International Journal for Parasitology 41 (2011) 835–841

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International Journal for Parasitology

journal homepage: www.elsevier .com/locate / i jpara

Extracellular Toxoplasma gondii tachyzoites do not require carbon source uptakefor ATP maintenance, gliding motility and invasion in the first hour of theirextracellular life

San San Lin a, Martin Blume b, Nicolas von Ahsen c, Uwe Gross a, Wolfgang Bohne a,⇑a Institute of Medical Microbiology, University Medical Center Göttingen, Germanyb Department of Molecular Parasitology, Humboldt University Berlin, Germanyc Department of Clinical Chemistry, University Medical Center Göttingen, Germany

a r t i c l e i n f o

Article history:Received 9 December 2010Received in revised form 8 February 2011Accepted 2 March 2011Available online 7 April 2011

Keywords:Toxoplasma gondiiExtracellular stageEnergy metabolismGlycolysisMotilityATP

0020-7519/$36.00 � 2011 Australian Society for Paradoi:10.1016/j.ijpara.2011.03.005

⇑ Corresponding author. Address: Institute of MedMedical Center Göttingen, Kreuzbergring 57, Götting+49 551 395869; fax: +49 551 395861.

E-mail address: wbohne@gwdg.de (W. Bohne).

a b s t r a c t

Apicomplexan parasites undergo metabolic shifts in adaptation to environmental changes. Here, weinvestigate the metabolic requirements which are responsible for ATP homeostasis in the extracellularstage of Toxoplasma gondii. Surprisingly, we found that freshly released tachyzoites are able to maintaina constant ATP level during the first hour of extracellular incubation without the acquisition of externalcarbon sources. We further demonstrated that the extent of gliding motility and that of host cell invasionis independent from the availability of external carbon sources during this one hour extracellular period.The ATP level and the invasion efficiency of extracellular parasites were severely decreased by treatmentwith the glycolysis inhibitor, 2-deoxy-D-glucose, but not by the F0F1-ATPase inhibitor, oligomycin. Thissuggests that although the uptake of glucose itself is not required during the 1 h incubation period, extra-cellular parasites depend on the activity of the glycolytic pathway for ATP homeostasis. Furthermore,active glycolysis was evident by the secretion of lactate into the culture medium, even in the absenceof external carbon sources. Together, our studies suggest that tachyzoites are independent from externalcarbon sources within the first hour of their extracellular life, which is the most relevant time span forfinding a new host cell, but rely on the glycolytic metabolisation of internal carbon sources for ATP main-tenance, gliding motility and host cell invasion.

� 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Toxoplasma gondii encodes all of the genes required for glycoly-

Apicomplexan parasites can replicate within a wide spectrum ofmammalian cells and have been implicated in many important hu-man and veterinary diseases. One startling feature in many of theapicomplexan parasites is their niche adaptability, which includesthe capacity to refine the energy metabolism in response to envi-ronmental changes (Ginger, 2006). In Plasmodium spp., for exam-ple, metabolism for energy production shifts from glycolysis inthe intraerythrocytic stage to oxidative phosphorylation in game-tocytes (Daily et al., 2007; Kawahara et al., 2009; Polonais andSoldati-Favre, 2010). In Toxoplasma gondii, at least three phasesin the life cycle with differences in energy metabolism were iden-tified, including dormant bradyzoites, replicating intracellulartachyzoites and egressed extracellular tachyzoites (Tomavo,2001; Dzierszinski and Knoll, 2007; Weiss and Kim, 2007; Pomelet al., 2008; Polonais and Soldati-Favre, 2010).

