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J Biol Chem, Vol. 274, Issue 35, 24888-24895, August 27, 1999

The Protozoan Parasite Toxoplasma gondii Expresses Two Functional Plant-like Glycolytic Enzymes
IMPLICATIONS FOR EVOLUTIONARY ORIGIN OF APICOMPLEXANS^*

Florence Dzierszinski^§, Octavian Popescu^¶, Catherine Toursel, Christian Slomianny^**, Bilel Yahiaoui^§, and Stanislas Tomavo

From the  Laboratoire de Chimie Biologique, CNRS UMR 111, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq, France and ^** INSERM U.42, Domaine du Certia, 369 rue Jules Guesde, 59650 Villeneuve d'Ascq, France

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The recent discovery of a vestigial, nonphotosynthetic plastid ("apicoplast") in the Apicomplexa has considerably modified our perception of the evolutionary origin of these parasites. Phylogenetic analysis and the presence of four surrounding membranes of the apicoplast provide important support for the hypothesis that apicomplexans have acquired their apicoplast by secondary endosymbiosis, probably from a green alga. This suggests that genes encoding predicted homologs of proteins of green algae or related photosynthetic lineages could have entered the nucleus of apicomplexan parasites by transfer from the ancestor harboring the apicoplast. We describe here complementary DNAs encoding two Toxoplasma gondii glycolytic enzymes, glucose-6-phosphate isomerase (G6-PI) and enolase, which have considerable identities with land plant counterparts. Both cDNAs of T. gondii complement Escherichia coli mutants lacking G6-PI and enolase genes and lead to the expression of active enzymes. In the drug untreatable encysted bradyzoites of T. gondii, G6-PI and enolase genes are overexpressed or exclusively expressed at both transcriptional and protein levels. Moreover, three-dimensional models and protein phylogeny confirmed that G6-PIs and enolases of T. gondii, Plasmodium falciparum, and land plants are closely related. Because these glycolytic enzymes are plant homologs, which differ from those of animals, they will be useful to trace the evolutionary origin of Apicomplexa and might offer novel chemotherapeutic targets in diseases caused by apicomplexan parasites.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Among 5000 species of apicomplexan parasites are numerous pathogens such as Toxoplasma gondii (an important opportunistic pathogen associated with AIDS and congenital birth defects), Plasmodium species (causative agents of malaria), Eimeria (agent of coccidiosis), and Cryptosporidium (opportunistic intestinal pathogen). These parasites possess a third genetic element, a 35-kilobase circular DNA contained within a nonphotosynthetic plastid in addition to the nuclear and mitochondrial genomes (1). Based on the phylogenetic analysis of the tufA gene encoded by the apicoplast genome, the presence of four membranes, and the analogy to present day chrysophytes, cryptomonads, diatoms, and chlorarachniophytes, it is postulated that apicomplexan parasites have acquired this organelle by the ingestion of another eukaryotic plastid-containing alga (2-5). However, these recent data provided unexpected and contradictory evidence for the origin of apicomplexan plastids because previous classifications showed the closest relationship between apicoplast and plastids of red alga (6) and euglenoids (7). Moreover, ultrastructural and biochemical studies revealed that most apicomplexan parasites synthesize amylopectin, a glucose storage form in cytosol like most of red algae and glaucophytes (8). These observations together with the acquisition of the apicoplast raise many key questions regarding the evolutionary origin of apicomplexan parasites. Identification of genes encoding glycolytic enzymes should promote investigations of the evolutionary origin of apicomplexans and the biology of parasitism, because these enzymes are particularly suitable for theories of enzymes evolution (9). Additionally, a better understanding of the evolutionary origin of apicomplexan parasites could also pave the way for the search of novel and specific chemotherapeutic targets.

