric assay to measure the extent of bradyzoite induction. We found that cell types vary widely in their capacity to facilitate bradyzoite differentiation, being either permissive or resistant for conversion. Furthermore, resistant cells could inhibit conversion within permissive cells in trans by the release of small molecular weight metabolites. The glycolytic metabolite lactate was identified by hydrophilic interaction (HILIC) chromatography and mass spectrometry as an inhibitory component of supernatants from resistant cells. Additionally, induction of glycolysis, by addition of glucose or expression of a dominant-active Akt gene, converted permissive cells to a resistant phenotype. To our knowledge, these results are the first to demonstrate that non-immune secreted metabolites can inhibit parasite conversion and suggest that the metabolic state of infected cells, and non-infected neighboring cells, can influence tachyzoite growth and bradyzoite differentiation.

2. Materials and methods 2.1. Cell culture Parasites of the Type II Pru strain, expressing red fluorescent protein (RFP) under the control of the gra8 promoter and yellow fluorescent protein (YFP) under the control of the bag1 promoter, or YFP under control of the tubulin promoter were provided by D. Roos (University of Pennsylvania, Philadelphia, Pennsylvania, USA). All cell lines were cultured in DMEM (Mediatech, Inc, USA) containing 10% heat inactivated FBS (Hyclone Laboratories, Inc., USA), 1% penicillin/streptomycin mix (Invitrogen, USA) and 0.1% fungizone (Invitrogen), and passaged when > 90% confluency was achieved. These media conditions were used throughout for all media conditions noted as ‘‘non-converting’’ or ‘‘normal’’. Tachyzoites were maintained by serial passage in human foreskin fibroblasts (HFFs) in DMEM + 10% FBS, 1% penicillin/streptomycin mix and 0.1% fungizone media as previously described (Roos et al., 1994). To purify tachyzoites for infection assays, infected HFF monolayers were scraped from culture flasks, needle passaged, and filtered using a 3-micron Nucleopore membrane. All cell lines

were routinely tested for Mycoplasma contamination and found to be negative.

2.2. Parasite conversion and growth assays Unless otherwise stated, parasite conversion was induced by culture in minimal essential media (MEM, Invitrogen) without bicarbonate, with 25 mM HEPES (Invitrogen), 2 mM L-glutamine (Invitrogen), 1% FBS and 1% penicillin/streptomycin mix, pH 7.0, at ambient CO2 as previously described (Dzierszinski et al., 2004). Host cells were trypsinized, counted and placed into 15 mL polypropylene tubes in normal media. Purified parasites were added to host cells at a multiplicity of infection (MOI) of 4:1, mixed thoroughly by gentle vortexing, then pelleted together for 10 min at 2, 000g and incubated for 30 min at 37 °C in 5% CO2. Cells and parasites were resuspended, pelleted at 1, 000g and washed once with normal media to wash away extracellular parasites. Infected cells were then placed into bradyzoite-inducing culture conditions in six or 12 well plates. After 36–72 h, infected cells were trypsinized and analyzed via flow cytometry using either a Beckman Coulter FC-500 or a Coulter EPICS XL, and data were plotted using FlowJo software, version 4 (Tree Star, USA).

2.3. Expression plasmids and transduction All genes were expressed in the MSCV retroviral vector containing an IRES-Thy1.1 surface marker protein. Plasmid DNA was transfected into Bosc23 packaging cells, and 48 h later supernatant was harvested and used to transduce target cells. The Thy1.1 marker was used to purify transduced cells using a biotinylated antiThy1.1 antibody (BD Pharmingen, USA) and streptavidin-coated magnetic beads (Miltenyi Biotec, USA). Thy1.1 was also used as a marker for transduced cells during infection of mixed cell populations. Infected cells were stained using biotinylated anti-Thy1.1 antibody followed by strepavidin-conjugated PE-Cy5 (BD Pharmingen) to allow for analysis of conversion within transduced and untransduced cell populations.

2.4. Supernatant collection and treatments Cells (293T and NIH3T3) were extensively washed prior to incubation at a concentration of 3  107 per ml in Hank’s Balanced Salt Solution without calcium or magnesium, pH 7.0 (HBSS; Invitrogen) for 6 h in a 12 well tissue culture dish after which supernatants, together with a mock cell control, were collected and stored at 4 °C. The supernatant treatments were performed as follows: (i) supernatants were spun through Amicon 3 kD cutoff columns (Merck Millipore, USA) at 3, 700g until less than 50 ll volume remained. (ii) Boiling: supernatants were boiled in 1.5 ml eppendorf tubes for 10 min, together with buffer control, and allowed to cool before addition to cultures. (iii) Proteinase K and trypsin: supernatants were incubated with 0.1 mg/ml of proteinase K (Invitrogen) or 0.25% trypsin (Invitrogen) for 1 h at 37 °C, then spun through a 3 kD cut-off column to retain the enzymes. (iv) Dialysis (1 kD): supernatants were dialyzed using Spectra/Por1 kD dialysis tubing (Cole-Palmer, USA) for 24 h at 4 °C into 1 L of MEM used in conversion assay. For the inhibitor treatment experiment, 293T cells were plated at 2  107 per 15 cm2 dish and incubated with or without sodium oxamate for 48 h. Phloretin was added to cells 4 h prior to harvest. After 48 h, cells were trypsinized and washed extensively, then incubated in HBSS as described above. For all experiments, unless otherwise indicated, supernatant or control was added to converting cultures at 5% total media volume.

D.R. Weilhammer et al. / International Journal for Parasitology 42 (2012) 947–959

2.5. Cell cycle analysis HFFs (1  106) were harvested, washed twice and resuspended in 500 ll of PBS. To fix cells, 5 ml of ice-cold ethanol was added drop-wise while lightly vortexing and incubated on ice for 1 h. Cells were then pelleted, washed twice with PBS, then resuspended in 300 ll of 69 lM propidium iodide (Sigma–Aldrich, USA) solution containing 0.6 lg/ml of RNAse A (Qiagen, USA) and incubated at 37 °C for 45 min. Cells were then analyzed for DNA content using a Beckman Coulter EPICS XL and data were plotted using FlowJo software (Tree Star). 2.6. HILIC columns HILIC Macro-spin SPE columns were purchased from the Nest Group, Inc (Southborough, MA, USA). Columns were wetted with methanol, rinsed with distilled water, conditioned with 0.2 M sodium acetate, 0.3 M sodium phosphate buffer, for 2 h, and rinsed with 90% acetonitrile (Fisher, USA)/10% aqueous 30 mM ammonium formate (Fisher). Supernatant (500 ll) in 30 mM ammonium formate was diluted to 90% acetonitrile, then spun over columns in 0.5 ml aliquots. Columns were washed twice with 90% acetonitrile/ 10% aqueous 30 mM ammonium formate and retained compounds were eluted using a 10% step-wise gradient of acetonitrile/aqueous 30 mM ammonium formate, 250 ll per elution. Column fractions were lyophilized and resuspended in water.

