Host-derived glucose and its transporter in theobligate intracellular pathogen Toxoplasma gondiiare dispensable by glutaminolysisMartin Blumea, Dayana Rodriguez-Contrerasb, Scott Landfearb, Tobias Fleigec, Dominique Soldati-Favrec,Richard Luciusa, and Nishith Guptaa,1

aDepartment of Molecular Parasitology, Humboldt University, Philippstrasse 13, 10115, Berlin, Germany; bDepartment of Molecular Microbiology andImmunology, Oregon Health and Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97239; and cDepartment of Microbiology andMolecular Medicine, University of Geneva, 1 Rue Michel Servet, 1211 Geneva, Switzerland

Edited by Thomas E. Wellems, National Institutes of Health, Bethesda, MD, and approved June 4, 2009 (received for review April 7, 2009)

Toxoplasma gondii, as an obligate intracellular and promiscuouspathogen of mammalian cells, utilizes host sugars for energy andto generate glycoconjugates that are important to its survival andvirulence. Here, we report that T. gondii glucose transporter(TgGT1) is proficient in transporting mannose, galactose, andfructose besides glucose, and serves as a major hexose transporterat its plasma membrane. Toxoplasma harbors 3 additional putativesugar transporters (TgST1–3), of which TgST2 is expressed at itssurface, whereas TgST1 and TgST3 are intracellular. Surprisingly,TgGT1 and TgST2 are nonessential to the parasite as their ablationsinflict only a 30% or no defect in its intracellular growth, respec-tively. Indeed, Toxoplasma can also tolerate the deletion of bothgenes while incurring no further growth phenotype. Unlike �tgst2,the modest impairment in �tggt1 and �tggt1/�tgst2 mutants isbecause of a minor delay in their intracellular replication, which isa direct consequence of the abolished import of glucose. The�tggt1 displays an attenuated motility in defined minimal mediathat is rescued by glutamine. TgGT1-complemented parasites showan entirely restored growth, motility, and sugar import. The lack ofexogenous glucose in �tggt1 culture fails to accentuate its intrinsicgrowth defect and prompts it to procure glutamine to sustain itsmetabolism. Unexpectedly, in vivo virulence of �tggt1 in miceremains unaffected. Taken together, our data demonstrate thatglucose is nonessential for T. gondii tachyzoites, underscore glu-tamine is a complement substrate, and provide a basis for under-standing the adaptation of T. gondii to diverse host cells.

glucose transport � glutamine metabolism � genetic manipulation

Toxoplasma gondii is an obligate intracellular parasite of thephylum Apicomplexa that causes infections in humans and in

animals. Infection is usually asymptomatic in immunocompetentindividuals, but it may cause severe complications or even befatal in immunocompromised people. Unlike other pathogens,Toxoplasma has adapted to replicate in most nucleated cells ofvertebrates, regardless of their cellular metabolism (1), and thusdisplays an exceptional metabolic robustness.

Most apicomplexan parasites reside and replicate in a nonfuso-genic parasitophorous vacuole, which protects them from lysosomaldegradation and confers a membrane interface with the nutrient-rich host cytosol. Its membrane acts as a molecular sieve, allowingthe permeation of soluble host metabolites below 1.4 kDa, such asamino acids, sugars, and nucleotides (2). The hexoses, includingglucose, being central to the cellular metabolism, are deemed to bevital for the replication of intracellular parasites. To facilitate theimport of glucose, these parasites express one or more sugarpermeases (www.membranetransport.org). Plasmodium speciesharbor sugar transporters mediating the uptake of glucose, and oneof these has been validated as a drug target (3, 4). Glucose and itspermeases are also indispensable for the survival of kinetoplastidparasites, Leishmania and Trypanosoma species (5).

The tachyzoite stage of T. gondii, responsible for an acuteinfection, rapidly metabolizes glucose via glycolysis (6). Glycolysisserves as a carbon source for the fatty acid synthesis (7), and hasbeen suggested to be essential for driving parasite motility and hostcell invasion (8). The glycosylation of proteins and the biogenesis oflipids in T. gondii are among the other vital processes that use sugars(9). To fulfill these cellular requirements, T. gondii harbors thenecessary pathways (www.ToxoDB.org), and a glucose transporter(TgGT1) that has been considered indispensable (8). The promi-nent metabolic role of glucose and other hexoses, has motivated usto examine the significance of sugar transport in T. gondii.

This study demonstrates that TgGT1, the major hexose trans-porter at the parasite plasma membrane, is not essential for the invitro survival and in vivo virulence of T. gondii tachyzoites. Theparasite harbors a set of 3 additional putative sugar permeases, only1 of which resides at its surface. Unexpectedly, T. gondii can toleratethe deletion of its surface transporters and thrives by catabolizingglutamine, despite its impaired access to host-derived glucose.