The infection by T. gondii can lead to severe syndromes including congenital malformations such as blindness, mental retardation, and hydrocephaly in children exposed in utero. Recently, more attention has been given to T. gondii because toxoplasmosis is the most common opportunistic infection in immunocompromised patients with AIDS or in transplant patients (10). Although the parasite has a sexual cycle occurring in cats, the infection is usually transmitted asexually by ingestion of undercooked meat from latently infected animals (e.g. pork or lamb). In mammalian nonfeline hosts, T. gondii is found in two haploid asexual forms, the rapidly replicating tachyzoites and the slowly dividing, quiescent encysted bradyzoites. The tachyzoites differentiate into encysted bradyzoites in response to the immune system attack during disease progression. They will remain in the brain and other organs during the lifetime of infected hosts. The reactivation of encysted bradyzoites into actively replicating and cytolytic tachyzoites is the cause of fatal toxoplasmic encephalitis in AIDS patients (11). Understanding stage conversion could be helpful in designing novel targets and strategies to overcome the disease. We and others have found that mitochondria are functionally impaired in encysted bradyzoites of T. gondii, suggesting that these parasitic forms rely predominantly on anaerobic glycolysis (12-14). Stage-specific lactate dehydrogenase homologs and two expressing sequence tags encoding short sequences of enolase have been identified in T. gondii (15, 16). Glucose 6-phosphate isomerase (D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) and enolase (2-phospho-D-glycerate hydrolase, EC 4.2.1.11) are two important enzymes of glycolysis and catalyze the interconversions of fructose 6-phosphate to glucose 6-phosphate or 2-phospho-D-glycerate to phosphoenolpyruvate, respectively.

Here, we report for the first time the full-length cDNAs, deduced amino acid sequences, and functional expression of cytosolic G6-PI¹ and enolase of T. gondii. We show several lines of evidence demonstrating that G6-PI and enolase of both T. gondii and Plasmodium falciparum are homologous to their land plant counterparts using extensive analyses of predicted primary sequences, three-dimensional structures, and molecular phylogeny. We further discuss the implications of these findings regarding our understanding of the evolutionary origin of apicomplexan parasites and for developing novel chemotherapeutic strategies.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Parasite Growth-- The 76K strain of T. gondii was used because of the ease in culturing these parasites in vitro or to obtain encysted bradyzoites from chronically infected mice. Encysted bradyzoites were purified and freed by pepsin digestion (0.05 mg ml¹ pepsin in 170 mM NaCl, 60 mM HCl) for 5-10 min at 37 °C (17). Tachyzoites were grown in human foreskin fibroblasts (HFF) using Dulbecco's modified Eagle's medium (Biowhittaker, Belgium) supplemented with 10% fetal calf serum (Dutscher), 2 mM glutamine (Sigma), and 0.05% gentamicin (Sigma). Parasites were harvested from the monolayer HFF cells and purified by filtration on a glass wool column and finally through a 3.0-µm pore size filter (Nucleopore) (18).

Isolation of RNA, DNA Extraction, and Southern Blots-- Total RNA was isolated from parasites using Trizol reagent according to the manufacturer's instructions (Life Technologies, Inc.). Poly(A⁺) RNA was purified using the oligo(dT)-cellulose columns (Life Technologies, Inc.). Genomic DNA isolation, recombinant DNA purification, and the analysis of nucleic acids by blots were carried out according to standard procedures (19). Specific probes were randomly labeled DNAs generated from the complete ORFs of G6-PI and enolase using the digoxigenin method (DIG-High Prime, Roche Diagnostics).

Rapid Amplification of cDNA Ends (RACE) and Sequencing-- To obtain full-length cDNAs, RACE techniques were performed using a Marathon cDNA Amplification Kit (CLONTECH) with the Adaptor Primer 1 and specific oligonucleotides of either G6-PI (5'-AACGCCAGCAGCTGGCCAATATGG-3' for 5'-RACE and 5'-CTTTGCTCAGC CAGATGCGCTGGC-3' for 3'-RACE) or enolase (5'-CAGAATATCGTCCCCTATAACCTG-3' for 5'-RACE and 5'-CGAGGGGTGGCTGAAAAAGTATCC-3' for 3'-RACE). Nested PCR was performed using Adaptor Primer 2 and internal primers of either G6-PI (5'-GGTGAGATCTCCGGAAAAAGAAGC-3' for 5'-RACE and 5'-GACACCCGAAGAACTCCGCAAGGA-3' for 3'-RACE) or enolase (5'-TATAACCTGTGTTTTCTCACCCAC-3' for 5'-RACE and 5'-CGACCAAGATGACTTCGCAAGTTT-3' for 3'-RACE). PCR products were cloned into the TA-Cloning pCRII vector (Invitrogen) and sequenced using an ALFexpress automated sequencer (Amersham Pharmacia Biotech).