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converting conditions, bicarbonate, glucose and FBS were added to final concentrations of 3.7 g/L, 4 g/L and 10%, respectively. Perchloric acid was added to supernatants at a final concentration of 0.6% before measurement was performed to precipitate serum protein. Lactate measurement was performed enzymatically as previously described (Gutmann and Wahlefeld, 1974). 2.9. Chemicals The following chemicals were all purchased from Sigma and were added to bradyzoite-inducing media at the following concentrations: uridine (0.1 mM), thymine (0.4 mM), thymidine (0.4 mM), adenosine (0.5 mM), choline (0.5 mM), alanine (10 mM), ammonium chloride (0.75 mM), nicotinic acid (0.4 mM), glutamine (5 mM), creatinine (0.5 mM), sodium oxamate (20 mM), and phloretin (40 lM). Lactate was used either as lactic acid (Fisher Scientific, USA) neutralized with 10 N sodium hydroxide or as Lactated Ringer’s solution (Baxter Healthcare, USA). Pyruvate (Invitrogen) was used at 5 mM. Three-methyl adenine (3-MA), adenine, and Akt VIII inhibitor were all purchased from Calbiochem and were used at 5 mM, 100 lM, and 100 nM, respectively. Glucose (Fisher, USA) was added to a final concentration of 4 g/L. 3. Results 3.1. Stage conversion assay

2.7. Mass spectrometry Supernatant fractions were analyzed using an LTQ Orbitrap XL hybrid mass spectrometer equipped with an Ion Max electrospray ionization source (ESI; Thermo Fisher Scientific, Waltham, MA, USA) that was connected in-line with an Agilent 1200 series autosampler and quaternary pump (Santa Clara, CA, USA). Sample solutions contained in autosampler vials sealed with septa caps were loaded into the autosampler compartment prior to analysis. An injection volume of 75 lL was used for each sample. The injected sample aliquot was pumped to the ESI probe of the mass spectrometer for a period of 8 min using a flow of 99.9% acetonitrile/0.1% formic acid (v/v) delivered at a flow rate of 50 lL/min. Solvent (Milli-Q water) blanks were run between samples, and the autosampler injection needle was rinsed with Milli-Q water after each sample injection, to avoid cross-contamination between samples. The connection between the autosampler and the ESI probe of the mass spectrometer was made using PEEK tubing (outer diameter 0.005 in., inner diameter 1/16 in.; Western Analytical, Lake Elsinore, CA, USA). External mass calibration was performed prior to analysis using the standard LTQ calibration mixture containing caffeine, the peptide MRFA, and Ultramark 1621 dissolved in 51% acetonitrile/25% methanol/23% water/1% acetic acid solution (v/ v). The ESI source parameters were as follows: ion transfer capillary temperature 275 °C, normalized sheath gas (nitrogen) flow rate 25%, ESI voltage 2.0 kV, ion transfer capillary voltage 20 V and tube lens voltage 45 V. Mass spectra were recorded in the positive ion mode over the range m/z = 80 to 1, 500 using the Orbitrap mass analyzer, in profile format, with a full MS automatic gain control target setting of 5  105 charges and a resolution setting of 6  104 (at m/z = 400, full width at half-maximum height). Mass spectra were processed using Xcalibur software (version 4.1, Thermo Fisher Scientific). 2.8. Lactate measurement Samples for lactate measurements were collected in MEM without phenol red or bicarbonate (Sigma), with 25 mM HEPES, 2 mM L-glutamine, 1% penicillin/streptomycin mix and 1% FBS. For non-

To study T. gondii stage conversion, we utilized a previously described in vitro system that reproduces many aspects of early in vivo bradyzoite differentiation (Dzierszinski et al., 2004). Briefly, conversion is induced by culturing HFF cells infected with a Type II Pru strain of parasites at ambient CO2 in bicarbonate-free media, thereby limiting formation of carbamoyl phosphate, an intermediary metabolite in the urea cycle and pyrimidine synthesis. Carbon dioxide restriction provides a metabolic stress that has been utilized as a standardized condition to study T. gondii gene regulation (see toxodb.org), and reproducibly induces bradyzoite differentiation with minimal toxicity to host cells (Dzierszinski et al., 2004). Conversion was assessed by the induction of a fluorescent reporter gene driven by the bradyzoite-specific bag-1 promoter, and while bag-1 expression represents an early event in bradyzoite differentiation, transgenic reporter expression was tightly correlated to later expression of multiple markers of bradyzoite conversion including Dolichos lectin cyst wall staining (Dzierszinski et al., 2004). In our adaptation of this in vitro system, induction of conversion was measured using a dual transgenic Type II Pru strain with a constitutive RFP reporter gene driven by the gra8 promoter and an inducible YFP reporter gene driven by the bag-1 promoter. Flow cytometric analysis of infected host cells allowed accurate quantitation of parasite load via RFP expression and specific induction of bradyzoite differentiation under CO2 restriction via YFP expression (Fig. 1A). Thus, the level of conversion was determined by calculating the ratio of (YFP+ host cells)/(total RFP+ host cells). Because bradyzoite differentiation is both dependent upon parasite replication and ultimately leads to decreased parasite growth (Dubey et al., 1998), bradyzoite conversion was quantitated at a time point of 48 h after imposing CO2 restriction. Although at later time points YFP+RFP+ bradyzoites ceased division and began to form stable cysts, YFP-RFP+ tachyzoites continued dividing, resulting in lysis of initial host cells and significant reinfection of new host cells. Thus, at 48 h, the alternative parasite-fate decision between remaining a YFP RFP+ tachyzoite or converting to a YFP+RFP+ bradyzoite could be most readily visualized and accurately measured.