ResultsTgGT1 Can Transport Major Sugars in L. mexicana Null Mutant. Joet etal. (3) have shown that TgGT1 can facilitate the transport of glucoseand fructose in Xenopus oocytes. However, it remains unknownwhether TgGT1 can also mediate the import of galactose andmannose, required for glycolipid and glycoprotein synthesis.TgGT1 was tested in the L. mexicana sugar transporter null mutant,�lmgt, which is defective in importing glucose, mannose, fructose,and galactose (10). TgGT1 restored the ability of �lmgt cells to takeup all 4 hexoses, whereas the control cells (pX63NeoRI) were unableto import sugars (Fig. 1A). All parasites incorporated [3H]ade-nosine, indicating that all transgenic lines were viable and compe-tent in transporting other substrates. Substrate saturation curvesrevealed an apparent Km of �50 �M for glucose (Fig. 1B). To assessthe affinity of TgGT1 toward other hexoses, we tested their abilityto inhibit the glucose uptake (Fig. 1C). Glucose inhibited thetransport of glucose with a Ki of �53 �M that is similar to its Km,and mannose exhibited a 5-times higher Ki of �250 �M. In contrast,fructose and galactose did not significantly prevent TgGT1-mediated glucose transport up to 5 mM, but it was reduced by�50–70% at 50 mM inhibitors. These results confirm that TgGT1

Author contributions: N.G. and M.B. designed research; M.B., N.G., D.R.-C., and T.F. per-formed research; S.L., D.S.-F., R.L., and N.G. contributed new reagents/analytic tools; M.B.,S.L., D.S.-F., and N.G. analyzed data; and N.G. and M.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

DataDeposition:Theproteinsequences reported inthispaperhavebeendeposited intheNCBIGenBank database, www.ncbi.nim.nih.gov (accession nos. TgST1, EF198053; TgST2, EF427938;TgST3, EF427939).

1To whom correspondence should be addressed. E-mail: Gupta.Nishith@staff.hu-berlin.de.

This article contains supporting information online at www.pnas.org/cgi/content/full/0903831106/DCSupplemental.

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has high affinity for glucose and mannose, and fructose andgalactose are low-affinity ligands.

TgGT1 Is Not Essential to the Survival of T. gondii. TgGT1 has beenconsidered as the only glucose transporter in T. gondii, and pre-sumed to be indispensable for parasite survival. Our data on theimport of major hexoses by TgGT1 consolidate this conjecture. Totest it, the TgGT1 gene was deleted by double homologous recom-bination using the knockout construct pTUB8CAT (Fig. S1A).Following selection of stable transformants, chloramphenicol-resistant clones were analyzed for disruption of the TgGT1 locus(Fig. S1B). The knockout construct-specific PCRs revealed thepresence of the CAT resistance cassette adjacent to the 5�- and3�-UTRs in the genome of the �tggt1 parasites and in the controlplasmid, but not in the parental cells. Similarly, PCRs with 5� and3� recombination-specific primers yielded DNA bands of the ex-pected sizes in TgGT1-ablated parasites, but not in the parental andplasmid samples. Sequencing of the obtained UTRs confirmed theoccurrence of the recombination events exactly at the TgGT1 locus.The absence of the TgGT1 transcript in the �tggt1 (Fig. S1C)indicated its specific deletion with no apparent influence on themRNAs of TgST1–3. The successful disruption of TgGT1 geneproves its nonessential nature for T. gondii tachyzoites.

T. gondii Tachyzoites Express 3 Additional Novel Sugar Transporters.To test whether the dispensability of TgGT1 is because of functionalredundancy of sugar transport in T. gondii, we searched theToxoplasma Gene Database (www.ToxoDB.org) for the presenceof potential transporters. Three genes, named as TgST1, TgST2, andTgST3 were identified and their full-length ORFs were cloned fromtachyzoite mRNAs (Fig. S2A). All of the novel sugar permeasesharbor a sugar transport domain (Pfam 00083) of the conservedmultifacilitator SLC2A family and typical transmembrane/hydrophobic regions (Fig. S3). TgGT1 is 37.2% and 25% identicalto Plasmodium (PfHT1) and human transporters (HsGlut1) (Fig.S2B). TgST1–3 proteins are only �20% identical to TgGT1,PfHT1, and HsGlut1, but they show a higher degree of mutualresemblance. TgST1 and TgST2, with 34.4% identity, are mostproximal. The sequence divergence of TgST1–3 proteins is alsosupported by phylogenetic analysis, which revealed their clusteringdistant from protozoan, plant and mammalian permeases (Fig.S2C). In addition, the N termini of TgST1–3 extend beyond TgGT1

(see Fig. S2A). Although similar to each other, none of theseextensions is homologous to a protein with known function. Ourattempts to find the substrates for TgST1–3 in Saccharomycescerevisiae and L. mexicana have met with no success, so far.

