ABSTRACT
Toxoplasma gondii is a major food pathogen and neglected parasitic infection that causes eye disease, birth defects, and fetal abortion and plays a role as an opportunistic infection in AIDS. In this study, we investigated pantothenic acid (vitamin B5) biosynthesis in T. gondii. Genes encoding the full repertoire of enzymes for pantothenate synthesis and subsequent metabolism to coenzyme A were identified and are expressed in T. gondii. A panel of inhibitors developed to target Mycobacterium tuberculosis pantothenate synthetase were tested and found to exhibit a range of values for inhibition of T. gondii growth. Two inhibitors exhibited lower effective concentrations than the currently used toxoplasmosis drug pyrimethamine. The inhibition was specific for the pantothenate pathway, as the effect of the pantothenate synthetase inhibitors was abrogated by supplementation with pantothenate. Hence, T. gondii encodes and expresses the enzymes for pantothenate synthesis, and this pathway is essential for parasite growth. These promising findings increase our understanding of growth and metabolism in this important parasite and highlight pantothenate synthetase as a new drug target.
INTRODUCTION
Toxoplasmosis is considered the primary cause of death due to food-borne illness and the second greatest food-borne pathogen for disease burden in the United States, with the United Kingdom Food Standards Authority reporting that the full risks in the food chain remain unknown (1). The seroprevalence in the population is estimated to be 10 to 30% worldwide, although it is unclear how many of these cases are chronic infections. Toxoplasmosis plays a role as an opportunistic infection in AIDS patients, can be congenitally transmitted, possibly leading to miscarriage or neurological damage, and can cause ocular disease. The etiological agent, Toxoplasma gondii, is a ubiquitous obligate intracellular protozoan parasite that infects a range of tissues in warm-blooded animals. T. gondii is an apicomplexan parasite related to coccidia of veterinary importance, Eimeria spp. and Sarcocystis spp., and the parasite responsible for malaria, Plasmodium.
T. gondii is passed to the fetus through the placenta at early stages of pregnancy in humans and other mammals (2); it can cause tissue destruction and congenital malformation, including microcephaly and intracerebral calcification, and can often lead to sudden abortions in humans and other mammals (3–5). Transmission may also occur via blood transfusions that contain tachyzoites, transplants with tissue cysts, or even unpasteurized milk from sheep, cows, or goats (6–9). The disease can be severe in immunosuppressed individuals. Most T. gondii infections are chronic but are considered asymptomatic in healthy immunocompetent hosts, although immunoprevalence is correlated with neuropsychiatric disorders (10, 11).
The most common treatment for active toxoplasmosis infections is an antifol combination (pyrimethamine and sulfadiazine) (12–14). Atovaquone may be given to pyrimethamine- and sulfonamide-intolerant patients (15). With long-term treatment, atovaquone and pyrimethamine resistance in patients has been reported (16, 17). For Toxoplasma infections during pregnancy, the antibiotic spiramycin is also given as a treatment (14, 18), minimizing the possibility of transplacental transmission (19). Although success in managing the symptoms of infections has often been achieved with these treatments, there is no known cure for the chronic Toxoplasma cyst stages.
T. gondii contains many metabolic pathways found in microorganisms, such as the aromatic (shikimate) pathway, that may serve as drug targets (20, 21). These might have been retained from the free-living ancestor or obtained through horizontal transfer (22). Little is known about pantothenate metabolism in T. gondii, but pantothenate biosynthesis has been highlighted as a target for the discovery of novel antibiotic and herbicidal compounds (23). Pantothenate (vitamin B5) is metabolized to coenzyme A (CoA) and acyl carrier protein (ACP) in all organisms in which it serves as an essential cofactor, in 4% of enzymatic energy-yielding reactions involving multiple pathways (e.g., carbohydrate metabolism, the tricarboxylic acid cycle, and fatty acid biosynthesis) (24, 25). Animals cannot synthesize pantothenate and therefore must scavenge pantothenate from their diets or endogenous gut bacteria (26).