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bag 1-YFP Fig. 1. Cell types exhibit both conversion-resistant and conversion-permissive phenotypes. (A) Assay for induction and measurement of Toxoplasma gondii bradyzoite conversion. Human foreskin fibroblasts (HFF) cells were infected with a Pru strain engineered with a constitutive gra8-red fluorescent protein (RFP) transgene and a bradyzoite-specific bag 1-yellow fluorescent protein (YFP) transgene and cultured in bicarbonate-free media with or without 5% CO2. Infected cells were analyzed for RFP and YFP expression by flow cytometry 48 h p.i. Bar graphs represent bradyzoite conversion as the percentage ratio of (YFP+ cells)/(RFP+ cells) calculated from flow cytometry data. (B) Bradyzoite conversion occurs readily in permissive HFF and Vero cells, but not in resistant NIH3T3 and 293T cells. Conversion of infected cell lines was measured under bradyzoite-inducing conditions as described in (A). RFP+ and YFP+ gates are set using uninfected populations of each cell type, due to differences in inherent autofluorescent properties. Bar graphs represent the average bradyzoite conversion calculated from five independent experiments.

3.2. Cell types are either permissive or resistant for conversion In order to explore the impact of host cell environment on stage conversion, the efficiency of conversion in cell types with different metabolic properties was determined (Pavlides et al., 2009): fibroblasts (HFF and NIH3T3 cells) and tumor cell lines (Vero and 293T). Interestingly, it was found that cells were either permissive or resistant for conversion (Fig. 1B). Conversion in permissive cells ranged from 30% to 60%, whereas conversion in resistant cells was consistently less than 5% (Fig. 1B). One prediction, based upon previous findings that slowing of host cell growth was coincident with slowing of parasite growth and subsequent induction of bradyzoite differentiation (Radke et al., 2006), and the fact that bradyzoite cysts in vivo are found within cells with low to absent

replicative capacity (Coppens and Joiner, 2001), was that faster growing tumor cells would be less efficient at facilitating parasite conversion. Surprisingly, conversion efficiency did not correlate with growth properties of the host cell, as Vero and 293T cells, both tumor cell lines with a fast doubling time, displayed very different abilities to facilitate conversion (Fig. 1B). Fibroblast HFF and NIH3T3 cells also displayed dissimilar abilities to facilitate conversion, with HFFs providing a permissive cellular environment while NIH3T3 cells provided a resistant cellular environment for conversion. The discrepancy between permissive and resistant cells was observed only under bradyzoite-inducing conditions; spontaneous conversion was not detected within any of the four cell lines under tachyzoite growth conditions (data not shown). Parasites were similarly able to replicate within the four cell types (Fig. 1B, based

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on total levels of RFP+ cells and observations made by fluorescent microscopy, data not shown), therefore the differences in conversion could not be attributed to slower parasite growth within permissive cell types. These results suggested an unappreciated characteristic of the host cell environment was significantly impacting upon bradyzoite development and prompted exploration of the molecular basis for the conversion-resistant phenotype of NIH3T3 and 293T cell lines. Interestingly, the conversion-resistant phenotype of NIH3T3 and 293T cell lines was cell non-autonomous. Co-culture of infected permissive cells with increasing numbers of uninfected resistant cells resulted in dose-dependent inhibition of bradyzoite differentiation (Fig. 2A). In contrast, co-culture with increasing numbers of uninfected conversion-permissive cells did not inhibit bradyzoite differentiation. In order to assess the relative strength of the inhibition of bradyzoite differentiation by resistant cells, conversion within the same number of total infected cells was examined while varying the ratio of permissive to resistant cells (Fig. 2B). The level of conversion within both permissive and resistant cells decreased steadily with increasing numbers of resistant cells, suggesting that inhibition by resistant cells was mediated by a titratable, yet saturable diffusible factor. Furthermore, these results suggested that the low levels of conversion typically seen within resistant cells were not due to an inability of those cells

to respond to the conversion stimulus. When the source of this diffusible factor was limited, the level of conversion within resistant cells increased dramatically, from less than 5% when seeded as 100% resistant cells, to nearly 20% when seeded as 10% resistant/ 90% permissive cells, for both 293T and NIH3T3 cell types (Fig. 2B). 3.3. Soluble metabolites inhibit bradyzoite differentiation and maintain parasite growth We next sought to confirm whether the inhibition of bradyzoite induction by resistant cells was indeed mediated by a soluble factor or required cell-cell contact for its action. Supernatants collected from NIH3T3 and 293T cells were added to converting cultures at 5% of the total media volume and were found to potently inhibit conversion in both HFF and Vero cells compared with the addition of mock supernatant of collection buffer alone (Fig. 3A). These results indicate that the inhibition was mediated by soluble factors and attention was focused on characterizing the inhibitory activity of supernatants from resistant cells. The ability of supernatants from resistant cells to inhibit expression of an early bradyzoite-specific reporter suggested that factors present in the supernatant could support growth of more rapidly dividing tachyzoites under converting conditions. To determine whether parasite growth was enhanced by addition of 293T

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Fig. 2. Conversion-resistant cells inhibit Toxoplasma gondii bradyzoite conversion in trans. (A) Conversion within permissive cells is inhibited by co-culture with uninfected resistant cells. Infected human foreskin fibroblasts (HFF) or Vero cells (1  105) expressing a retroviral Thy1.1 marker were cultured alone (black bar), or with the addition of 1, 2 or 3  105 uninfected wild type HFF or Vero cells (hatched bars), or NIH3T3 or 293T cells (white bars) per six well dish and conversion was determined in Thy1+ cells. (B) Inhibition by resistant cells is titratable and saturable. Individual cell types were infected separately (as described in Section 2.2), then plated at different ratios of resistant to permissive cells to a total of 4  105 infected cells per six well dish, and conversion within both populations was measured using a unique Thy1.1 retroviral marker on permissive cells to distinguish infected cell populations. Experiments shown in (A) and (B) are representative of a minimum of three independent experiments.