TgGT1 and TgST2 Localize to the Surface of T. gondii. The transportof major hexoses by TgGT1 in L. mexicana mutant and uniquefeatures of TgST proteins prompted us to examine their subcellularlocalization in T. gondii to obtain an insight into their function.Therefore, we performed the ectopic over-expression of theirepitope-fusion constructs in intracellular tachyzoites (Fig. 2).TgGT1-HA under the control of the NTPase3 gene promoterlocalizes in the plasma membrane as confirmed by its colocalizationwith TgGAP45, a protein adjacent to the inner membrane complexof T. gondii. Its surface targeting in the TgGT1-HA-expressing�tggt1 strain that is governed by its endogenous promoter corrob-orates these data (see Fig. 2A). TgGT1 expression appears to behomogeneous, indicating a uniform uptake of hexoses by theparasite. The localization of the myc-TgGT1 resulted in punctuateintracellular mislocalization that suggests the prerequisite of its freeN terminus for sorting to the parasite surface (Fig. S4A). Onlypermeabilized tachyzoites were stained for TgGT1-HA, confirmingthe cytosolic orientation of its C terminus (Fig. S4B).

N-terminally HA-fused TgSTs were expressed transiently underthe control of the NTPase3 promoter (see Fig. 2B). TgST2 co-localizes with TgGAP45 to the periphery of T. gondii. In contrast,TgST1 and TgST3 were only detected within the parasite, andshown to partially localize with a dense granule protein, TgGRA3.To rule out the risk of a mislocalization because of over-expressionunder a strong promoter, such as pNTPase3 or N-terminal tag asobserved for TgGT1, we tested C-terminal Ty1-tagged permeaseswith the TUB8 promoter in stably transgenic parasites (see Fig. 2C).TgST2-Ty1 is targeted to the parasite surface, and TgST1 andTgST3 were again intracellular. To further assess the puzzlinglocalization of TgST1 and TgST3, we introduced an internal Ty1 tagbetween their sixth and seventh transmembrane helices (see Fig.2D). Yet again, TgST1 and TgST3 were present in intracellularcompartments. Further microscopic analysis demonstrated thatirrespective of the epitope fusion, the localization pattern is com-parable in all 3 cases, suggesting that a mislocalization because oftag interference or over-expression is quite improbable. Definitiveassessment of their subcellular location will await a confirmation

Fig. 1. TgGT1canmediate the importofglucose,mannose, fructose,andgalactose in L.mexicananullmutant (�lmgt). (A) TgGT1-complementedpromastigoteswereassayed for their ability to transport 100 �M D-[3H]glucose, D-[3H]mannose, D-[14C]fructose, or D-[14C]galactose. The pX63NeoRI vector was used as the control. (B)Substrate saturation curve for D-[3H]glucose. Uptake was determined over a 30-s period for a range of substrate concentrations (0.01–2 mM). (C) Inhibition of 100 �MD-[3H]glucose uptake in the presence of sugar inhibitors (0–50 mM). The Km and Ki were determined by Michaelis–Menten and nonlinear regression analyses.

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with specific antibodies recognizing the endogenous proteins.These data also confirm that the sorting of TgST2 to the parasitesurface does not require its free N and C termini. Both termini ofall 4 permeases should project into the parasite cytosol with theirputative substrate-binding site facing the parasitophorous vacuole(TgGT1, TgST2) or intracellular organelles (TgST1, TgST3) (Fig. S5).

TgST2 Is Dispensable and Nonredundant with TgGT1 in T. gondii. Toexamine whether the surface-associated potential sugar permeaseTgST2 is vital for T. gondii, we deleted the TgST2 gene. Upontransfection of the RH hxgprt- tachyzoites with p2854 knockoutconstruct, the pool of pyrimethamine-resistant parasites wasscreened to identify clones, in which the TgST2 had been replacedby the dihydrofolate reductase (DHFR) cassette (Fig. S6A). Thepresence of 5�- and 3�-UTRs was validated in the �tgst2 parasitesand in the plasmid, but not in the parental strain by using theconstruct-specific primers (Fig. S6B). Likewise, the 5� and 3�recombination-specific primers amplified the expected PCR bandsin the �tgst2 mutant but not in the parental strain and controlplasmid. Sequencing of the UTRs and the absence of TgST2 but notof the TgST1, TgST3, and TgGT1 transcripts corroborated thesuccessful ablation of the TgST2 locus (Fig. S6C). As with theTgGT1 gene, disruption of TgST2 confirms its dispensability for thesurvival and propagation of tachyzoites in tissue culture. However,the localization of both permeases at the parasite surface raised theobvious question of their functional redundancy. Hence, we gen-erated a �tggt1/�tgst2 double-deletion mutant by ablating the TgST2gene in the �tggt1 strain. Further PCR screening yielded themutants that had undergone double homologous recombination.The mRNAs of TgGT1 and TgST2 were not detectable in thismutant, whereas the TgST1 and TgST3 transcripts were present(Fig. S6D). The in vitro viability of the �tggt1/�tgst2 mutantconfirms the collective nonessentiality as well as the nonredundantfunctions of TgGT1 and TgST2 in T. gondii tachyzoites.