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sis and the tricarboxylic acid (TCA) cycle (Fleige et al., 2007, 2008)and possesses a functional respiratory chain (Vercesi et al., 1998;Lin et al., 2009). However, it remains unresolved how these meta-bolic pathways are connected, given the absence of a mitochon-dria-localised pyruvate dehydrogenase (PDH) complex (Fothet al., 2005; Fleige et al., 2007), the key enzyme that links glycolysisand TCA cycle by converting pyruvate into acetyl coenzyme A(acetyl-CoA). Nevertheless, the supply of acetyl-CoA in T. gondiiand in Plasmodium spp. is apparently accomplished via the catabo-lism of branched-chain amino acids, given that both genomes en-code branched-chain keto acid dehydrogenases (BCKDH) (Seeberet al., 2008; Vaidya and Mather, 2009). To date, previous workusing biochemical assays has provided clear evidence that oxida-tive phosphorylation is functional in T. gondii (Vercesi et al.,1998). Additionally, in intracellular T. gondii, it has been demon-strated that oxidative phosphorylation is required for the produc-tion of ATP, showing a severe depletion of the parasitic ATP levelupon treatment of the ATPase inhibitor, oligomycin (Lin et al.,2009).

It has recently been suggested that extracellular parasites shifttheir energy metabolism towards glycolysis, based on the observa-

Ltd. All rights reserved.

836 S.S. Lin et al. / International Journal for Parasitology 41 (2011) 835–841

tion that a relocation of glycolytic enzymes from the cytosol to thepellicle occurs at the time when tachyzoites egress from host cells(Pomel et al., 2008). In the present study, we sought to define themetabolic requirements for ATP homeostasis in extracellular T.gondii. We surprisingly found that T. gondii is capable of maintain-ing a short-term stabilised ATP level without the acquisition ofexternal carbon sources during its first-hour extracellular lifespan.We further revealed that motility and invasion efficiency are notaffected by the lack of external carbon sources and that the para-sites depend on glycolysis to maintain the ATP level, even in theabsence of glucose. Collectively, these findings suggest that extra-cellular T. gondii can utilise their own internal carbon sources topower the ATP demanding gliding motility and thus possess a highdegree of independence from environmental conditions to estab-lish new host cell infections.

2. Materials and methods

2.1. Parasite cultivation

Toxoplasma gondii tachyzoites of the RH, Dtggt1 and its parentalRH/Dhxgprt strain as well as the NTE strain (type-II) and the C56strain (type-III) were propagated in human foreskin fibroblasts(HFFs) as previously described (Roos et al., 1994). Parasites weremaintained in DMEM containing 4.5 g/l glucose supplementedwith 1% FCS. For several experiments, extracellular parasites wereincubated in glucose/amino acid/pyruvate-free DMEM (Biochrom,Germany) without FCS, referred to as carbon source-free medium.For cultivation of parasites without glucose during intracellulargrowth, glucose-free DMEM was used (Invitrogen) and dialysedFCS was added to a final concentration of 1%.

2.2. Determination of parasitic ATP concentration

Measurement of the parasitic ATP level was described in detailin our previous work (Lin et al., 2009). In brief, freshly harvestedextracellular parasites (�1 � 107 per ml) were incubated at 37 �Cwith indicated supplements, treatments and time points. After-wards, parasites were centrifuged, resuspended in 250 ll of phenolred-free DMEM, and an aliquot of 20 ll of parasites was used forcounting. A control sample, harvested at time (t) = 0 h, was usedas a reference for determining the initial ATP level. The parasiticATP level was measured by using luciferase-based BacTiter-Glo re-agents (Promega) and quantified as photon per second (CPS), de-tected by luminometry (Wallac 1420, Perkin Elmer). Standardsamples with known ATP concentrations were included in themeasurement and the CPS results were used for the standard curveplot. The ATP concentrations of the samples were derived from thelinear equation of the standard curve and finally normalised withthe counted parasites.