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Fig. 3. Resistant cells release soluble factors that inhibit Toxoplasma gondii bradyzoite differentiation and promote continued parasite growth without altering host cell-cycle progression. (A) Supernatants collected from conversion-resistant cells inhibit parasite conversion. Supernatants collected in conversion media from washed 293T or NIH3T3 cells were compared with mock supernatant for their ability to inhibit conversion in human foreskin fibroblast (HFF) or Vero cells. Supernatant or media control was added to converting cultures at 5% total media volume. Bar graphs show mean values of triplicate samples with S.D. error bars from a representative experiment performed a minimum of four times. (B) Supernatant from 293T cells promotes continued parasite growth under bradyzoite-inducing conditions. HFF cells infected with a Pru strain containing a yellow fluorescent protein marker under transcriptional control of the tubulin promoter (tub-YFP2), allowing accurate determination of parasite load/host cell, were cultured under non-converting or bradyzoite-inducing conditions with (heavy black line) and without (shaded) addition of 293T cell supernatant and analyzed by flow cytometry at 36 h for YFP expression. The percentage of infected HFF cells falling within the individual YFP peaks corresponding to the indicated parasite load/cell (1, 2, 4, etc. parasites per cell; noted on histograms) was determined. The fold change in host cells containing the indicated parasite loads induced by addition of 293T supernatant was calculated from the ratio of (% host cells with X parasites in presence of 293T supernatant)/(% host cells with X parasites in presence of mock control supernatant). Bar graphs show mean values of three independent experiments with S.D. error bars. ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001 (Student’s t-test). (C) Supernatants from 293T cells do not alter host cell-cycle progression under bradyzoite-inducing conditions. Mock control media, 293T cell supernatant, or 10% FBS was added to uninfected HFF cells pre-cultured for 2 days under converting conditions. After an additional 2 days, cells were stained with propidium iodide (PI), cell-cycle analysis was performed by flow cytometry for DNA content and the percentages of cells in G1, S and G2/M phases were determined by Watson (Pragmatic) analysis (Watson et al., 1987). The experiment shown is representative of three independent experiments.

supernatant, a Pru strain with a YFP reporter gene driven by a tubulin promoter that allows accurate quantitation of parasite load per host cell by flow cytometry was employed. Under nutrient-rich non-converting conditions, addition of the supernatant had no effect upon parasite growth; however, there was significant enhancement of parasite growth under bradyzoite inducing conditions, as evidenced by the approximately 1.5 and threefold increase in the number of host cells containing eight and 16 parasites, respectively (Fig. 3B). These results are consistent with the ability of supernatants from conversion-resistant cells to maintain parasite growth as tachyzoites under conditions of metabolic stress. Next, it was determined whether inhibitory supernatants were potentially driving host cells into enter the cell-cycle and thereby inhibiting bradyzoite conversion, given the recent demonstration that cell-cycle arrest of host cells induced by human CDA-1 gene expression triggers bradyzoite differentiation (Radke et al., 2006). Supernatants from 293T cells did not trigger host cell-cycle

progression under bradyzoite-inducing conditions, compared with the addition of serum (Fig. 3C). These results indicate that the enhancement of parasite growth induced by 293T supernatant was not driven by changes in the host cell-cycle status. Biochemical characterization of the inhibitory activity of 293T supernatants indicated that all of the activity partitioned in the flow through of 3 kD molecular weight cut-off columns (Fig. 4A). Conversely, all activity was lost after removal of compounds less than 1 kD by dialysis. Inhibitory activity was not diminished by protease treatment, boiling nor lyophilization. These results indicate that the active inhibitory compounds were entirely small molecular weight, non-protein molecules. Additionally, the lack of effect of lyophilization on activity is an important control that indicates that the inhibitory compound is not CO2 itself. The involvement of non-protein molecules less than 1–3 kD in molecular weight suggested that the parasite senses metabolite/s released from NIH3T3 and 293T cells that support continued

D.R. Weilhammer et al. / International Journal for Parasitology 42 (2012) 947–959

A

engineered to be defective in pyrimidine synthesis (Fox and Bzik, 2002). Thus, despite its dependence upon metabolic stress, the conversion assay was not overly sensitized to non-specific inhibition by indiscriminate additional metabolites. (Note: there is a switch in the presentation of the data in this figure from % conversion to fold inhibition of conversion. Normalizing the level of conversion under experimental conditions to conversion under mock untreated conditions allows more accurate comparison between experiments).

125

% activity (vs control)

953

100 75 50 25

3.4. Inhibitory factors also block 3-MA induced bradyzoite differentiation try pr ps ot in ei na se K lyo boi l i n ph iliz g at io n

>

<

3k D 1k D

0

B uridine adenine thymidine choline thymine ammonium glutamine nicotinic acid creatinine alanine adenosine 0.25x

0.5x

1x

2x

4x

Inhibition of conversion induced by CO2 restriction Fig. 4. Soluble activity is mediated entirely by low molecular weight compounds. (A) Biochemical properties of soluble activity are consistent with non-protein compounds less than 1–3 kD in molecular weight. Supernatants from 293T cells were fractionated to purify components less than 3 kD by centrifugation through molecular weight cutoff columns, or greater than 1 kD by dialysis, treated to destroy protein activity by protease treatment or boiling, or lyophilized and rehydrated to eliminate volatile compounds. The percentage of activity was calculated from the ratio of conversion in human foreskin fibroblasts (HFF) cells in the presence of treated versus untreated 293T supernatants. Results are representative of three independent experiments. (B) Uridine specifically inhibits conversion induced by CO2 restriction. The indicated metabolites were tested for their ability to modulate Toxoplasma gondii bradyzoite conversion induced by CO2 restriction in HFF cells. The fold change in inhibition induced by different metabolites was calculated from the ratio of (% conversion in the presence of metabolite)/(% conversion without metabolite). Bar graphs show mean values of triplicate experiments with S.D. error bars.

growth as a tachyzoite under conditions of metabolic stress. This prompted us to test the effects in this conversion assay of a panel of commonly secreted metabolites involved in either host or parasite metabolism (Fig. 4B). These metabolites included alanine, adenosine and ammonium released from metabolically active host cells, as well as choline for which T. gondii is functionally auxotrophic. Although the addition of a number of metabolites, such as adenosine, could modestly augment bradyzoite conversion, uridine was the only metabolite initially tested that inhibited bradyzoite conversion at levels approaching the activity of supernatants from resistant cells. The inhibition of bradyzoite conversion by uridine was a predicted result given its ability to complement the defect in pyrimidine synthesis induced by the metabolic stress of CO2 restriction, and its use in the selection of parasites genetically