Measurement of mRNA Expressions of T. gondii G6-PI and Enolase Genes Using Reverse Transcriptase-PCR-- cDNAs were synthesized from total RNA isolated from tachyzoite and bradyzoite forms using reverse transcriptase. Serial dilutions of cDNAs (1:1, 1:10, and 1:100) were used to amplify ORFs of T. gondii -tubulin (20), G6-PI, and enolase by PCR. Primers were as follows: for -tubulin, 5'-ATGAGAGAGGTTATCAGCATC-3' and 5'-TTAGTACTCGTCACCATAGCC-3'; for G6-PI, 5'-GGGCGGATCCATGGCGCCGACACAGCTGGAACAG-3' and 5'-GGGCGGATCCAGCCTTCGACTGCTGCACGTAGTG-3'; and for enolase, 5'-GGGCCCATGGTGGTTATCAAGGACATCGTT-3' and 5'-GGGCAGATCTTTTTGGGTGTCGAAAGCTCTCTCC-3'.

Expression of Recombinant Proteins and Immunological Analysis-- The complete ORFs of G6-PI and enolase were tagged to oligonucleotide encoding a polyhistidine peptide and cloned inframe into the pQE-60 expression vector (Quiagen) designated pQE-G6PI and pQE-Enolase, respectively. Under induction with isopropyl-1-thio--D-galactopyranoside, transformed Escherichia coli (XL1-Blue strain) was lysed, and recombinant proteins were purified on Ni²⁺-nitrilotriacetic acid-agarose columns using an imidazole gradient elution. Polyclonal antisera were from BALB/c mice immunized with purified recombinant proteins. Western blots were performed using polyclonal antisera diluted 1:1000 and secondary antibodies conjugated to alkaline phosphatase or to peroxidase.

Functional Complementation in E. coli Mutants Lacking G6-PI and Enolase-- E. coli G6-PI/glucose-6-phosphate dehydrogenase-deficient DF214 strain (21) and enolase-deficient DF261 strain (22) were obtained from the E. coli Genetic Stock Center, Department of Biology, Yale University. These mutants, which grow normally in nonselective media composed of Luria broth (Life Technologies, Inc.) containing 100 µg ml¹ ampicillin for DF214 strain or M9 minimal medium containing 12.5 mM glycerol and 25 mM malate for the DF261 strain, are unable to grow on the selective media consisted of M9 minimal medium (Life Technologies, Inc.) supplemented with 4 mg ml¹ glucose and 1 mM isopropyl-1-thio--D-galactopyranoside (21, 22). Bacteria were transformed by electroporation (BTX, 2.45 kV, 129 ohms, 5 ms), plated onto selective medium, and incubated at 30 °C for 3 days.

Enzyme Assays-- The G6-PI assay was performed with 2 µg of purified G6-PI in 980 µl of a mixture containing 100 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 10 mM MgCl₂, 2 mM fructose 6-phosphate (Sigma), 0.2 mM NADP, and 0.1 unit glucose-6-phosphate dehydrogenase at 37 °C. Purified enolase (2.55 µg) was mixed with 990 µl of assay buffer containing 50 mM Tris-HCl, pH 7.5, 1 mM MgCl₂, 1 mM 2-phospho-D-glycerate (Sigma) and incubated at 37 °C. The G6-PI and enolase activities were detected by spectrophotometry at 230 and 340 nm, respectively (23).