2.3. Determination of lactate

Freshly released parasites (�1 � 107 per ml) were resuspendedin glucose/amino acid/pyruvate-free DMEM at 37 �C in a 5% CO2

humidified atmosphere with indicated treatments for 24 h. Culturesupernatants were collected by centrifugation at 14,000g for 2 minat 4 �C, filtered and immediately stored at �80 �C until used. Se-creted lactate was measured in duplicate using a colourimetric teston an automated clinical chemistry analyzer (Modular P, RocheDiagnostics). In this test, L-lactate is oxidised to pyruvate by thespecific enzyme lactate oxidase. Peroxidase is then used to gener-ate a coloured product by reacting with the hydrogen peroxidegenerated in the first reaction. Afterwards, a peroxidase catalysed

reaction generates a coloured product by using hydrogen peroxideas a substrate, which was generated in the first reaction.

2.4. Invasion assay

An equal amount of freshly released parasites were infectedonto HFFs in regular DMEM or glucose/amino acid/pyruvate-freemedium. The invasion rate was determined at 15 min or 24 h p.i.on HFFs. For the 15 min invasion assay, a two-colour IFA protocolwas applied to precisely determine the intracellular parasites(Wetzel et al., 2004). For 24 h invasion, samples were simply fixedwith 4% paraformaldehyde/PBS (PFA) followed by permeabilisationwith 0.25% Triton X-100/PBS for 15 min each. Samples were exam-ined under phase contrast microscopy and five randomly selectedfields from each sample were counted for the numbers of parasi-tophorous vacuoles (NV). A control sample was included in eachexperiment. Invasion (%) of the samples was determined after nor-malisation with the control (= NV of sample/NV of control � %).Relative invasion (%) is defined as the invasion (%) for those in-fected at t = 1 h with normalisation with those at t = 0.

2.5. Gliding motility

Freshly harvested parasites were resuspended in carbon source-free medium with or without glucose. An aliquot of parasites(250 ll; �2 � 106) from each duplicated sample was pipetted ontocoverslips placed in a 24-well plate and incubated first at roomtemperature (RT) for 10 min and for another 15 min at 37 �C. Sam-ples were first fixed with 4% PFA/0.05% glutaraldehyde/PBS for15 min, then blocked with 1% BSA/PBS for 1 h. Afterwards, sampleswere probed with a monoclonal anti-SAG1 (DG52; 1:500) followedby a Cy3-conjugated secondary antibody (1:300; Dianova) for 1 heach. Images from five fields per coverslip were taken and parasiteswith trails (P3 lm) were defined as motile. At least 250 parasiteswere determined. All samples were duplicated and at least twoindependent experiments were performed.

2.6. Drug treatments

Parasites were treated with oligomycin (Sigma), 2-deoxy-D-glu-cose (2-DOG) (Sigma) or a combination of both at indicatedconcentrations.

2.7. Statistics

Values were expressed as mean ± S.E.M. unless otherwise indi-cated. The variations of the data were analysed with the GraphPadPrism software, using an unpaired Student’s t test. P < 0.05 wasconsidered significant.

3. Results

3.1. Extracellular tachyzoites maintain a high ATP level withoutacquiring external carbon sources for a 1 h period

In order to investigate the energy metabolism in extracellulartachyzoites, we determined the dependence of the parasitic ATP le-vel on uptake and metabolisation of external carbon sources.Freshly harvested parasites, obtained by syringe passage, wereincubated in a medium without any carbon sources or in regularDMEM containing glucose, sodium pyruvate and amino acids ascarbon sources. The parasitic ATP concentrations were determinedimmediately after isolation (t = 0) and at time points 1, 3, 6 and24 h, respectively. As illustrated in Fig. 1, parasites exhibited atime-dependent ATP depletion, indicating that ATP consumption

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Fig. 1. Kinetics of ATP decrease in extracellular Toxoplasma gondii tachyzoites in theabsence or presence of carbon sources. Freshly released parasites were equallydivided into two samples, centrifuged and resuspended in regular DMEM with4.5 g/l glucose (+ carbon sources) and in DMEM without any carbon sources (�carbon sources), respectively. Parasites were incubated at 37 �C in a 5% CO2

atmosphere and an aliquot of �1 � 107 parasites was removed from each sample atthe indicated time points, t = 0, 1, 3, 6 and 24 h. Parasitic ATP was normalised withthe ATP content of the sample collected at t = 0 h (100%). Results are expressed asmeans ± S.E.M. at least from two independent experiments.