Only TgGT1 Deletion Confers a Growth Defect in T. gondii. To establishthe impact of TgGT1 and TgST2 gene deletions, we evaluated the

phenotype of all mutants by plaque assays that recapitulate all of theevents of several parasite lytic cycles. Their representative images inFig. 3A confirm that the plaques formed by the �tgst2 were similarin size to those of the parental strain. The �tggt1 and �tggt1/�tgst2mutants, by contrast, showed a reduction in their plaque sizes. Thequantitative measurements, shown in Fig. 3B, corroborated thesedata and established a 30% growth defect in the �tggt1. Notably, thegrowth of the �tggt1/�tgst2 strain was similar to the �tggt1, and noapparent accentuated defect was observed. This impairment wasentirely restored in the TgGT1-HA-harboring �tggt1 strain. Toexamine whether the reduced plaques of the �tggt1 were because ofa decrease in its motility, we performed motility assays in buffersmimicking the culture conditions. No reduction was observed inmotility of the mutant when compared to the parental strain (Fig.S7).

Although the plaque assay is a good indicator of the overallfitness of T. gondii, it is not suitable to assess selective defects duringits intracellular replication. Therefore, in vitro replication assayswere performed to estimate the intracellular replication rate (seeFigs. 3C and 4B). The �tgst2 mutant exhibited a doubling time of�8.8 h, and thus demonstrated no significant delay of replicationwhen compared to its parental strain (�8.6 h). The �tggt1 and�tggt1/�tgst2 mutants with comparable rates of �9.8 h and �9.5 h,however, divide �10 to 12% slower than the control parasites. Thisdefect was entirely restored in the TgGT1-complemented �tggt1strain, exhibiting a doubling rate of �8.4 h that is similar to theparental strain. Taken together, whereas TgGT1 contributes to—but is not essential for—the in vitro growth of tachyzoites, TgST2is absolutely expendable. It can also be concluded that the collectivedeletion of both surface transporters does not exert a syntheticlethal or cumulative growth phenotype.

The �tggt1 but Not �tgst2 Mutants Display Attenuated GlucoseMetabolism. To investigate whether the replication defect in the�tggt1 and �tggt1/�tgst2 strains is a result of attenuated importof host-derived glucose, we measured the utilization of sugar by

B TgGRA3HA-TgST1 Merge Phase

TgST1-3PNTPase3 HA

TgGap45HA-TgST2 Merge Phase

TgGRA3HA-TgST3 Merge Phase

C TgGRA3TgST1-Ty1 Merge Phase

TgGap45TgST2-Ty1 Merge Phase

TgGRA3TgST3-Ty1 Merge Phase

TgST1-3PTUB8 Ty1

TgST1/3: TMD 1-6PTUB8 TgST1/3: TMD 7-12Ty1

D TgST1-Ty1 TgGRA3 Merge

TgST3-Ty1 TgGRA3 Merge

A

TgGT1PNTPase3PTgGT1 HA

TgGT1-HA TgGAP45 Merge Phase

TgGT1-HA in Δtggt1

TgSag1 Merge Phase

Fig. 2. TgGT1 and TgST2 localize in theplasmamembraneofT.gondii,whereasTgST1and TgST3 reside in intracellular vesicles. (A)The parental or �tggt1 strains were trans-fected with TgGT1-HA-pNTP3 and TgGT1-HA-pGT1 constructs, respectively. (B) Transientlytransfected parasites expressing N-terminallyHA-tagged TgST1, TgST2, or TgST3 in pNTP3vector. (C) C-terminally Ty1-fused and TUB8-regulated T. gondii sugar permeases in stablytransfected parasites. (D) TgST1 and TgST3were tagged with Ty1 between sixth and sev-enth transmembrane domains in pTUB8.