A conserved pathway for pantothenate biosynthesis in bacteria, fungi, and plants has been described (Fig. 1). The vitamin is synthesized de novo from pyruvate (supplied from glycolysis) or valine (23). The pantothenate biosynthetic pathway observed in bacteria consists of three enzymatic steps. The initial step is the formation of ketopantoate from α-ketoisovalerate by 3-methyl-2-oxobutanoate hydroxymethyltransferase (EC 2.1.2.11), followed by the reduction of ketopantoate to pantoate by 2-dehydropantoate 2-reductase (EC 1.1.1.169). Pantothenate is synthesized by condensation of pantoate and β-alanine in an ATP-dependent reaction catalyzed by pantothenate synthetase (EC 6.3.2.1; also termed pantoate-β-alanine ligase). The crystal structures of the Escherichia coli enzymes have been resolved (27–29).
Pantothenate and coenzyme A biosynthetic pathway. The pathway for pantothenate biosynthesis, based on that in E. coli, is above the dashed line with the terminal enzyme pantothenate synthetase (pantoate-β-alanine ligase). This pathway is found in bacteria, fungi, algae, and plants. Pantothenate is metabolized to coenzyme A through the conserved pathway shown. The UniProt protein sequence accession numbers for the T. gondii, N. caninum, and human sequences are included for reference.
Bioinformatic analyses in our earlier study were the first indication of a de novo pantothenate biosynthetic pathway in coccidia, with a description of genes encoding the enzymes in the genome sequence in Eimeria tenella (30). Similar genes were identified by genome searches of T. gondii (G. A. McConkey, unpublished observations) and are annotated in the ToxoDB database (http://toxodb.org). This contrasts with studies of Plasmodium falciparum, which found that pantothenate in media is essential for growth (31). This study describes the presence of the conserved pantothenate biosynthetic pathway in T. gondii and explores its origin and the necessity of the pathway for proliferation.
MATERIALS AND METHODS
Cultivation of cell and parasite strains.T. gondii RH (a kind gift from D. Roos) and RH-YFP (stably expressing yellow fluorescent protein [YFP] fused to chloramphenicol acetyltransferase under the dihydrofolate reductase [DHFR] promoter; generously provided by Boris Striepen) (32) strains were used for all experiments. Hs27 human foreskin fibroblast (HFF) cells were purchased from the European Collection of Cell Cultures (ECACC), a Health Protection Agency culture collection.
HFF cells were maintained by serial passage and cultured in T25 (25-cm2) vented flasks until they reached confluence. HFF cells were maintained by continuous passage and used at passages 16 to 35. To passage host cells, 80 to 90% confluent monolayers were rinsed with phosphate-buffered saline (PBS) (pH 7.0) and treated with 0.25% trypsin-EDTA for 1.5 min at 37°C. The cells were then centrifuged at 2,500 × g for 5 min and resuspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated iron-supplemented fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) antibiotic solution at pH 7.2. All medium components were from Life Technologies (Paisley, Scotland). Cell cultures were grown at 37°C in 5% CO2. The medium of the cells was changed to fresh medium prior to parasite invasion.
T. gondii tachyzoites were cultured and maintained by serial passage on adherent confluent monolayers of HFF cells. Infected HFF monolayers were trypsin treated and the parasites released from the host cells by forceful passage through a 27-gauge needle several times. Released parasites were purified by centrifugation at 2,500 × g for 10 min. The supernatant was discarded, and the parasite pellet was resuspended in PBS, followed by centrifugation as described above. The parasite pellet was resuspended in DMEM.
Custom DMEM was prepared following standard formulations. Medium for experiments lacking pantothenate, which contained the same formulation as normal DMEM except without pantothenate supplementation, was custom-made. The DMEM was adjusted to pH 7.2 with 10% NaHCO3, and filter-sterilized medium was supplemented with 10% dialyzed heat-inactivated fetal bovine serum and 1% PS.