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exceeds ATP synthesis in extracellular tachyzoites. The ATP de-crease was less pronounced in samples incubated in medium withcarbon sources, suggesting that extracellular parasites importnutrients and use it for ATP synthesis. However, even in the com-plete absence of any carbon sources, parasites were able to main-tain an ATP level of >90% for at least 1 h. This implies thatextracellular parasites take advantage of external carbon sourcesfor ATP synthesis if they are available. On the other hand, theycan sustain a remarkably stable ATP level without the metabolisa-tion of external carbon sources for a 1 h period, which is probablythe most critical time for finding a new host cell.

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Fig. 2. Effect of external carbon sources on Toxoplasma gondii invasion. (A) Freshlyreleased parasites were incubated in regular DMEM with 4.5 g/l glucose (+ carbonsources) or in DMEM without any carbon sources (� carbon sources). An aliquot of30 ll (�2 � 105) was removed from each sample at the indicated time points (t = 1,3 and 6 h), mixed well with regular DMEM medium and then inoculated onto aconfluent monolayer of human foreskin fibroblasts (HFFs) grown on 24-well plates.Samples were analysed at 24 h p.i. A control, which was inoculated at t = 0 h, wasused as a reference for the initial invasion rate. (B) Freshly released parasites wereincubated in DMEM without carbon sources and at the indicated time pointsaliquots were used to infect HFFs in either regular DMEM (black bars) or in DMEMwithout carbon sources (white bars). (C) Freshly released parasites were incubatedin regular DMEM or in DMEM without carbon sources for 1 h. Infections of HFFswere performed in the same medium as used for extracellular incubation.Remaining extracellular parasites were removed after 15 min by extensive washing.All samples were performed in duplicate. Unless indicated, results are expressed asmeans ± S.D. from duplicated wells from a representative experiment. At least twoindependent experiments were performed.

3.2. Short-term deprivation of carbon sources in extracellular parasitesdoes not affect invasion and motility

Extracellular parasites need ATP as an energy source to fuelgliding motility and thus invasion. The invasion efficiency of extra-cellular parasites was tested by incubation of parasites in mediumwith or without carbon sources. Identical numbers of parasiteswere taken from the parasite suspension at the same time pointsas for the ATP kinetics and were used to infect a HFF monolayerin regular DMEM. The results are illustrated in Fig. 2A and showa strong correlation between the parasitic ATP level and invasionefficiency. The invasion rates decreased to less than 10% duringthe 24 h incubation period and the drop was less pronounced insamples which had been incubated in the presence of carbonsources. As for ATP, the invasion efficiency was almost unaffectedby a 1 h incubation period in medium without any carbon sources.Since the infection process in this experiment took place in regularmedium, we could not exclude the possibility that the invasionability in carbon source-free medium itself is low after 1 h, but isrebuilt with the aid of the carbon sources from the infection med-ium. We thus investigated whether the invasion efficiency is lowerwhen the medium used for infection is also deprived of any carbonsources. After an incubation period of 0, 1, 3 and 6 h in carbonsource-free medium, parasites were divided and used to infectHFFs in medium either with or without carbon sources. Invasionrates were independent of the absence or presence of carbonsources during the infection process itself, but were only depen-dent on the length of the previous incubation time in the carbon

source-free medium (Fig. 2B). To further exclude the possibilitythat host cells might provide nutrients during the infection period,for example by secretion, we shortened the infection time to15 min. Again, parasites deprived of external carbon sources dur-ing the 1 h extracellular incubation period and the 15 min infectionperiod, showed no significant reduction in invasion (Fig. 2C). Insummary, our invasion data imply that parasites can efficiently in-vade host cells without requiring external carbon sources duringthe first hour of extracellular life.