Primary Sequences, Three-dimensional Structures, and Protein Phylogeny-- The G6-PI or enolase sequences were aligned using Clustal V and W (24, 25). Using Swiss-Model and Swiss-PDBViewer (26, 27), three-dimensional models were generated from the data of the three-dimensional structure (x-ray diffraction at 2.0 Å resolution) of Saccharomyces cerevisiae enolase published previously (28). Phylogenetic trees were constructed using maximum likelihood, neighbor joining, and maximum parsimony algorithms (29, 30). Bootstrap analysis and trees were performed using 100 replicates in MOLPHY 2.2 (30) and Tree View software (31).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Characterization of T. gondii G6-PI and Enolase Genes-- Recently, we have cloned two fragments of cDNAs encoding the glycolytic enzymes G6-PI and enolase using a subtractive library devised for the isolation of cDNAs that are specifically expressed by the encysted bradyzoites of T. gondii (32). These two cDNAs of 242 and 491 bp enabled appropriate PCR primers to be designed for the generation of 5'-end and 3'-end DNA fragments by using the RACE method. The reconstruction of full-length cDNAs of the two enzymes was achieved by RACE. For G6-PI, the nucleotide sequence of 2656 bp revealed an ORF of 1689 bp (nucleotides 142-1833), which encodes a 563-amino acid protein with a relative molecular mass (M_r) of 63,122. The enolase cDNA is 2016 bp long with an 1332-bp ORF (nucleotides 321-1652), which encodes a 444-amino acid protein (M_r = 48,341). To assess the copy number of both genes in the nuclear genome of T. gondii, Southern blots were carried out using G6-PI (Fig. 1A) or enolase (Fig. 1B) probes. Under stringent conditions, the blots gave mostly a single band except when predicted internal restriction sites were present. The data were consistent with a single copy number G6-PI and enolase genes in the T. gondii genome.

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Southern blot analysis with digoxigenin-labeled G6-PI (A) and enolase (B) probes. C, semiquantitative reverse transcriptase-PCR is used for the amplification of ORFs of G6-PI, enolase, and the housekeeping -tubulin using serial dilutions of cDNAs prepared from in vivo encysted bradyzoites or from tachyzoites of T. gondii.

Developmental Expression of G6-PI and Enolase Genes-- Because cDNAs of these enzymes were isolated from the in vitro bradyzoite library, we verified the level of their transcripts in encysted bradyzoites isolated from mice. Semiquantitative reverse transcriptase-PCR was performed because there are limitations in obtaining sufficient cysts in vivo for Northern blots. Levels of G6-PI and enolase transcripts are increased 10- and 1000-fold, respectively, in the encysted bradyzoites (Fig. 1C). These conclusions were strongly supported by the nearly constant level of the housekeeping -tubulin amplified in both tachyzoite and bradyzoite forms (Fig. 1C). These results indicate that G6-PI and enolase genes are transcriptionally overexpressed in the encysted stage of T. gondii. To investigate whether these mRNA abundances increase the expression of G6-PI and enolase proteins, recombinant proteins tagged to the C-terminal polyhistidine peptide were produced in E. coli transformed with pQE-G6PI and pQE-Enolase plasmids. Fig. 2 shows the purification of G6-PI (Fig. 2A) and enolase (Fig. 2B) recombinant proteins. Purified recombinant proteins (lanes 4a and 4) were used to raise polyclonal antisera in mice. By comparing the reactivity of monoclonal antibody specific to the tachyzoite protein SAG1 (Fig. 2E, lane 4), Western blots of polyclonal antibodies revealed proteins of 63 (Fig. 2C, lane 3) and 48 kDa (Fig. 2D, lane 3) corresponding to the expected sizes of G6-PI and enolase only in the encysted bradyzoite of T. gondii. However, the polyclonal antibody against enolase cross-reacted with a faint band of a 50-kDa protein present in brain cells (Fig. 2D, lane 2) but absent in HFF cells (lane 1). The cross-reactivity can be explained by the high degree of homology (65%) between all enolases. Together with the increase of mRNA abundance, the data demonstrate the expression of stage-specific G6-PI and enolase by T. gondii. Finally, immunofluorescence assays were used to determine the localization of G6-PI and enolase in T. gondii. The two glycolytic enzymes were found exclusively in the cytosol of the parasite.²