HFF cell stocks were grown in standard DMEM supplemented with 10% FBS and 1% penicillin-streptomycin and were infected when monolayers were >80% confluent. Prior to use, the medium was replaced with pantothenate-deficient DMEM supplemented with 10% dialyzed FBS and 1% PS. The lack of pantothenate had no observable effect on the viability of the HFF cell monolayers.
Cloning of pantothenate genes.The open reading frames of the pantothenate pathway genes were reverse transcribed with SuperScript II reverse transcriptase (RT) (Life Technologies, Paisley, Scotland), from 3 μg of total RH strain RNA purified from isolated trophozoites using RNAgents (Promega Corp., Madison, WI), to synthesize cDNA, according to the manufacturer's instructions. Gene-specific forward and reverse primers were designed for PCR from the cDNA using primer3 software (http://primer3.ut.ee). Sequences for these primer sets were as follows: 3-methyl-2-oxobutanoate hydroxymethyltransferase gene: forward primer, 5′-ATGAAAGCACACCACACGAA-3′; reverse primer, 5′-CCATGACGACGACTCTCACA-3′; pantothenate synthetase gene: forward primer, 5′-AAGCTGCTCTCTCAGGTTCG-3′; reverse primers, 5′-GCGCAAGTGGAAGCCGAAAGACGA-3′ and 5′-TCAGGTCCGACGGCCGCAGC-3′. When possible, these primers were designed across exon-intron boundaries. Ten percent of the synthesized first-strand cDNA was used as the template for amplification in PCRs, using a standard PCR protocol and Phusion polymerase (New England BioLabs, Ipswich, MA). PCR products were blunt-end cloned by terminal addition of an adenine nucleotide with Taq polymerase and ligation to the pCRII vector using a TA cloning kit (Life Technologies, Paisley, Scotland), according to the manufacturer's instructions. The correct insert was confirmed by digestion with EcoRI and agarose gel electrophoresis. Sequencing of RT-PCR product clones confirmed the coding sequences (including intron-exon borders) that metaTIGER predicted with clones sequenced in the forward and reverse directions using standard vector primers.
Inhibitor assays.The synthesis of the pantothenate synthetase inhibitors will be described elsewhere. Pyrimethamine was purchased from MP Biomedicals. Inhibitors were diluted in culture medium from 100 mM stock solutions dissolved in dimethyl sulfoxide (DMSO), and aliquots were stored at −20°C.
Black, 96-well, tissue culture-treated, polystyrene microplates with optically clear flat bottoms were seeded with HFF cells, and the cells were cultured in normal (commercial) DMEM supplemented with 10% FBS and 1% PS until they reached confluence. At that point, the cell cultures were washed with several rinses of DMEM without pantothenate.
Free T. gondii strain RH-YFP parasites were isolated by lysis of infected cells in cultures by passage through a 27-gauge needle, centrifugation at 2,500 × g for 10 min, and then resuspension in PBS for counting, followed by recentrifugation at 2,500 × g for 10 min. The supernatant was discarded, and the parasites were resuspended in prepared DMEM containing 10% dialyzed FBS and 1% PS. The washed HFF cells in the microplates were infected with 200 to 250 tachyzoites per well. Triplicate 10-fold serial dilutions of inhibitors, covering the range of concentrations of 0.1 mM to 10 nM, were applied to infected cells. The cultures were incubated in a humidified incubator with 5% CO2 at 37°C for 48 h. A fluorometric plate reader (POLARstar Optima; BMG LabTech, Offenburg, Germany) was used to measure fluorescence intensity with excitation and emission wavelengths of 492 nm and 520 nm, respectively (33). A single flash from a xenon lamp for each well was used for excitation, and emission signals were recorded with a gain setting of 90. The fluorescence intensities of uninfected cells and untreated parasite culture controls were recorded in parallel. The measurements for triplicate samples were averaged and compared with values for untreated parasite cultures. The 50% inhibition values were determined by linear regression analysis using Prism5 (GraphPad Software Inc., La Jolla, CA).