838 S.S. Lin et al. / International Journal for Parasitology 41 (2011) 835–841

In order to investigate whether the observed independencefrom external carbon sources is restricted to the utilised type-IRH strain, or whether it is a general feature of extracellular T. gon-dii, we analysed the kinetics of the ATP level and of the invasionrate in the NTE strain (type II) and in the C56 strain (type III).Freshly harvested, extracellular parasites of both strains displayedsimilar properties to the RH strain, possessing a stable ATP leveland a stable invasion rate after 1 h incubation in culture mediumwithout carbon sources (Supplementary Figs. S1 and S2). As inthe RH strain, the ATP level and the invasion rated decreased atthe 3 and 6 h time points. We thus conclude that independencefrom external carbon sources within the first extracellular hour ap-pears to be a general phenomenon in T. gondii.

Despite the high invasion rate which parasites display, indepen-dent of the availability of external carbon sources, it might still bepossible that motility is reduced in medium depleted of nutrients.We thus compared gliding motility of parasites incubated inmedium with or without carbon sources. Gliding trails, which areconstituted by the secreted surface proteins, were analysed by

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Fig. 3. Gliding motility does not depend on external carbon sources, but on glucose uToxoplasma gondii motility performed in medium with or without carbon sources. Scalgliding motility inhibition control, T. gondii were pretreated with 1 lM of cytochalasin Dfrom a representative experiment with duplicate samples. Three independent experimparental parasites in DMEM without carbon sources (� carbon sources) and in carbon soglucose). Results are expressed as means ± S.E.M. from two independent experiments.intracellular growth. RH/Dhxgprt parasites were cultured in human foreskin fibroblastgroups in the absence and presence of glucose during the motility assay with the sameexperiments. ⁄P < 0.05.

anti-SAG1 immunostaining and the motile fraction of the parasitesthat were cultured with or without carbon sources was assessed(Fig. 3A and B). Parasites incubated in medium with carbon sourcesdisplayed the same degree of motility as parasites incubated inmedium without carbon sources, suggesting that motility is inde-pendent of the uptake of external carbon sources. To further ex-plore a putative link between carbon source availability andmotility we took advantage of a recently described T. gondii knock-out mutant, which lacks the only known functional plasma mem-brane glucose transporter TgGT1 (Blume et al., 2009).Interestingly, extracellular Dtggt1 parasites possessed a stronglyreduced gliding motility in carbon source-free medium (Blumeet al., 2009). We compared gliding motility of Dtggt1 parasiteswith the parental strain in media without carbon sources and withglucose as the only carbon source. As expected, gliding motility ofDtggt1 parasites was lower than that of the parental strain andcould not be increased by addition of glucose to the medium, con-firming the earlier results (Blume et al., 2009). However, we alsofound this independence of gliding motility from external glucose

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ptake in the intracellular stage. (A) Representative immunofluoresence images ofe bars = 5 lm. (B) Quantification of the motility of T. gondii as described in A. As a

(CytD) for 5 min prior to the motility assay. Results are expressed as means ± S.D.ents were performed. (C) Analysis of gliding motility for Dtggt1 and RH/Dhxgprturce-free DMEM to which glucose was added to a final concentration of 25 mM (+⁄P < 0.05. (D) Dependency of gliding motility on the availability of glucose durings using DMEM with or without glucose. Gliding motility was determined for both

media used in C. Results are expressed as means ± S.E.M. from three independent

S.S. Lin et al. / International Journal for Parasitology 41 (2011) 835–841 839

in parental RH/Dhxgprt parasites (Fig. 3C), thereby confirming thedata shown in Fig. 3B, which were obtained from wild type RH par-asites. Thus, the parental strain possesses a higher gliding motilitythan Dtggt1 parasites, but this difference does not depend on glu-cose uptake during the extracellular stage. A possible explanationfor this result is that the lack of glucose uptake during the extracel-lular stage is not responsible for reduced motility, but that glucoseuptake and metabolism during intracellular growth is required toachieve the wild type motility. To test this hypothesis, we deter-mined the gliding motility of freshly harvested RH/Dhxgprt para-sites, which were cultured during intracellular growth either inregular DMEM or in DMEM that lacks glucose but contains gluta-mine. Gliding motility was indeed decreased when parasites weredeprived of glucose during their intracellular growth (Fig. 3D). Insummary, these assays show that the initial extent of glidingmotility in freshly harvested tachyzoites does not depend on theavailability of external carbon sources but on the availability ofglucose during intracellular growth.