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intracellular parasites. These results (Fig. 4A) demonstrated�80% reduction in labeling of the �tggt1 when incubated with[14C]glucose, which was restored in the TgGT1-HA-comple-mented �tggt1 strain, confirming the specificity of the phenom-enon. The ablation of TgST2 did not exert any measurabledecrease in glucose import, whereas the �tggt1/�tgst2 cellsexhibited a comparable diminution in its labeling as observed for�tggt1. To deduce the origin of residual labeling in �tggt1, weused a 20-fold excess of 2-deoxy-D-glucose (2-DOG) to inhibitthe glucose metabolism in host cells. 2-DOG is also known toaffect the glucose transport by TgGT1; however, more than3,000-fold excess of inhibitor over glucose has been applied toabolish the process (3). These assays resulted in a complete lossof metabolic counts in the �tggt1 parasites that is consistent withthe import of other glucose-derived metabolites from the hostcell. Collectively, these data reveal that the hampered growth of�tggt1 strain is a direct consequence of attenuated uptake of hostglucose, and TgST2 does not participate in glucose transport.

In Vitro Survival of T. gondii Does Not Require Host-Derived Glucose.The modest contribution of host glucose for the replication of �tggt1mutants motivated us to test whether the parasite can tolerate a lackof glucose in culture media. All strains were able to replicate underthe conditions where glucose was omitted in the medium a daybefore infection. In glucose-free culture, �tgst2 replicates at ratesthat are similar to the �tggt1 and �tggt1/�tgst2 strains in glucosemedia and parental strain in nonglucose media (see Fig. 3C and4B). Together with our data on parasite glucose import, theseresults imply that tachyzoites can tolerate a substantial depletion ofglucose in the host cytosol. More importantly, the absence ofhost-derived glucose failed to further delay the replication of the

�tggt1 and �tggt1/�tgst2, confirming the aforesaid notion. In accor-dance, akin to the parental tachyzoites, the �tggt1-TgGT1 strainregained its modest dependence on exogenous glucose. The dis-pensable nature of glucose for these mutants implies the depen-dence of tachyzoites on alternative host-derived nutrients.

The �tggt1 Mutant Utilizes Glutamine as a Major Alternative Nutrient.Glycolysis has been shown to drive parasite motility (8); hence, weimplemented this assay in a defined media to deduce the source ofalternative nutrients for the �tggt1 mutant (Fig. 5). As shown in Fig.5A, the number of mutants forming a secretory trail as well as itslength (detected by anti-Sag1 antibody) in the absence of glucose ismuch lower compared to the parental strain. The fraction of motile�tggt1 parasites is below 10% in salt buffers and was not influencedby external glucose, indicating �tggt1 is unable to use the sugar (seeFig. 5B). As predicted, the TgGT1-complemented mutant dis-played normal motility and its dependence on external glucose.Next to the sugar, glutamine and pyruvate are the most abundantcarbon sources in culture media, and the former has been shown toserve as the major nutrient in transformed or cancer cells (11, 12).Notably, the motility of the mutant is completely rescued inminimal media containing glutamine but not when the media issupplemented with pyruvate. Intracellular �tggt1 parasites incor-porated 60% more glutamine than control strain, a phenomenonthat was reversed in the complemented strain (see Fig. 5C),indicating glutamine acting as an alternative nutrient. Collectively,these results confirm that glutamine acts as a supplement bioen-ergetic substrate in T. gondii, and reveal that glucose contributes to,but is not essential, to empower the parasite motility.

In Vivo Virulence of T. gondii Does Not Depend on Glucose Import.Because the �tggt1 mutant cannot use external glucose and displaya 30% growth defect together with a markedly reduced motility inminimal media, we conducted in vivo assays to test its virulence inBALB/c mice (Fig. S8). All mice infected with 50 wild-type ormutant tachyzoites exhibited severe defect at day 8 and were killed.

B

∆tggt1/∆tgst2∆tgst2

∆tggt1Parental ∆tggt1-TgGT1

CParasite Strains

Intracellular Replication(hrs in standard media)

Intracellular Replication (hrs in glucose-free media)

Parental (hxgprt-) 8.6 ± 0.2 9.4 ± 0.3Δtggt1 9.8 ± 0.1 9.8 ± 0.3Δtgst2 8.8 ± 0.3 9.5 ± 0.1Δtggt1/Δtgst2 9.5 ± 0.1 8.9 ± 0.2Δtggt1-TgGT1 8.4 ± 0.5 10.6 ± 0.2

A

Fig. 3. The �tggt1 but not �tgst2 strain of T. gondii demonstrates a protractedin vitro growth. (A) Representative images of the parental, �tggt1, �tgst2, and�tggt1/�tgst2 strains as generated by plaque assays. (B) The images were digi-tized and plaques were manually encircled to calculate the area of 80 plaquesformed by the individual strains by using the ImageJ program. (C) Replicationrates of T. gondii tachyzoites in human foreskin fibroblasts

A

B

Fig. 4. The �tggt1 but not �tgst2 strain of T. gondii is compromised in usinghost-derived glucose and exhibits a delayed replication. (A) Glucose utilizationassays were performed with parasite-infected human foreskin fibroblasts (HFF)monolayers (multiplicity of infection � 4) at 24-h postinfection. (B) HFFs wereinfected for 40 h in glucose-free or normal media, and the inoculum-normalizedparasite yield was calculated.