For cytotoxicity assays, Hs27 human foreskin fibroblast (HFF) cell monolayers (ECACC number 94041901) were grown in DMEM supplemented with 10% fetal bovine serum, in 96-well microtiter plates. When confluent, the cells were treated with a range of concentrations of inhibitors (1 mM to 1 μM) and incubated as described above. Cell viability was measured using the CellTiter-Blue assay (Promega), and data were processed as described above.
RESULTS
Toxoplasma gondii encoding of enzymes for de novo pantothenate and coenzyme A biosynthesis.Earlier studies indicated that enzymes for pantothenate biosynthesis are encoded in coccidian parasites, with sequences for these enzymes being detectable in the Eimeria tenella genome (30). With software developed to identify metabolic enzymes in parasitic protozoa, i.e., metaSHARK, the genes were found in the coccidian parasites E. tenella and T. gondii (unpublished observations). The algorithm for searching for homology, based on PRIAM profiles, is a highly sensitive method to detect orthologous enzymes. Similar genes in T. gondii were annotated in the ApiDB genome database (34) and are summarized in a review (35). Genes encoding 2-dehydropantoate 2-reductase, 3-methyl-2-oxobutanoate hydroxymethyltransferase, and pantothenate synthetase are detectable in the genome of T. gondii (Fig. 1). Of interest, the first two enzymes are encoded in a single bifunctional gene. The expression of the genes in T. gondii was confirmed by RT-PCR of RNA from replicating tachyzoites (data not shown). These genes are annotated in ToxoDB/EuPathDB (accession numbers TGME49_257050 and TGME49_265870), with patterns of detectable protein expression in tachyzoites and constitutive gene expression throughout the life cycle (i.e., bradyzoites, tachyzoites, and oocysts). The enzyme-encoding genes are also detectable in Neospora caninum (36).
The remaining enzymes of the pathway that leads to the synthesis of coenzyme A from pantothenate are also encoded in T. gondii, as found with E. tenella. All steps for synthesis except for the terminal step with dephospho-CoA kinase were identified in metabolic reconstructions (in EuPathDB and the Library of Apcomplexan Metabolic Pathways), and our software detected orthologs with high levels of similarity (E values of 2.9e−49 for T. gondii and 1.6e−51 for N. caninum) that are found in the genome sequences (36). Interestingly, the related apicomplexan parasite Plasmodium falciparum, the etiological agent of malaria, encodes the enzymes for the metabolism of pantothenate to coenzyme A, but genes encoding pantothenate biosynthetic enzymes have not been found and were not detectable in our analysis using SHARKhunt (30). The coccidia Sarcocystis neurona and Hammondia hammondi are also likely to have the complete pantothenate pathway, as sequences with high levels of similarity (E values of ≤1e−57) to the enzymes are detectable in the genome sequences in EuPathDB searches. Hence, it appears that de novo biosynthesis of pantothenate is specific to the coccidia among Apicomplexa species, whereas pantothenate metabolism to coenzyme A, as found in eukaryotes, appears to be conserved among Apicomplexa species.
Pantothenate synthetase in T. gondii.The coding sequence of T. gondii pantothenate synthetase was determined from sequencing of the mRNA. The sequence matches the coding sequence predicted from the genome sequence (ToxoDB accession number TGME49_265870). The T. gondii sequence (Fig. 2) contains the conserved domains found in orthologous pantothenate synthetases (37). The conserved regions of pantothenate synthetase were found in the predicted sequences of the three coccidian parasites in a multiple-sequence alignment with Mycobacterium tuberculosis and Saccharomyces cerevisiae. Pantothenate synthetase is considered a potential drug target in M. tuberculosis (38–40). The T. gondii ortholog contains 34 strictly conserved residues and 14 of 15 active site residues, including the HIGH and KMSKS sequence motifs. Dimerization sequences identified in plant and bacterial pantothenate synthetases could not be detected. The catalytic residues identified in M. tuberculosis and Escherichia coli pantothenate synthetase structures are conserved (28, 41).