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Expression and purification of recombinant proteins corresponding to G6-PI (A) and enolase (B) of T. gondii. Lanes 6 and 7, uninduced and isopropyl-1-thio--D-galactopyranoside-induced E. coli transformants, respectively. B, affinity purified recombinant proteins: lane 1, 50 mM; lane 2, 100 mM; lane 3, 150 mM; lane 4, 200 mM; lanes 5a and 5b, 250 mM imidazole. Western blot analysis of an equal number (7.5 × 104) of tachyzoites, in vivo bradyzoites, HFF, and brain cells using polyclonal antibodies against G6-PI (C) and enolase (D) or monoclonal antibodies against tachyzoite-specific surface protein, SAG1 (E). Lane 1, HFF cells; lane 2, brain cells; lane 3, bradyzoites; lane 4, tachyzoites.

Demonstration of Isolated cDNAs Encoding Enzymatically Active G6-PI and Enolase-- To determine whether pQE-G6PI and pQE-Enolase plasmids encode active enzymes, we have chosen to complement E. coli mutants lacking G6-PI and enolase genes and to perform enzymatic assays on purified recombinant proteins. The transformation of these E. coli mutants with three different concentrations of pQE-G6PI and pQE-Enolase plasmids gave an increasing number of colonies (Fig. 3, A and D). The presence of pQE-G6PI and pQE-Enolase plasmids was confirmed by PCR (Fig. 3B, lanes 2-5, and Fig. 3E, lanes 4-7). These clones expressed the 63-kDa G6-PI (Fig. 3C, lanes 2-5) or the 48-kDa enolase (Fig. 3F, lanes 4-7) recognized specifically by the polyclonal antibodies that cannot react with any protein in noncomplemented E. coli mutants, as expected (Fig. 3C, lane 1, and Fig. 3F, lane 3). Interestingly, although the antibodies reacted against T. gondii G6-PI and enolase recombinant proteins purified on a Ni²⁺-nitrilotriacetic acid-agarose column (Fig. 3C, lane 6, and Fig. 3F, lane 1), no reactivity could be observed against G6-PI (Fig. 3C, lane 7) or enolase (Fig. 3F, lane 2) from rabbit, confirming that our polyclonal antibodies were specific to the enzymes of the parasite. Moreover, the purified recombinant G6-PI and enolase displayed specific activities comparable to the rabbit enzymes (data not shown). In conclusion, the data show convincingly that these isolated cDNAs encode functional G6-PI and enolase that are specifically expressed in encysted bradyzoites of T. gondii.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Functional complementations of E. coli mutants lacking G6-PI and enolase by pQE-G6PI (A) or by pQE-Enolase (D) plasmids. The presence of plasmids was demonstrated by PCR amplification of G6-PI (B) and enolase (E) ORFs in four E. coli transformants. The expression of G6-PI (C, lanes 2-5) and enolase (F, lanes 4-7) was confirmed by Western blots. Lane 1 in C or lane 3 in F corresponds to untransformed E. coli mutants. C and F (lane 6 and 1) show antibody reactivity to purified recombinant G6-PI and enolase. The polyclonal antibodies failed to recognize G6-PI (panel C, lane 7) and enolase from rabbit (panel F, lane 2).

G6-PIs and Enolases from T. gondii and P. falciparum Are Land Plant Homologs-- The amino acid sequences of G6-PI and enolase from T. gondii (this study) and P. falciparum (33, 34) were aligned with those of several other species. The G6-PIs of T. gondii and P. falciparum displayed four amino acid insertions (residues 286, 423, 425-426, and 429) and four deletions (residues 21-22, 32-33, 47-50, and 403-404) like land plants G6-PIs (Fig. 4A). Similarly to G6-PIs, the two apicomplexan enolases revealed also two short insertions (residues 96 and 263-264) and one deletion (residues 82-83) like plant and green alga enolases (Fig. 4B). In addition, a plant enolase synapomorphy (a pentapeptide EWGWC insertion) is also found in T. gondii (35, 36). However, in contrast to higher plant enolases, the green alga Chlamydomonas reinhardtii lacks this plant signature. The pentapeptide, first reported in apicomplexan parasites for P. falciparum (33), is composed of EWGYS amino acids (residues 103-107) in T. gondii and EWGWS in P. falciparum instead of EWGWC. We have recently cloned another isoform of enolase, which contains the true EWGWC motif of land plant in the tachyzoite stage of T. gondii.²