To examine whether the phenomenon of trans inhibition of conversion was specific to the particular metabolic stress of CO2 restriction, we sought to determine whether 293T supernatant could inhibit conversion induced by a different method. Other published methods of inducing conversion, such as high pH and heat shock (Soete et al., 1994), did not result in robust conversion in our hands; therefore, we sought to identify an alternative method of induction. We reasoned that inhibition of autophagy, which is triggered by parasite infection of host cells to provide an increased source of amino acids and other nutrients (Wang et al., 2009), might provide a distinct metabolic stress that could trigger bradyzoite differentiation. Addition of the autophagy inhibitor, 3-MA, to infected Vero cells cultured under non-converting conditions (DMEM + 10% FBS + 5% CO2) successfully triggered bradyzoite conversion (Supplementary Fig. S1A). Consistent with 3-MA treatment representing a distinct metabolic stress from CO2 restriction, addition of adenine, but not uridine, preferentially reversed the induction of bradyzoite differentiation (Supplementary Fig. S1B). Importantly, addition of 293T supernatant inhibited bradyzoite conversion mediated by either CO2 restriction or 3-MA at levels equivalent to the maximal inhibition that could be achieved with the addition of uridine or adenine (Fig. 4B and Supplementary Fig. S1), which are predicted to be specific inhibitors of those respective assays. 3.5. Identification of lactate as an inhibitory factor We next sought to biochemically identify the metabolite(s) released in cellular supernatants that inhibited bradyzoite conversion. As a starting point for purification, supernatants from washed 293T cells was collected in HBSS and then high molecular weight compounds were removed using 3 kD cutoff columns. Using HILIC chromatography, it was consistently found that activity from inhibitory supernatants was retained and eluted in the fraction corresponding to 70% acetonitrile/30% aqueous ammonium formate (Fig. 5A). Significant inhibitory activity was not retained on HILIC columns as evidenced by the activity observed in the flow through and initial column washes prior to elution, suggesting the presence of multiple inhibitory compounds with different hydrophilic properties in the cellular supernatants. Using an Orbitrap mass spectrometer equipped with an electrospray ionization source, a singly charged positive ion was measured at m/z = 135.003 from active fractions of 293T cells which was not measured from either neighboring inactive fractions or parallel fractions of control media alone supernatants (Fig. 5B). Supernatant collected from NIH3T3 cells was similarly fractionated by HILIC chromatography and analyzed by mass spectrometry, and the same singly charged ion at m/z = 135.003 was identified in active fractions (data not shown). Due to the small molecular weight and accuracy of the mass determination, this positive ion corresponded uniquely to an (M H + 2Na)+ ion, where M is the chemical formula C3H6O3. While there are a number of different metabolites with this formula, a disodium adduct was consistent

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A 70

conversion (%)

60 50 40 30 20 10 0

S FT 1 2 1 wash

2 3 4 elution

S FT 1 2 1 wash

5

100

135.003

5

293T

control

B

2 3 4 elution

(M - H + 2Na) + M = C3H6O3

80

relative abundance (%)

60

Active fraction

40 20

173.079 116.071

216.923

413.267

270.978

0 100 80 60

Inactive fraction

40 20

173.078 142.948 132.102

0 100

140

413.266

180

220

260

300

340

380

420

460

500

m/z Fig. 5. Biochemical identification of lactate as a component of inhibitory 293T supernatant fractions. (A) Fractionation of an inhibitory component of 293T cell supernatants by hydrophilic interaction (HILIC) chromatography. Supernatant from 293T cells and buffer control were passed over HILIC columns and supernatant (S), flow through (FT), washes and elution fractions were tested for inhibitory activity on Toxoplasma gondii bradyzoite conversion. The experiment shown is representative of four independent experiments. (B) Active fractions contain a major singly-charged positive ion corresponding to an acidic compound, C3H6O3, consistent with lactate. Mass spectra were measured for active and inactive fractions of inhibitory 293T cell supernatants from HILIC chromatography. The singly charged positive ion at m/z = 135.003 (active fraction) corresponds to the (M – H + 2Na)+ ion of M = C3H6O3.

with a hydroxyacid, making lactate the best candidate for this peak. Lactate was next tested directly for the ability to inhibit conversion in permissive cells (Supplementary Fig. S2A). Lactate was found to exhibit a dose-dependent inhibition of conversion within Vero cells at physiologically relevant concentrations of 1–3 mM, but had little to no effect on inhibiting conversion within HFF cells. The selective inhibition of conversion within Vero, but not HFF, cells strongly indicates that lactate was not fully responsible for the inhibitory activity of supernatants from resistant cells, consistent with the lack of complete retention of inhibitory activity by HILIC chromatography. While lactate cannot account for all of the activity of the supernatant, it was determined whether lactate accounted for a significant portion of the inhibitory activity on Vero cells. Two inhibitors that block lactate release by different mechanisms were utilized (sodium oxamate inhibits the lactate dehydrogenase (LDH) enzyme that converts pyruvate to lactate, and phloretin blocks extracellular lactate release via cell-surface monocarboxylate transporters), and it was determined that both inhibitors block lactate release by 293T cells under both non-converting and

bradyzoite-inducing culture conditions (Supplementary Fig. S2B). Pretreatment of 293T cells with either sodium oxamate or phloretin decreased the inhibitory activity of 293T cell supernatants on bradyzoite formation (Supplementary Fig. S2C). Calculation of the relative activity of supernatants collected from inhibitor-treated cells versus control untreated cells indicated that lactate is responsible for approximately 30% of the inhibitory activity of 293T supernatant on conversion within Vero cells. 3.6. Natural induction of glycolysis shifts permissive cells to a resistant phenotype The identification of the glycolytic metabolite lactate as an active inhibitory component of 293T and NIH3T3 supernatants prompted us to explore the role of glycolysis in the regulation of conversion within permissive HFF and Vero cells. We first addressed whether induction of glycolysis in permissive cells could render them resistant to conversion by switching them from the normal physiological concentration of glucose of 1 g/L used in the conversion assays to a high glucose concentration of 4 g/L used in high-glucose media formulations. High glucose enhanced lactate

D.R. Weilhammer et al. / International Journal for Parasitology 42 (2012) 947–959

release in HFF but not Vero cells (Fig. 6A), indicating that HFF cells responded to increased extracellular glucose by upregulating glycolysis, while Vero cells did not. This difference in induction of gly-

A lactate (mM)