Alignment of pantothenate synthetase (pantoate-β-alanine ligase) sequences, including coccidian parasites. T. gondii, Eimeria tenella (GI number 557147758), and Neospora caninum (GI number 401401059) sequences were aligned with Mycobacterium tuberculosis (GI number 490013547) and Saccharomyces cerevisiae (GI number 37362661) sequences using the ClustalW2 multiple-sequence alignment program, and the conserved region is shown. The T. gondii sequence was confirmed by sequencing. Residues shown in bold are conserved among these coccidia, bacteria, and yeast. Consensus levels are annotated beneath the sequences (asterisk, fully conserved; colon, highly conserved; period, weakly conserved), and the motif sites are indicated. Boxed residues, glutamines predicted to bind pantoate in the homology model.
Sensitivity of T. gondii to pantothenate synthetase inhibitors.To assess whether pantothenate synthesis is required for growth, preliminary experiments were performed to examine the requirement for pantothenate in the medium. Unlike observations with P. falciparum, parasites cultured in medium deficient in pantothenate and coenzyme A grew at near-normal rates (31). Exogenous pantothenate was not required for growth, suggesting that T. gondii supplies pantothenate for coenzyme A through de novo biosynthesis. It remains a possibility that T. gondii imports coenzyme A from host cells to fulfill its requirements, as the fibroblasts were washed free of pantothenate and cannot synthesize pantothenate. Therefore, the effects of pantothenate biosynthesis inhibitors on T. gondii growth were measured.
A panel of 13 pantothenate synthetase inhibitors developed to target M. tuberculosis pantothenate synthetase were screened for their effects on the growth of T. gondii (39). In this assay, serial dilutions of inhibitors were tested for their effects on the growth of parasites infecting human fibroblasts. Following cultivation for 48 h, the growth of YFP-expressing parasites was measured by monitoring fluorescence. All experiments were repeated at least three times. The toxoplasmosis drug pyrimethamine (42) served as a positive control. Pyrimethamine plus sulfadiazine is the recommended treatment for acute T. gondii infections. The 50% effective concentration (EC50) values for the tested inhibitors and pyrimethamine were measured in parallel (Table 1).
Inhibitory values of pantothenate synthetase inhibitors for T. gondii parasite growth
The inhibitors exhibited a range of inhibitory activities (Table 1). The most potent inhibitors (SW413 and SW404) inhibited T. gondii growth with EC50s in the nanomolar range (0.02 ± 0.07 μM and 0.13 ± 0.07 μM, respectively) and exhibited exemplary dose-response curves (Fig. 3). Pyrimethamine exhibited an EC50 similar to published values, which range between 0.067 μM and 12 μM (43–46). In cytotoxicity tests, a range of effects was observed (Table 1). The inhibitors were specific for the parasites, as the 50% lethal concentration (LC50) values for human cells were considerably higher than the EC50s, with selectivity indices of up to >50,000-fold for the most potent inhibitors. Of interest, the most active inhibitors were at least as potent as pyrimethamine in inhibiting parasite growth. This indicates that de novo pantothenate biosynthesis is essential for T. gondii growth and can be targeted.
Dose-response curve for the effects of pantothenate synthetase inhibitor SW404 on T. gondii growth. The inhibition of the number of parasites is expressed as a percentage of values for control cultures in medium deficient in pantothenate. The percent inhibition is plotted against the log concentration of inhibitor.
Selective targeting by pantothenate synthetase inhibitors of pantothenate biosynthesis in T. gondii.To assess whether the selected pantothenate synthetase inhibitors specifically inhibit the pantothenate biosynthetic pathway in T. gondii, growth inhibition assays were performed in the presence of supplemental pantothenate. The inhibitors SW404 and SW314 were examined for selectivity. Parallel cultures of parasites lacking or supplemented with 50 mg/liter d-pantothenic acid were tested to assess the specificity of inhibition for the target. Experiments were performed in triplicate and repeated at least twice.