View larger version (123K): [in this window] [in a new window]   Fig. 4.   Comparative alignment of primary sequences between G6-PIs (A) from T. gondii, Arabidopsis thaliana, Z. mays, Oryza sativa, P. falciparum, Homo sapiens, and S. cerevisiae or between enolases from T. gondii, P. falciparum, Lycopersicon esculentum, Z. mays, C. reinhardtii, and H. sapiens  form (B). *, residues perfectly conserved; ·, well conserved; !, insertions or deletions. The amino acid residues corresponding to the two loops (I, 101-107; II, 260-266) of the three-dimensional models of enolase in Fig. 5 are overlined. The percent of identity between T. gondii and other enzymes is shown at the end of each sequence.

Superimposition of Three-dimensional Structures Confirmed Similarities between T. gondii, P. falciparum, and Plant Enolases-- These studies revealed the almost complete matches, as assessed by the perfect superimposition of three-dimensional structures of T. gondii and human enolases (with a C root mean square of 1.05 Å), except for domains I and II (Fig. 5A). These two loops were also found when P. falciparum and human enolases were superimposed (Fig. 5B). Domain I contains the pentapeptide EWGWC insertion only found in plants and in apicomplexans, whereas a 2-amino acid (EK or NK) insertion is observed in domain II. These short insertions of only five and two amino acids seem to be responsible for the increased length of the two loops. The superimposition of T. gondii and Zea mays enolases indicates similarities between the two loops in plant and apicomplexan enolases (Fig. 5C). These two domains evidenced by the superimposition of all four T. gondii, P. falciparum, maize, and human enolases (Fig. 5D, domain II, and Fig. 5E, domain I) confirmed that enolases from apicomplexan parasites are more closely related to plants than to human and other enolases. Finally, two other features distinguish the three-dimensional models of the human enzyme from those of the apicomplexan counterpart: 1) one -sheet is present in domain II of human enolase, whereas none is found in apicomplexan and plant enolases (Fig. 5D); and 2) two -sheets are present in loop I of P. falciparum, whereas none are seen in that of plants and T. gondii (Fig. 5E). The biological significance of these -sheets in the loops is presently unknown.

View larger version (54K): [in this window] [in a new window]   Fig. 5.   Superimposition of three-dimensional structures between T. gondii and human (A), P. falciparum and human (B), and T. gondii and Z. mays enolases (C). The loops (arrows) correspond to domains I and II. D and E show the superimposition of domains II and I from all four enolases. D is presented after a 90° rotation of loop II. Some amino acid numbers (for example, Lys206, Arg412, and Leu79) are indicated according to their positions in Fig. 4B. Hs, H. sapiens; Zm, Z. mays; Pf, P. falciparum; Tg, T. gondii.