0.75

HFF Vero

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ns

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4 1 glucose (g/L)

4

B 1 g/L glucose 103

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101

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100

gra 8-RFP

103

100

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101

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100

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10-1 -1 10

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18

22

Vero

100

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103

bag 1-YFP ns

conversion (%)

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60

***

50

3.7. Akt expression inhibits bradyzoite differentiation

HFF

10-1 103 10-1

102

6.1

40

10-1 103 10-1

102

17

colysis correlated with the ability of high glucose to inhibit bradyzoite conversion in HFF, but not Vero, cells (Fig. 6B). These results indicate that the presence of increased extracellular glucose was not by itself sufficient to inhibit parasite conversion; rather, the induction of host glycolysis as evidenced by lactate release was also required to inhibit bradyzoite differentiation within permissive cells. The complete insensitivity of conversion within Vero cells to extracellular glucose levels allowed examination of whether induction of glycolysis in HFF cells now rendered those capable of inhibiting conversion in trans. The addition of uninfected HFF cells to cultures of infected Vero cells induced to undergo conversion in high glucose conditions resulted in inhibition of conversion within Vero cells compared with conversion within Vero cells alone under high glucose conditions (Fig. 6C). Similar to results in Fig. 2A, the addition of uninfected Vero cells had no effect upon conversion under high glucose conditions (Fig. 6C). These results demonstrate that natural induction of glycolysis in permissive HFF cells was sufficient to recapitulate the conversion-resistant phenotype of 293T and NIH3T3 cells, both in terms of lower conversion within HFF cells themselves, and the ability to inhibit conversion in trans within Vero cells.

4 g/L glucose 103

102

10-1 10-1

955

40 30 20

To confirm that enhanced glycolysis can convert permissive cells to a resistant phenotype, a dominant-active form of Akt was utilized to force induction of glycolysis within HFF and Vero cells. Akt is a serine/threonine kinase involved in the regulation of many diverse host cellular functions, including growth and proliferation, survival and cellular metabolism (Manning and Cantley, 2007). Myristolated Akt (myrAkt) acts as a dominant-active kinase via constitutive localization to the plasma membrane and expression of myrAkt has previously been shown to enhance glycolysis, with a corresponding increase in lactate production in both transformed and non-transformed cell lines (Rathmell et al., 2003; Elstrom et al., 2004). Akt activity is also physiologically relevant to T. gondii biology as parasite infection induces host Akt activation and promotes parasite survival by inhibition of host cell apoptosis (Kim and Denkers, 2006). Expression of myrAkt enhanced lactate production in both HFF and Vero cells (Fig. 7A), indicating that glycolysis was induced in both cell types. The effect of myrAkt expression on conversion was assessed in mixed populations of cells that were either transduced or untransduced with the myrAKT-IRES-Thy1.1 vector or a control IRES-Thy1.1 vector. Infected cells were cultured under bradyzoite-inducing conditions and conversion was assessed within both transduced and untransduced cells using an anti-Thy1.1

10 0

3 1

4 1 glucose (g/L)

4

Inhibition of conversion (Vero)

C ***

2x

ns 1x

0.5x

+

+

+ glucose

-

+

+ HFF - Vero

Fig. 6. Natural induction of glycolysis by high glucose shifts permissive human foreskin fibroblasts (HFF) cells to a conversion-resistant phenotype capable of cellextrinsic inhibition of Toxoplasma gondii bradyzoite conversion. (A) Added glucose enhances glycolysis in HFF cells, but not Vero cells. HFF and Vero cells were cultured under bradyzoite-inducing conditions at either 1 g/L or 4 g/L of glucose. Lactate concentration in supernatants was measured at 24 h. (B) Added glucose inhibits bradyzoite conversion within HFF cells, but not Vero cells. Conversion was measured as described in Fig. 1 in infected HFF and Vero cells under bradyzoiteinducing conditions at either 1 g/L or 4 g/L of glucose. Bar graphs in (A) and (B) show mean values of triplicate samples with S.D. error bars from a representative experiment performed three times, ⁄⁄⁄P < 0.001 (Student’s t test). ns; not significant. (C) High glucose renders HFF cells capable of cell-extrinsic inhibition of bradyzoite conversion. Infected Vero cells (1  105) expressing a Thy1.1 retroviral marker were cultured under converting conditions at 1 g/L of glucose and at 4 g/L of glucose alone or 4 g/L of glucose with the addition of 9  105 uninfected Vero or HFF cells in a 10 cm2 dish. The fold change in inhibition of conversion was calculated from the ratio of (conversion at 4 g/L of glucose, +/ uninfected cells)/(conversion at 1 g/L of glucose). Bar graphs in (C) show mean values of triplicate experiments with S.D. error bars, ⁄⁄⁄P < 0.001 (Student’s t-test). ns, not significant.

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A lactate (mM)

1.5

HFF Vero

***

1.0

*** 0.5

0

–

+

–

+

myrAkt IRES Thy1.1

B

C Thy1.1 – 10

16

10 1

10 1

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IRES Thy1.1

10 -1

10 -1 10 0

10 1

10 3 10 -1

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7

10 3

10 2

10 2

10 1

10 1

10 0

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Thy1.1 + 33

5.2

myrAKT IRES Thy1.1

untransduced myrAkt IRES Thy1.1 4x

10 1

10 2

10 3 10 -1

*

* **

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Vero

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co-culture glucose – –

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bag 1-YFP

D

E

antiapoptotic

myr-Akt z-VAD bcl-2 + z-VAD p65 c-Rel IGTP LRG-47 GTPI 0.25x

0.5x

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conversion (%)