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From the group infected with 500 parasites of the mutant, 1 mousehad to be killed at day 7, whereas all of the other animals were killedat day 8 because of their physical impairments. Briefly, no differ-ence in virulence of the �tggt1 mutant was observed in comparisonto the RH strain. These data are rather unexpected in light of theaforementioned facts, and that sugars are used to produce keyvirulence molecules in T. gondii.

DiscussionToxoplasma as an obligate intracellular pathogen procures vitalsugars from the host cell to satisfy its metabolic needs. In thisarticle, we demonstrate TgGT1 as the only surface-residentglucose transporter in T. gondii, and its deletion abolishes thesugar import to the parasite interior. Impaired utilization ofhost-derived glucose by �tggt1 mutants in standard media slowsT. gondii replication to a degree comparable to the effectinflicted by the absence of external glucose on parental and the�tgst2 strains. If sugar is indeed essential for T. gondii, then thedepletion of exogenous glucose should severely compromise orabolish the growth of parental and mutant parasites. Our resultsdo reveal the contribution of glucose to the parasite growth,albeit modest. Minor influence of the lack of glucose on theparental and �tgst2 strains can be anticipated, as these parasitesmay fulfill their metabolic needs by TgGT1-mediated uptake ofresidual sugar from glucose-depleted host cytosol that mightsustain their growth. However, the �tggt1 strain is alreadyimpaired in glucose import, and subsequent glucose exclusionvirtually abolishes the parasite’s access to host glucose. Thesustained existence of the �tggt1 mutant, despite this sugardeficiency, strongly argues for the expendable nature of glucosefor its in vitro culture. The limited requirement for glucose in T.

gondii may confer it the ability to replicate in most cell types,regardless of their glucose levels.

The �tgst2 strain still acquires the normal amounts of host-derived glucose, indicating that TgST2 does not partake in glucoseimport and cannot functionally replace TgGT1 in intracellulartachyzoites. Its nonredundant nature is validated further by collec-tive deletion of TgST2 and TgGT1. Our futile attempts to find anyapparent growth defect in the �tgst2 also reflect its absolutenonessential function in tachyzoites. The presence of TgST1 andTgST3 isoforms in distinct intracellular compartments implies theirfunctional specialization. It is noteworthy that in some organisms,such as in S. cerevisiae, glucose transporter-like proteins have beenshown to participate in environmental sensing (13). Hence, oneplausible explanation is that TgST1 and TgST3 function as intra-cellular sugar sensors. Whether the deletions of their genes willinfluence the parasite phenotype is being investigated.

It has been suggested that glycolysis powers the parasite motility(8), and that impairment in gliding motility is associated with theabsence of plaque formation (14). Our results on the �tggt1 mutantare in agreement with the former report as the fraction of motiletachyzoites is substantially reduced in defined minimal mediairrespective of the presence of glucose. However, motility of the�tggt1 is not affected in nutrient-rich media, and it is rescued in thebuffers containing glutamine. These results advocate the existenceof compensatory mechanisms in T. gondii, such as glutaminolysisand gluconeogenesis that can maintain the energy and carbonrequirements in the absence of TgGT1. In fact, glutamine has beenshown to be the key nutrient that fulfills the most biosyntheticrequirements in transformed and cancer cells (11, 12). The rapidmultiplication of T. gondii and tumor cells imposes a high metabolicburden, indicating their analogous biosynthetic needs that areapparently not dictated by glucose. Indeed, the absence of glu-tamine, but not of glucose, in the �tggt1 culture is detrimental to itsgrowth (data not shown). Furthermore, the genomic annotationsconfirm the presence of glutamine catabolism and of gluconeo-genesis in T. gondii that should result in utilization of substrates,such as amino acids, pyruvate, lactate, and glycerol (15). In fact, the�tggt1 mutant displayed a 2- to 4-fold induction of its glutamineaminotransferase and glutamate dehydrogenase transcripts (datanot shown). Based on our preliminary data, we assume thatglutamine can feed into the tricarboxylic acid cycle and gluconeo-genesis and acts as a bioenergetic substrate to T. gondii. In addition,there are also annotations for the syntheses and import of sugar-phosphates that should sustain lipid and protein glycosylation.Glycosylated proteins and lipids contribute to the virulence in T.gondii and to the host immunity (16, 17). It is rather unanticipatedand salient that the in vivo virulence of the �tggt1 mutant in miceremains unaltered, and it is probable that as observed in cancercells, glutamine can compensate for all parasitic requirements. Itremains to be seen if other nutrients can also contribute to thecellular needs of T. gondii. The deployment of motility assays usingthe �tggt1 and its derivative mutants will facilitate the search ofauxiliary nutrients. Whether the �tgst2 and/or �tggt1/�tgst2 mutantsdisplay an altered in vivo immunogenic potential would also be atopic of future research. All experiments undertaken in the presentwork were performed on the tachyzoite stage. It will be interestingto determine whether glucose is essential for other stages of T.gondii, including bradyzoites.