Phylogenetic Analyses Support Apicomplexans and Plants as Sister Clades-- To gain further phylogenetic support for our assumption, extensive molecular phylogenetic analyses were conducted. For G6-PIs and independently to the algorithm used, maximum likelihood (ML), neighbor joining (NJ), and maximum parsimony phylogenetic trees showed that apicomplexans and plants are unequivocally sister lineages (Fig. 6A). The apicomplexans plant clade is supported by bootstrap values of 88, 94, and 96% for ML, NJ, and P, respectively. When the enolase trees were generated, only the maximum parsimony analysis indicate that T. gondii, P. falciparum, and plants are most closely related to each other (Fig. 6B). The data showed an apicomplexans plant clade supported by a bootstrap value of 92%, and there is no way to root the parsimony tree of enolases without having apicomplexans and higher plants as a monophyletic group. However, the ML and NJ analyses gave fragile branchings, with bootstrap values of 38 and 40%, respectively (data not shown). Therefore, the ML and NJ data of enolase trees do not provide any direct statistical support for the apicomplexans plant clade. It should be noticed that bootstrap values above 70% are considered as true clades at >95% probability (37). Thus, it appears that the evolutionary relationships between apicomplexan parasites and plant enolases evidenced by the parsimony tree are weakly supported by the NJ and ML trees, indicating that the phylogenetic links are not as congruent as those found for the G6-PIs. This argues for the placement of Apicomplexa as a sister group closer to plants than animals, fungi, Trypanosomatidae, and bacteria.

View larger version (72K): [in this window] [in a new window]   Fig. 6.   Phylogenetic relationships between G6-PIs (A) and enolases (B). Maximum likelihood, neighbor joining, and maximum parsimony trees are shown with bootstrap confidence values >50%.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have cloned and expressed two functional plant-like glycolytic enzymes, G6-PI and enolase of T gondii. Three lines of evidence support our conclusion. First, the amino acid sequences show considerable homology to known G6-PIs and enolases. Second, these cDNAs complement and restore the ability of E. coli mutants lacking endogenous enolase and G6-PI to utilize glucose. Third, and most importantly, E. coli transfected with the cDNAs encode active recombinant G6-PI and enolase. These two glycolytic enzymes are localized in the cytosol of T. gondii. Consistent with their cytosolic localization, G6-PI and enolase lack either signal sequences or organelle-targeting presequences of the T. gondii apicoplast (38). Both T. gondii and P. falciparum G6-PIs and enolases displayed amino acid insertions and deletions like their plant homologs. It is unlikely that these amino acids were inserted or removed randomly and independently in apicomplexans and plants. The more direct interpretation is that these events occurred in ancestral glycolytic enzymes inherited by both apicomplexans and plants. The plant signatures found in the amino acid sequences and the phylogenetic results of G6-PIs and enolases provide support for apicomplexans being a sister group of the plant clade. However, the ML and NJ analyses of enolase trees do not firmly establish this evolutionary link because of the weakness of the bootstrap values. Altogether, these data raise several hypotheses about the evolutionary origin of apicomplexan parasites.

First, these cytosolic G6-PIs and enolases of apicomplexan parasites could be of algal origin. This assumption is supported by the recent discovery of a nonphotosynthetic remnant plastid ("apicoplast") in apicomplexan parasites (1-3). In light of these observations and on the basis of previous analyses of the evolution of chloroplast cytosol isoenzymes (39-41), the closer evolutionary relationship existing between G6-PIs and enolases of apicomplexans and higher plants could indicate an endosymbiotic gene transfer from the cyanobacterial ancestors of the green algal plastid. By analogy to higher plants, which display in the cytosol a eubacterial type of glycolytic pathway (40-41), our data suggest that the archaebacterial enzymes of the ancestor cell, which gave rise to modern apicomplexan parasites, have been replaced by the products of genes that were donated to the nucleus from cyanobacterial symbionts within green algae (3). Most importantly, plastidial origin for G6-PI has been previously hypothesized (42). It seems possible to root the ML and the maximum parsimony trees described in this study in such a way that monophyly can be obtained between the apicomplexans plant and bacteria cyanobacteria clades. However, it appears that enolases of apicomplexans are closer to those of land plants rather than those of green algae from which the apicoplast might be derived. This interpretation should be taken with caution because the enolase phylogeny provides solid statistical proof only for the parsimony analysis. In addition, the C. reinhardtii enolase is the only green algal sequence presently available. Therefore we feel that more green algal enolase sequences are required for extensive phylogenetic analyses.

Second, apicomplexans may have evolved from a plant parasite of protist nature, which has acquired several genes by lateral transfers. In this case, the ancestor has completely disappeared or has not been yet identified in modern protists.