3.3. The glycolytic pathway is required for ATP homeostasis inextracellular tachyzoites

The data obtained indicated that an almost stable parasitic ATPlevel is maintained in the first hour of extracellular incubation incarbon source depleted medium, although an ATP expensive glid-ing motility occurs. A possible explanation for this observation isthat ATP might be generated from internal energy stores. We nextattempted to identify the energy generating pathway which isresponsible for ATP homeostasis. The individual contribution ofglycolysis versus oxidative phosphorylation on ATP production inextracellular parasites deprived of external sources was examinedwith the aid of the F0F1 ATPase inhibitor, oligomycin, and the hexo-kinase inhibitor, 2-DOG. Parasites treated with oligomycin retainedan unaltered ATP level (Fig. 4, bar 5), suggesting that oxidativephosphorylation is not important for ATP production under the ap-plied conditions. In contrast, the ATP level was decreased by �85%after 2-DOG treatment (Fig. 4, bar 6). The 2-DOG-mediated ATPdepletion could be restored when glucose was supplemented

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Fig. 4. Glycolysis is required for ATP production in Toxoplasma gondii extracellularparasites. A pool of freshly released parasites was resuspended in a medium thatwas deprived of carbon sources and parasites were then equally divided into 1.5-mltubes (�5 � 106 per ml). Samples were supplemented with the indicated carbonsources and inhibitors, namely glucose (25 mM), glutamine (2 mM), oligomycin(1 lM) and 2-deoxy-D-glucose (2-DOG; 5 mM). Incubation took place for 1 h at37 �C in a 5% CO2 humidified atmosphere. Parasites were harvested by centrifuga-tion and resuspended in 250 ll of DMEM. A control sample, harvested at time point,t = 0 h, was included for comparison. ATP levels were normalised with the numberof counted parasites and are expressed as means ± S.E.M. from duplicate samplesfrom three independent experiments. ⁄P < 0.0001; ⁄⁄P < 0.005; #P < 0.09.

(Fig. 4, bar 7), confirming the action of 2-DOG as a competitiveinhibitor. It is noteworthy that a combination of 2-DOG and oligo-mycin did not synergistically decrease the ATP content (Fig. 4, bar10). Taken together, these data suggest that glycolysis, but not oxi-dative phosphorylation, is required for ATP homeostasis in the firsthour of extracellular incubation in carbon source depletedmedium.

A recent study has clearly demonstrated that glutamine can beutilised as an alternative carbon source in intracellular parasiteswhen glucose uptake is prevented (Blume et al., 2009). We thusexamined whether glutaminolysis also contributes to ATP produc-tion in extracellular parasites when glycolysis is inhibited. We ob-served that glutamine combined with 2-DOG led to an increasedATP level (Fig. 4, bar 8), which was oligomycin sensitive (Fig. 4,bar 9), suggesting that oxidative phosphorylation is responsiblefor the additional ATP production. However, this twofold increasedATP level was still very low compared with the untreated control(Fig. 4, bar 2). Taken together, these data suggest that glycolysis,but not oxidative phosphorylation, is the major contributor forATP synthesis in the extracellular stage.

To confirm that glycolysis is indeed active in extracellular par-asites, we determined the secretion of lactate, the major end prod-uct of aerobic glycolysis, after 24 h of extracellular cultivation.Lactate secretion was detectable in the presence of glucose, butalso in the complete absence of carbon sources in the medium(Fig. 5). Importantly, lactate secretion was completely undetect-able when parasites were treated with 2-DOG (Fig. 5, bar 4), butit could be restored by the addition of glucose (Fig. 5, bar 5). Thesedata again provide evidence that glycolysis is an active pathway inextracellular T. gondii, even in the absence of external glucose.