Taken together, our data confirm that TgGT1 protein is expend-able in T. gondii, despite it being the major hexose transporter at theparasite plasma membrane. TgGT1 is also not functionally redun-dant with other surface-localized sugar permease present in T.gondii. The genetic disruption of TgGT1 abolishes the import ofhost-derived glucose by T. gondii, which resorts to glutaminecatabolism to sustain its cellular requirements. The �tggt1 strain,with its restricted access to external glucose, underlines the meta-bolic robustness of T. gondii and provides an excellent model to

C

B

AParental (+Glucose) ∆tggt1 (+Glucose)∆tggt1 (+Glutamine) ∆tggt1 (+Pyruvate)

Fig. 5. Glutamine fulfills the metabolic needs of the �tggt1 mutant. Freshlyharvested tachyzoites were used to perform the motility assays. (A) Representativeimages of the parental and �tggt1 strains in Hanks’s balanced salt solution supple-mentedwith the indicatedreagents. (B)Quantitativediagramof themotile fractionin three independent experiments. (C) Glutamine (0.5 �Ci/mmol of [3H]glutamine)incorporation assays were executed with parasite-infected HFF monolayers (multi-pliciy of infection � 4) at 24 h postinfection.

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dissect the intricate redundancies of nutrient acquisition by T. gondiias an obligate intracellular pathogen of diverse host cells.

Materials and MethodsChemical and Biological Resources. Cell culture chemicals and radioactive sugarswere procured from Biotherm and Hartmann Analytic. Other reagents, includingsecondary antibodies, and mouse anti-HA antibody were purchased from Invitro-gen and Sigma, respectively. Anti-TgGRA3, DG52 anti-TgSag1, and anti-Ty1 an-tibodies were kindly provided by J.-F. Dubremetz (University of Montpellier,France), J. Boothroyd (Stanford University School of Medicine), and K. Gull(University of Oxford, U.K.), respectively.

Cell Culture and Cloning of Sugar Transporters. All strains of T. gondii tachyzoitesand the derived mutants were maintained in confluent monolayer cultures of theHFF, as described previously (18). Host cells were cultured and infected in DMEMsupplemented with 10% FCS, 2 mM glutamine, 1� MEM nonessential amino acids,1 mM sodium pyruvate, 100 units/mL penicillin, and 100 �g/mL streptomycin (37 °C,10%CO2).Tachyzoites (108)weresubjectedtomRNAextractionorfirst-strandcDNAsynthesisusing�MACSmRNAisolationandcDNAsynthesiskits (MiltenyiBiotec).ThemRNA or cDNA pool was used to generate the cDNAs of TgST1–3 and TgGT1 byPfu-Ultra FusionII Polymerase (Stratagene) and cDNA-specific primers (Table S1).

Generation of Immunolocalization Constructs. The N- or C-terminus epitope-taggingofall sugarpermeaseswasperformedusingtheindicatedprimers (seeTableS1). The N terminus HA-fusion of TgST1–3 in pNTP3 and of TgGT1 in pTetO7Sag1vector was executed with their respective F2/R2 primers. Likewise, the COOH-fusionof Ty1-epitope in TgST1–3 in pTUB8 and of TgGT1 in pNTP3 plasmid was performedusing F3/R3 primers. Internal tagging of Ty1 epitope between the sixth and seventhtransmembrane helices of TgST1 and TgST3 in pTUB8 was done by 2 independentcloning for each cDNA by consecutive use of F3/R3.1 and F4/R4 primer sets.