Third, if the origin of these plant-like glycolytic enzymes is not linked to the horizontal gene transfer events, it should then be hypothesized that Apicomplexa and plants are derived from a common ancestor (8). This ancestor could be a member of the phylum Protalveolata because of common features such as the presence of ultrastructural similarities between the inner membranous complex of apicomplexans and the cortical alveoli in Dinozoa (that include dinoflagellates) and Ciliophora phyla. These cortical alveoli are also present in Glaucophyta, which have starch-like granules in their cytosol like most apicomplexan parasites. To further investigate these hypotheses, molecular cloning and analysis of G6-PIs and enolases from cyanobacteria, algae, dinoflagellates, ciliates, and other protists need to be determined. Nevertheless, it appears that the ancestor of apicomplexan parasites might belong to the photosynthetic lineage and has switched from photoautotrophy to endocellular parasitism. Specific targets for novel chemotherapeutic agents should emerge from these fascinating facets of the evolutionary origin of apicomplexan parasites (38, 43). Even though it was postulated that high degrees of identity between glycolytic enzymes would hamper the development of specific inhibitors directed solely against the enzymes of the parasite, it has been recently reported that glycolytic enzymes remain good candidates for chemotherapy (44-46). For instance, species-specific inhibition has been achieved for triose-phosphate isomerase of Trypanosoma brucei based on the evolutionary changes found in the amino acid sequences and three-dimensional structures (45). Furthermore, the selective and specific inhibition of four glycolytic enzymes from T. brucei has been described using suramin derivatives (46). The glycolytic enzymes identified in T. gondii could be evaluated as potential targets against which new chemotherapeutic agents could be developed. Glycolysis may represent a major or even the unique source of ATP production in encysted bradyzoites, whereas tachyzoites may depend on both glycolysis and mitochondrial oxidative phosphorylation (12-14). The expression of G6-PI and enolase is increased in the cysts of T. gondii, and similar increases of glycolytic enzymes are also observed in red blood cells infected by P. falciparum (47). These glycolytic enzymes could represent new therapeutic targets because of their resemblance to plant enzymes. Theoretically, rational drug design can be envisaged using information drawn from the three-dimensional structure of enolase such as the two enlarged loops located right in front of the channels used by the substrate (2-phosphoglycerate) and the end products, phosphoenolpyruvate and H₂O. To our knowledge, G6-PI and enolase isolated here are the first glycolytic enzymes of T. gondii to be functionally expressed in heterologous systems. The complemented E. coli expressing T. gondii glycolytic enzymes can be helpful for rapid screening of new drugs.

	ACKNOWLEDGEMENTS

We thank Drs. K. Séron and J. Vamecq for critical reading of the manuscript. We are grateful to Dr. I. Callebaut for analysis of the three-dimensional models.

	FOOTNOTES

^* This work was supported by grants from the Agence Nationale de Recherches sur le Sida (ANRS), Fondation pour la Recherche Médicale (FRM), and Ensemble contre le Sida (Sidaction) (to S. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF065160 (G6-PI) and AF051910 (enolase).

^§ Supported by fellowships from the Agence Nationale de Recherches sur le Sida (ANRS).

^¶ Supported by the Conseil Régional du Nord-Pas de Calais.

Supported by the Ministère de l'Enseignement Supérieur et de la Recherche (MESR).

To whom correspondence should be addressed. Laboratoire de Chimie Biologique, CNRS UMR 111, Bâtiment C9, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq, France. Tel.: 33 03 20 43 69 41; Fax: 33 03 20 43 65 55; E-mail: Stan.Tomavo@univ- lille1.fr.

² F. Dzierszinski, O. Popescu, C. Toursel, C. Slomianny, B. Yahiaoui, and S. Tomavo, unpublished data.

	ABBREVIATIONS

The abbreviations used are: G6-PI, glucose-6-phosphate isomerase; HFF, human foreskin fibroblast; ORF, open reading frame; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; bp, base pair(s); ML, maximum likelihood; NJ, neighbor joining.

	REFERENCES

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