We next evaluated the effect of glycolysis inhibition and of oxi-dative phosphorylation inhibition on the invasion rate of extracel-lular parasites. Extracellular parasites were treated with 5 mM 2-DOG and/or 1 lM oligomycin for 1 h and viability was determinedafterwards in an invasion assay. Oligomycin-treated parasites dis-played unchanged invasion rates. In contrast, 2-DOG-treated para-sites revealed a more than 30% reduction in invasion comparedwith the untreated control (Fig. 6). Adding glucose could reversethe inhibitory effect of 2-DOG on invasion, which is in agreementwith 2-DOG acting as a hexokinase inhibitor. The reason that 2-DOG did not possess a stronger inhibitory potential on invasionmight also be a direct consequence of its nature as a reversiblehexokinase inhibitor. The medium used for infection after the 2-DOG treatment contained glucose, which could have prevented a

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Fig. 5. Toxoplasma gondii extracellular parasites secrete lactate. Freshly releasedparasites (1 � 107) were resuspended in medium without any carbon sources andthen supplemented with the indicated carbon sources, glucose (25 mM) orglutamine (2 mM), or/and the treatment of 2-deoxy-D-glucose (5 mM 2-DOG) for24 h. Culture media were collected at 24 and 0 h for lactate measurement. Lactateconcentrations for the extracellular parasites were normalised with the internallactate concentration detected at 0 h. ⁄P < 0.01; ⁄⁄P < 0.0005.

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Fig. 6. Inhibition of glycolysis but not of oxidative phosphorylation decreasesinvasion efficiency. Freshly released Toxoplasma gondii extracellular parasites wereincubated under similar conditions as described in Fig. 4. An aliquot of 30 ll wastaken from each sample at time point t = 1 h, mixed well with regular DMEM andthen inoculated onto a confluent human foreskin fibroblast monolayer grown on24-well plates. Samples were assayed for invasion rates at 24 h post p.i. A controlsample, infected at t = 0 h, was used as a reference for initial viability (100%).Results are expressed as means ± S.E.M. from duplicate wells from three indepen-dent experiments. ⁄P < 0.01; ⁄⁄P < 0.05.

840 S.S. Lin et al. / International Journal for Parasitology 41 (2011) 835–841

more severe effect. Together these results confirm that glycolysis,but not oxidative phosphorylation, is important to maintain thevital functions of extracellular T. gondii.

4. Discussion

Protozoan parasites and many microbial organisms have beenwell documented as undergoing a metabolic shift in response toenvironmental changes (Saunders et al., 2010). In this study weinvestigated the metabolic requirements for ATP homeostasis,motility and invasion efficiency in extracellular tachyzoites andunexpectedly found that these vital functions are remarkably inde-pendent from the presence of external carbon sources during thefirst hour of the extracellular lifespan. Only at extended extracellu-lar exposure times of 3 h and longer, does the uptake of carbonsources leads to significantly higher ATP levels and invasion rates.However, in a natural infection most of the extracellular parasitesare expected to infect new host cells within a relatively short timespan (Chtanova et al., 2008) and it is thus reasonable to assumethat within 1 h in an in vivo situation, most of the freshly egressedparasites will have invaded new host cells. The observed dispens-ability of nutrient uptake during this critical phase of invasion pro-vides the parasite with great flexibility for host cell infection anduncouples the invasion process from the availability of certainextracellular carbon sources.