50

empty vector bcl-2

***

40 30 20 10 0

p47 GTPase

1x

2x

–

+

Akt VIII inhibitor

4x

host gene inhibition of conversion Fig. 7. Akt activity controls both cell-intrinsic and cell-extrinsic inhibition of bradyzoite conversion through induction of glycolysis. (A) Dominant-active Akt expression enhances glycolysis in both human foreskin fibroblasts (HFF) and Vero cells. MyrAkt expressing or wild type cells were cultured under bradyzoite-inducing conditions, and lactate concentration in supernatants determined at 24 h. The plus and minus signs are indicative of myrAkt expression. Bar graphs show mean values of triplicate samples with S.D. error bars from a representative experiment performed three times, ⁄⁄⁄P < 0.001 (Student’s t-test). (B) Dominant-active Akt expression results in both cell-intrinsic and cell-extrinsic inhibition of Toxoplasma gondii bradyzoite conversion. Bradyzoite conversion was assessed within mixed populations of HFFs that were retrovirally transduced with either a myrAKT-IRES-Thy1.1 or IRES-Thy1 empty vector control. Conversion was measured as described in Fig. 1, and was assessed within transduced and untransduced cell populations by using the Thy1.1 marker. Data shown is representative of a minimum of three independent experiments. (C) Inhibition of bradyzoite conversion by dominant-active Akt expression occurs via its role in activation of glycolysis. Conversion was assessed within untransduced and myrAkt-IRES-Thy1.1 expressing Vero and HFF cells in co-culture conditions indicated by brackets, in the presence of either high glucose (4 g/L) or normal glucose (1 g/L) indicated by the plus and minus signs, respectively. Conversion within specific populations was distinguished using the Thy1.1 marker, and the fold change in inhibition of conversion was calculated from the ratio of (conversion in indicated cell population under indicated co-culture conditions)/(conversion in wild-type cells alone under normal glucose conditions). (D) Inhibition of bradyzoite conversion by activated Akt does not correlate with its anti-apoptotic function. Conversion was assessed in HFFs transduced with retroviral vectors expressing host genes corresponding to anti-apoptotic function, NF-jB or p47 GTPase gene families. Anti-apoptotic function was additionally tested using the pan-caspase inhibitor z-VAD. The fold change in inhibition of conversion by different host genes was calculated from the ratio of (conversion in presence of retroviral gene expression)/(conversion in wild-type cells). Bar graphs in (C) and (D) show mean values of triplicate experiments with S.D. error bars. ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001 (Student’s t-test). (E) Inhibition of endogenous Akt activity enhances conversion. Conversion was measured in wild-type HFF cells the presence or absence of 100 nM Akt VIII inhibitor that specifically inhibits Akt activity. Bar graph shows mean values of triplicate samples with S.D. error bars from a representative experiment performed three times, ⁄⁄⁄P < 0.001 (Student’s t- test).

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antibody, similar to experiments conducted in Fig. 2. MyrAkt expression inhibited conversion both within transduced cells and within neighboring untransduced cells (Fig. 7B), indicating again that induction of glycolysis results in the release of soluble mediators that can inhibit conversion in trans. Interestingly, this phenomenon was observed not only within HFF cells but also Vero cells. Conversion was inhibited within Vero cells expressing myrAkt and within neighboring untransfected cells (Fig. 7C), providing a similar correlation between the enhancement of lactate production and the ability to inhibit conversion in this cell type that was observed with HFF cells (Fig. 6). Experiments examining the simultaneous effects of glucose and myrAkt on conversion suggest that the effect of myrAkt is due to its role in cellular metabolism, rather than one of the many other cellular functions Akt impacts upon. In co-cultures of infected wildtype and myrAkt expressing Vero cells, the inhibition of bradyzoite conversion was augmented in both cell populations by the addition of exogenous glucose (Fig. 7C). A significant increase in the inhibition of conversion was observed within myrAkt expressing Vero cells (pair a, P = 0.03), and within the wild-type Vero cells co-cultured with myrAkt expressing cells (pair b, P < 0.05) when high glucose was added. This was entirely dependent upon myrAkt expression, since conversion within wild-type Vero cells is otherwise completely insensitive to glucose (Fig. 6B; wildtype Vero cells cultured alone in high glucose analyzed in comparison with wildtype cells cultured with myrAkt expressing Vero cells in high glucose in Fig. 7C as pair c with P = 0.003). In parallel experiments with HFF cells (Fig. 7C), the induction of glycolysis by a combination of myrAkt expression and high glucose resulted in almost total abrogation of conversion. Thus, the additive effects of myrAkt and glucose, particularly in Vero cells that in the absence of myrAkt expression are non-responsive to glucose, suggest that the inhibitory effect of Akt on parasite conversion is due to its role in cellular metabolism, as glucose metabolism is intricately tied to this function of Akt (Manning and Cantley, 2007). A limited parallel screen of several host gene families involved in T. gondii pathogenesis provides further implication of the metabolic function of Akt. Of those tested, only genes involved in regulation of apoptosis markedly affected bradyzoite differentiation (Fig. 7D). Neither expression of NF-jB transcription factors regulating inflammatory responses, nor p47 GTPase family members involved in host defense, significantly altered conversion within HFF cells. Interestingly, anti-apoptotic responses induced by bcl-2 and myrAkt expression resulted in opposing effects on conversion. The effects of bcl-2 on enhancing conversion could be phenocopied by the pan-caspase inhibitor z-VAD, suggesting that prevention of apoptosis facilitates survival of bradyzoites within host cells. The tendency of anti-apoptotic treatments to enhance parasite conversion suggests that the inhibitory effect of Akt is likely due to its role in cellular metabolism, rather than its role in cell survival, which has also been shown to be dependent on glucose metabolism (Rathmell et al., 2003). Although host Akt activity is induced by parasite infection (Kim and Denkers, 2006), it is less clear how sustained this activation is, prompting us to explore whether inhibition of endogenous Akt in infected HFF cells would also alter bradyzoite conversion. Addition of a specific inhibitor of Akt enhanced conversion in HFF cells (Fig. 7E), indicating endogenous Akt may play a role in regulating the bradyzoite developmental switch. In summary, the inhibition of conversion by myrAkt expression and the enhancement of conversion by endogenous Akt inhibition suggest that activation of Akt by parasite infection may play a role in supporting parasite growth – not only by promoting host cell survival but also by altering host cell metabolism, leading to enhanced glycolysis that promotes the growth of tachyzoites.