In assays with added pantothenate, the inhibitor EC50s increased significantly, to 52 ± 25 μM and 104 ± 52 μM for SW404 and SW314, respectively (Table 2). The inhibition concentrations with pantothenate were >200-fold higher than those without pantothenate; this demonstrates specific inhibition of the pantothenate pathway in T. gondii parasites. In contrast, there were small differences in the sensitivity of parasites to pyrimethamine, with EC50s of 0.8 ± 0.3 μM and 6.5 ± 0.3 μM without and with pantothenate, respectively. The decreased sensitivity of T. gondii to pyrimethamine with supplemental pantothenate is intriguing and may indicate metabolic interactions between the folate and coenzyme A pathways.
Selectivity of pantothenate synthetase inhibitors, based on sensitivity in pantothenate-deficient and pantothenate-supplemented media
DISCUSSION
T. gondii contains a complete pathway for pantothenate synthesis and metabolism to coenzyme A, based on the canonical enzymes in bacteria and fungi, as shown in this study. The pathway enzymes are expressed and the pathway is essential for parasite growth, highlighting it as a target for chemotherapy. Using inhibitors of the terminal enzyme, the parasite pantothenate synthetase was found to be essential and a potential target. Further developments could lead to new treatments. Genomic evidence supports the presence of pantothenate biosynthesis among coccidian parasites, with evidence for N. caninum, E. tenella, and S. neurona, but it is not found in other branches of Apicomplexa.
T. gondii was found to proliferate without pantothenate and hence is autotrophic for pantothenate. This contrasts with P. falciparum, which depends on pantothenate for growth (31). Indeed, P. falciparum imports pantothenate using an H+-pantothenate symporter (47). An ortholog of the P. falciparum transporter is not detectable in the T. gondii genome. When we removed pantothenate from the medium, T. gondii continued to grow. Trace amounts remaining in the medium would be expected to be rapidly metabolized to coenzyme A by the host cells. One alternative explanation for the observations would be that T. gondii could import coenzyme A from the host cells using a novel mechanism. Hence, pantothenate synthetase inhibitors were used to demonstrate that T. gondii synthesizes pantothenate de novo and that pantothenate synthesis is required for growth.
Of a panel of inhibitors designed to inhibit M. tuberculosis pantothenate synthetase, several inhibited T. gondii growth. T. gondii pantothenate synthetase has considerable similarity to the M. tuberculosis ortholog and contains 14 of the 15 active site residues. The growth inhibition was disrupted by the addition of pantothenate, demonstrating the specificity of the inhibitors for the pantothenate pathway in T. gondii. It is unclear how the exogenous pantothenate is imported into the parasites, but a computationally annotated gene in the genome of T. gondii (ToxoDB accession number 541.m01156), with some similarity to the human Na+-pantothenate symporter, that could transport pantothenate was found (35). As the prediction of the specificity of membrane transporters is difficult and often impossible without testing, it is unclear whether this protein can take up pantothenate, but if it functions, even at low efficiency, then this could explain why the high levels of pantothenate used in these experiments could exacerbate inhibition.
The requirement for pantothenate biosynthesis by T. gondii and its absence from mammals provide the exciting possibility of this pathway as a target for future chemotherapy. Tissue levels of pantothenate in mammals are very low (rats, 109 ± 7 nmol/g; human colon, 5.9 ± 0.4 nmol/g) (48, 49). Inhibitors of E. coli and M. tuberculosis pantothenate synthetase have been designed, and M. tuberculosis auxotrophic mutants with mutations in pantothenate biosynthetic enzymes are attenuated in animals (50). It might be worth investigating the structural and kinetic similarities of the parasite and bacterial orthologs of pantothenate synthetic pathway enzymes, possibly starting with the key enzyme pantothenate synthetase. The concept of exploiting certain antibiotics to target pantothenate synthetase as anticoccidian drugs should be further investigated.