Although glucose uptake was not necessary for ATP homeo-stasis in the first hour of extracellular incubation, activity ofthe glycolytic pathway itself was found to be essential for ATPlevel maintenance, as demonstrated by the sharp ATP decreasein the presence of the glycolysis inhibitor, 2-DOG. Furthermore,the secretion of lactate into medium confirms the activity ofthe glycolytic pathway in extracellular tachyzoites. On the otherhand, an inhibition of oxidative phosphorylation by oligomycindid not significantly reduce the ATP level in extracellular para-sites, while for intracellular parasites oxidative phosphorylationwas shown to be essential for ATP homeostasis (Lin et al.,2009). The concept that glycolysis is the most important path-way for ATP production in extracellular parasites is supportedby the observation that glutaminolysis was insufficient to main-

tain the ATP level when glycolysis was inhibited. Although theATP level increased in the presence of glutamine, it was stillapproximately threefold lower than controls. Conversion of glu-tamine into alpha-ketoglutarate and subsequent metabolisationin the TCA cycle and respiratory chain is thus insufficient tocompensate for the loss of glycolysis in extracellular parasites.Together, the obtained results are consistent with a metabolicshift of the major ATP generating pathway from oxidative phos-phorylation in intracellular parasites towards glycolysis in theextracellular stage.

Interestingly, a relocation of glycolytic enzymes from thecytosol towards the pellicle was recently reported to occur afteregress of parasites from the host cell (Pomel et al., 2008). Toxo-plasma gondii is equipped with a highly ATP-demanding actin-myosin motility system for host cell invasion (Sibley, 2003)and the relocation of glycolytic enzymes are likely an adaptationto optimise the energy requirements in extracellular parasites,by bringing the place of ATP production into close proximityto the place of ATP consumption (Pomel et al., 2008). Anotherputative advantage of powering gliding motility by glycolysis in-stead of oxidative phosphorylation is the independence of theenergy metabolism from oxygen. This could potentially beimportant for bradyzoites, which after ingestion need to invadehost cells in the gut under anaerobic conditions. It will thus beof interest to investigate whether extracellular bradyzoites pos-sess an energy metabolism similar to extracellular tachyzoites.Furthermore, the sudden change in conditions when parasitesleave the intracellular compartment and are exposed to theextracellular environment constitutes an enormously stressfulsituation, which requires adaptive processes. It was recently re-ported that one of the mechanisms by which tachyzoites managethis extracellular stress situation exists in translation control,which is mediated by phosphorylation of the eukaryotic initia-tion factor-2a (Joyce et al., 2010). Shifting energy metabolismfrom oxidative phosphorylation towards glycolysis might be an-other adaptation which prevents the production of toxic oxygenradicals and thus minimises oxidative stress.

In a recent study, gliding motility was reported to depend onthe availability of glucose in the medium (Pomel et al., 2008), whilewe found that in freshly harvested parasites motility is indepen-dent from glucose uptake. Although this discrepancy cannot becompletely resolved here, we further analysed the correlation be-tween glucose uptake and motility with the aid of a glucose trans-porter knockout mutant (Dtggt1), which was previously shown topossess reduced motility in the absence of glucose (Blume et al.,2009). Our data confirmed this reduced motility of the mutantbut suggested that the lack of glucose import in extracellular par-asites is not important for the extent of gliding motility, but that alack of glucose import during intracellular growth leads to de-creased gliding motility.

In summary, extracellular tachyzoites were found to depend onglycolysis, but not on glucose uptake, for ATP maintenance and se-crete lactate into the medium, even in the absence of carbonsources. These observations can only be explained by the use ofputative internal carbon stores. We do not yet know what the nat-ure of these putative stores is, since the only described carbonstores in T. gondii are the amylopectin granules in bradyzoites(Guérardel et al., 2005). Although amylopectin metabolism is cer-tainly stage specifically regulated, minor amounts of amylopectinmight also be available in the tachyzoite stage.

Acknowledgement

We thank N. Gupta (Humboldt University, Berlin) for the T. gon-dii lines, Dtggt1 and RH/Dhxgprt.

S.S. Lin et al. / International Journal for Parasitology 41 (2011) 835–841 841

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ijpara.2011.03.005.

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