957

4. Discussion While evidence for host cell involvement is clear (Lüder et al., 1999; Radke et al., 2006; Ferreira da Silva et al., 2009a,b), the determinants of the host cell environment that favor differentiation of T. gondii into bradyzoites over sustained growth as tachyzoites, and vice versa, remain largely undefined. Using an in vitro system of bradyzoite induction, we identified a novel role for host metabolism in regulating the developmental switch of T. gondii from tachyzoites to bradyzoites. We showed that enhancement of host cell glycolysis can support tachyzoite growth under metabolic stress conditions and, thus, inhibit bradyzoite conversion. Furthermore, cell lines that are resistant to conversion, either intrinsically or due to the forced induction of glycolysis, release soluble mediators, exemplified by lactate, that can potently inhibit conversion in trans. This phenomenon of trans inhibition occurs not only in the context of conversion induced by the well-established metabolic stress condition of CO2 restriction (Bohne and Roos, 1997; Dzierszinski et al., 2004), but also in response to metabolic stress induced by 3-MA treatment that inhibits host cell autophagy induced by T. gondii infection (Wang et al., 2009). More recently, 3-MA has been shown to block parasite division, independently of its blockade of host cell autophagy (Wang et al., 2010), raising the issue of whether 3-MA induced bradyzoite differentiation was triggered through inhibition of parasite cell cycle or through inhibition of host cell autophagy. Interestingly, 3-MA did not trigger bradyzoite conversion in infected HFF cells (data not shown), suggesting that the effect of 3-MA was host-cell specific and likely not working directly on the parasite. In either scenario, 3-MA treatment represents a novel stress distinct from CO2 restriction that is both physiologically relevant to T. gondii biology and can be used to induce early bradyzoite differentiation. Meeting the large metabolic demands of apicomplexan parasites is critical to their growth and survival within host cells. Consequently, significant attention has been paid to how parasites acquire and utilize nutrients, particularly with respect to metabolic differences between the fast-replicating tachyzoite and quasi-dormant bradyzoite stages of the T. gondii life cycle (Tomavo, 2001). Several studies have demonstrated that T. gondii expresses glycolytic enzymes in a stage-specific manner (Yang and Parmley, 1997; Manger et al., 1998; Dzierszinski et al., 1999; Pomel et al., 2008). Interestingly, parasites utilize two distinct LDH isoforms with different kinetic properties (Dando et al., 2001) that are differentially expressed in tachyzoites and bradyzoites (Yang and Parmley, 1997), suggesting a potential role for lactate metabolism in the differentiation of bradyzoites. Studies aimed at broadly defining the sum total of host transcriptome and proteome changes in response to T. gondii infection have also identified host glycolytic enzymes as coordinately upregulated upon infection both at the RNA level (Blader et al., 2001) and protein level (Nelson et al., 2008), but the function or presumed benefit of this upregulation to T. gondii is not as well understood. This lack of understanding stems in part from an incomplete understanding of how T. gondii acquires energy sources from the host cell (Sinai, 2008). It has been postulated that mitochondrial recruitment to the parasitophorous vacuolar membrane upon T. gondii infection serves to provide the parasite with energy (Sibley, 2003), but whether that energy comes primarily in the form of glucose, glutamine or from other metabolic intermediates is not well understood (Polonais and Soldati-Favre, 2010). In fact, there is evidence that T. gondii may utilize a variety of energy sources, depending on metabolite availability. Mutant parasites that are defective in glucose transport exhibit a modest defect in tachyzoite growth and a marked defect in motility when extracellular in vitro (Blume et al., 2009). The defect in motility could be rescued by the addition of glutamine to culture media

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and intracellular mutant parasites were shown to incorporate 60% more glutamine than the control strain. Interestingly, these mutant parasites display no virulence defect in vivo (Blume et al., 2009). These results underscore the metabolic flexibility of T. gondii and in conjunction with our results, suggest the parasite may be able to utilize as yet unappreciated metabolites to support tachyzoite growth. Although the basis for upregulation of host glycolytic enzymes by T. gondii is not understood, it is consistent with studies demonstrating parasite-induced activation of two host regulatory proteins, Akt and HIF-1-a (Kim and Denkers, 2006; Spear et al., 2006). The significance of host Akt activation by parasite infection was initially ascribed to the anti-apoptotic function of Akt that appears to facilitate parasite survival in infected host cells (Kim and Denkers, 2006). Our data examining the consequences of modulating the anti-apoptotic state of infected host cells on conversion would suggest that an additional distinct consequence of Akt induction is to facilitate continued parasite growth through its induction of host-cell glycolysis. A key function of the transcription factor HIF-1-a which is induced by and coordinates cellular responses to hypoxia, is to upregulate glycolysis in response to the decreased availability of oxygen (Poellinger and Johnson, 2004). Toxoplasma gondii infection leads to the upregulation of host HIF-1-aactivity, and the induction of HIF-1-a-dependent host genes appears to be required for parasite growth at physiological oxygen levels (Spear et al., 2006). Intriguingly, induction of host HIF-1-aoccurs via a cellextrinsic mechanism where parasite secretion can induce HIF-1a activity in uninfected neighboring host cells (Spear et al., 2006). Taken together with our observation that host cell glycolysis in uninfected cells can inhibit conversion in neighboring infected cells, these results suggest that a reinforcing network for upregulation of host-cell glycolysis and continued parasite growth can be generated by the interplay between host and pathogen in infected tissues. Our initial goal in employing an in vitro assay of early bradyzoite induction was to gain molecular insight into the basis for preferential encystment within muscle tissue and neural tissues in vivo. However, our findings are arguably better described in the context of supporting tachyzoite growth rather than inhibition of bradyzoite conversion. This is perhaps not surprising, as parasite growth and differentiation are inextricably linked (Radke et al., 2003; Skariah et al., 2010), with a slowing of parasite growth always preceding the induction of the bradyzoite program. One striking, yet puzzling correlation with our data is that muscle and neural tissues are also highly glycolytic tissues that produce high levels of lactate (Winkler, 1981; Richardson et al., 1998; Brooks, 2002). Our in vitro data would predict that tachyzoite growth would be enhanced in highly glycolytic tissues producing lactate and currently we cannot explain this seeming contradiction. Certainly, the in vitro situation is not reflective of the entire story in vivo and much remains to be understood about the mechanism underlying the impact of glycolytic flux upon parasite growth and differentiation. There is not a strict correlation between the glycolytic properties of the host cell and permissiveness for conversion, as evidenced by the similar permissiveness for conversion of HFF and Vero cells despite their dissimilar glycolytic properties (Fig. 6A). More insight would undoubtedly be gained by determining the identity of other soluble factors, besides lactate, that possess inhibitory activity on bradyzoite conversion. The identification of glycolysis provides a starting point for the identification other such soluble factors. In summary, the notion that tachyzoite growth may be supported by the metabolism of not only the infected cell but also the surrounding tissue environment, provides a new framework in which to investigate preferential tissue encystment.

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