Intriguingly, two of the most potent M. tuberculosis pantothenate synthetase inhibitors, i.e., SW314 and SW404, were also two of the most potent compounds against T. gondii parasites (39). Inhibitors were optimized for binding to the M. tuberculosis pantothenate synthetase structure, with the highest affinity being with SW404. Cocrystallization of M. tuberculosis pantothenate synthetase with bound SW404 found the inhibitor docking within the ligand site, with hydrogen-bonding interactions with Met40 (RCSB Protein Data Bank [PDB] accession number 3IVX).
To further explore the interactions of the inhibitors with T. gondii pantothenate synthetase, a homology model was generated. The model was based on Yersinia pestis pantoate-β-alanine ligase (PDB number 3Q12), as this was the most similar enzyme in the database based on sequence alignment (28.6%). Ligands were added to the putative active site of the homology model following derivation of the model using the Swiss model workspace automated mode, in the same conformation as the initial cocrystal structure. The newly developed homology model with the bound inhibitor was minimized using the Schrodinger Maestro software suite and the OPLS2005 force field in water. In silico docking of the inhibitor molecules in the newly minimized homology model was performed in a region defined as a 10-Å area surrounding the ligand receptor, and the inhibitors were docked into the putative binding site using AutoDock. Of 40 putative poses produced by AutoDock, the most populated cluster in both energy and special similarity in a consensus docking approach, rather than the lowest energy, was chosen to represent the natural pose adopted by the ligands.
Interestingly, of the possible docking regions, all inhibitors bound within the pantoate binding site of the cocrystal structure (Fig. 4). Hydrogen-bonding interactions of the inhibitors with T. gondii residues Gln154 and Gln328, which form the hydrogen-bonding interactions with bound pantoate in the cocrystal structure 3Q12, homologous to Y. pestis residues Gln61 and Gln155 (and homologous to M. tuberculosis residues Gln72 and Gln164, involved in pantoate binding), were predicted. Several additional residues were consistently predicted to bind the docked ligands. The conserved residue Arg362 appears important for the binding of ALV553, whereas conserved Ser360 may be important for the binding of the compounds SW404 and SW414. Further experimentation is needed to assess whether the residues in the putative binding pose for these inhibitors in the in silico modeling are involved in binding, with particular attention to the conserved glutamine residues in the active site.
Predicted binding site for pantoate in the homology model of T. gondii pantothenate synthetase. This structural model is based on the cocrystal structure of PDB accession number 3Q12. The model predicts hydrogen bonds to conserved glutamine residues (GLN61 and GLN155, using Y. pestis numbering) (black dashed lines). Default colors of atoms in Swiss-PdbViewer are applied: C, white; O, red; N, blue; S, yellow; P, orange; H, cyan; other, gray.
Of interest, coenzyme A is likely to be essential in Apicomplexa species for fatty acid biosynthesis, which is mediated in the apicoplast (51). As targets for chemotherapy, lipid metabolism and fatty acid synthesis in Apicomplexa species, particularly T. gondii, have attracted considerable attention (52). Therefore, inhibiting pantothenate biosynthesis might result in disruption of fatty acid synthesis, further reinforcing the attractiveness of this pathway for drug discovery (53).
As de novo pantothenate biosynthesis is present in T. gondii and is essential for parasite growth, it represents a novel target for chemotherapy, particularly with the absence of the pathway in mammals. Mammals rely on dietary sources for pantothenate, and it is rapidly metabolized to coenzyme A. This study suggests that pantothenate synthetase is a possible drug target in T. gondii, with a lead candidate at least as potent as pyrimethamine being identified in this study. In vivo experiments are needed to examine its efficacy in animals.
ACKNOWLEDGMENT
Funding for this project was received from the Ministry of Higher Education and Scientific Research of Iraq, through the Iraq Cultural Embassy.
FOOTNOTES
- Received 3 March 2014.
- Returned for modification 14 April 2014.
- Accepted 10 July 2014.
- Accepted manuscript posted online 21 July 2014.
- Copyright © 2014, American Society for Microbiology. All Rights Reserved.