Knockout Constructs and Mutant Isolation. The TgGT1 knockout constructcontained 2.7 kb of the 5�-UTR and 3�-UTR of TgGT1 gene flanking the CATORF in pTUB8CAT vector (Table S2). To prepare the TgST2 deletion constructin p2854 vector harboring the DHFR expression cassette, 2.8 kb of its 5�- and3�-UTRs were cloned (Table S2). The RH hxgprt� tachyzoites (107) were trans-fected with 50 �g of the plasmid and selected for the chloramphenicol (10�g/mL) or pyrimethamine (1 �M)-resistant parasites as described elsewhere(19, 20). Individual clones were subjected to knockout screening.

Functional Expression of Parasite Transporters in L. mexicana. L. mexicana nullmutant, �lmgt (10), was transformed with all ORFs in pX63NeoRI plasmid, andmaintained inRPMImedium1640(pH7.2) containingthedrugG418(100 �g/mL).The transfectants were examined for the import of 100 �M [3H]glucose, [3H]m-annose, [14C]fructose, and [14C]galactose by an oil-stop method (21). To deter-mine the Ki, inhibition curves were generated with 100 �M glucose by using 0–50mM of each sugar as the inhibitor.

Glucose and Glutamine Uptake Assays. Glucose and glutamine uptake assayswere performed to examine the ability of the parasite mutants to import host-derived glucose or glutamine during their intracellular propagation in HFF (mul-tipliciy of infection � 4, 24 h postinfection). All samples were washed 2 times with4 mL of glucose-free DMEM before their labeling with 1 mL of [14C]glucose (0.2�Ci/mmol,5mM)or [3H]glutamine (0.5 �Ci/mmol2mM) inDMEMfor2hat37 °C.To isolate the labeled tachyzoites, the samples were washed 4 times with DMEM-glucose (25 mM) and treated (10 min, 37 °C) with 1.5 mL of the egression buffer(10 �M A23187, 25 mM glucose in Hanks’s balanced salt solution). Ionophore-egressed tachyzoites were collected (450 � g for15 min), washed once, andsubjected to scintillation and cell counting.

Plaque and Replication Assays. The parasite plaque assay recapitulates all of theeventsofparasite lytic cycle, includinghostcell invasion, intracellulargrowth,andegression followed by spreading by gliding motility (22). The HFF cells in 6-wellplateswere infectedwith2,000tachyzoites, culturedfor7days,fixedwith�80 °Cmethanol, stained with crystal violet, and scanned. The encircled area of eachplaquewasmeasuredandthemeansof80plaqueswascalculatedfor subsequentdata plots. For replication assays, infected HFFs (multipliciy of infection � 1.5)were cultured for 40 h, and then the parasites were egressed for 10 min by 10 �MA23187 inHBSS.Thenumberofegressedparasiteswasnormalizedtothenumberof invasive parasites, deduced by complementary plaque assays.

Indirect Immunofluorescence Assay. The ORF-transfected tachyzoites were sub-jected to immunofluorescence assay as reported previously (23). Briefly, theinfected HFF cells were fixed with 4% paraformaldehyde (PFA) or 4% PFA plus0.05% glutaraldehyde for 10 min or with �80 °C methanol for 2 min followed byneutralization in 100 mM glycine/PBS. The samples were permeabilized for 20min in 0.2% Triton-X100/PBS, preblocked with 2% BSA in 0.2% Triton X-100/PBS,and stained with antibodies (anti-TgGAP45 at 1:3,000 dilution; anti-TgGra3 at1:500; anti-HA at 1:1,500; anti-Ty1 at 1:50). After 3 washes with 0.2% TritonX-100/PBS, Alexa488, or Alexa568 antibodies (1:3,000 dilutions) were applied.The samples were washed 3 times before mounting with Fluoromount G.

Motility Assay. Tachyzoites were syringe-released from the HFF monolayers(30–36 h postinfection) and preincubated on a coverslip for 10 min at roomtemperature in serum-free Hanks’s balanced salt solution supplemented withindicated reagents. The samples were incubated (15 min, 37 °C) and fixed with4% paraformaldehyde/0.05% glutaraldehyde and stained with mouse monoclo-nal anti-TgSag1 (1:1,500) and Alexa488 (1:3,000) antibodies. Images were re-corded, and at least, 800 well-isolated parasites with visible trails (�3 �m) werecounted in 5 random fields to calculate the motile fraction.

ACKNOWLEDGMENTS. We thank Paco Pino for animal experiments and FrankSeeber for his scientific suggestions and manuscript appraisal. This work wassupportedbyGRK1121andSFB618grantsfromtheGermanResearchFoundation(to R.L. and N.G.), the Helmholtz Foundation PhD fellowship (to M.B.) in thelaboratory of N.G., and the European Molecular Biology Organization funding(to M.B.) for a 3-month research in the laboratory of D.S.-F. (COST 857-ProjectC05.0142). D.S.-F. and T.F.’s contributions were supported by SystemsX (LipidX).

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