Background
The rapid emergence of resistance in
Plasmodium falciparum to nearly all currently used anti-malarials makes control of falciparum malaria a difficult task. Identification of new drug targets for development of new anti-malarials is urgently needed. The malaria parasite lacks thymidine salvage pathway and depends solely on
de novo pyrimidine synthesis[
1,
2], in contrast to the human host, which utilizes both
de novo and salvage pathways. Serine hydroxymethyltransferase (SHMT) is one of three enzymes involved in dTMP cycle, namely, dihydrofolate reductase (DHFR) and thymidylate synthase (TS). SHMT has a pyridoxal phosphate as a cofactor and participates in one-carbon metabolism, in which SHMT converts serine and tetrahydrofolate (THF) to glycine and methylenetetrahydrofolate (MTHF) respectively. SHMT has been investigated as a possible drug target in cancer and microbial therapeutics, particularly as SHMT expression is tightly regulated with DNA replication during cell division and the enzyme catalyzes the rate-limiting step in dTMP synthesis cycle[
3‐
9].
Two forms of SHMT, cytosolic (c) and mitochondrial (m), can be found in eukaryotes[
10,
11]. Based on DNA sequence search in PlasmoDB, there are two genes encoding SHMT in
Plasmodium spp
.:
Plasmodium falciparum contains PFL1720w (PF3D7_1235600), a previously characterized cSHMT gene (
Pfcshmt), and PF14_0534 (PF3D7_1456100), a putative gene of mSHMT (
Pfmshmt). While the enzymatic function of recombinant
Pf cSHMT has been shown, the heterologously expressed
Pf mSHMT was found to be inactive[
9,
12,
13].
Pf mSHMT has been proposed to function in association with glycine cleavage components[
14], but experimental proof has yet to be provided. As for the cytosolic isoform, alignment of amino acid sequences of
Plasmodium cSHMT with human cSHMT shows an overall 44% homology and 80% similarity at the active site. In contrast to mammalian SHMTs,
Plasmodium SHMTs can convert D-serine, in addition to its physiological substrate L-serine, to glycine in the folate-dependent reaction[
9,
15]. Comparison between the crystal structure of human cSHMT and homology model of
Pf cSHMT has revealed differences at the substrate binding site, which could be exploited for the development of specific anti-malarial inhibitors that do not cross inhibit the human enzymes[
16].
Despite several lines of indirect evidence for the essential role of SHMT in malaria parasite growth, there is hitherto a lack of direct demonstration of this notion. Here, the study provides the genetic evidence confirming the two distinct compartmental localization of SHMT isoforms and demonstrates the indispensable role of cSHMT in growth and development of Plasmodium parasites.
Methods
Chemicals
All chemicals used were of the highest quality commercially available. The sequences of primers are listed in Additional file
1.
Semi-quantitative analysis of gene expression of SHMT isoforms in P. falciparum
Semi-quantitative reverse transcription PCR (semi-quantitative RT-PCR) was employed to measure expression levels of Pfcshmt (primers Bgl II 5′Pfcshmt and Eco RV 3′Pfcshmt) and putative Pfmshmt (primers Bgl II 5′Pfmshmt and Kpn I 3′Pfmshmt) relative to that of house-keeping gene Pfα-tubulin-2 (primers Pfα-tubulin-2 F and Pfα-tubulin-2 R). Total RNA was extracted from sorbitol-synchronized P. falciparum 3D7 strain at ring, early trophozoite, late trophozoite, and schizont stages using TRIzol® reagent (Invitrogen™, California, USA). Contaminating DNA was removed with RNase-free DNase I (New England Biolabs, Massachusetts, USA). cDNA was synthesized using oligo-dT primer and M-MuLV reverse transcriptase (New England Biolabs). PCR amplification was conducted using GoTaq® DNA polymerase (Promega, Wisconsin, USA) and the following thermal cycling conditions: 95°C for 3 minutes; 20 or 25 cycles of 95°C for 30 seconds, 50°C for 30 seconds, and 72°C for 2 minutes; and a final heating step of 72°C for 5 minutes. Amplicons were resolved by 2% agarose gel-electrophoresis, stained with ethidium bromide, and analysed for their intensities with ImageQuant™ Software (Molecular Dynamics, California, USA).
Plasmid constructions
Plasmids for the study of gene knockout in Plasmodium berghei ANKA strain were constructed based on the sequence of pL0017 vector (The Malaria Research and Reference Reagent Resource Center; MR4), which contains Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (Tgdhfr/ts) and green fluorescent protein gene (gfp) expression cassettes for pyrimethamine (PYR) selection and fluorescence detection of transfected parasites. The 553 and 1,018 bp of PCR amplicons, corresponding to 5′- and 3′UTR of Pbcshmt (PBANKA_145020) respectively, were produced initially from P. berghei genomic DNA (gDNA). The 5′UTR fragment was inserted into pL0017 at Hind III site, while the 3′UTR fragment was inserted at Kpn I and Sac II sites respectively. This construct, pL0017_Δshmt, was used in the knockout study. For allelic replacement construct, gfp in pL0017_Δshmt was replaced with Plasmodium vivax cshmt (Pvcshmt; PVX_100730) and named pL0017_(Pv)Δshmt.
Vectors for localization study were modified from the original pSSPF2/
Pf Hsp60-GFP vector (a gift from Shigeharu Sato, MRC National Institute for Medical Research, UK)[
17]. Initially, a short linker encoding 14 amino acids (SASKLGTSRATNNT) was inserted at
Avr II restriction site using two complementary oligonucleotides (Linker F and Linker R), which resulted in pSSPF2/
Pf Hsp60-GFP-Link vector. In order to determine the subcellular localization of
Pf cSHMT in malaria parasite,
gfp in pSSPF2/
Pf Hsp60-GFP-Link was replaced with the gene encoding red fluorescent protein DsRed generating pSSPF2/
Pf Hsp60-DsRed. Then, the coding sequence of
Pfcshmt was PCR amplified from cDNA and inserted into pSSPF2/
Pf Hsp60-DsRed replacing a mitochondrial targeting sequence of
PfHsp60 at
Bgl II and
Kpn I sites. For construction of the vector to enable study of putative
Pf mSHMT localization, human
dhfr in pSSPF2/
Pf Hsp60-GFP-Link was replaced with
blasticidin S deaminase (
bsd) at
Bam HI and
Hind III sites, after which the open reading frame region of putative
Pfmshmt was inserted at
Bgl II and
Kpn I sites. DNA sequences of the two constructs, named pRL_
Pf cSHMT and pGL_
Pf mSHMT, were confirmed by DNA sequencing (1st BASE, Singapore).
Parasite culture and transfection
All animal experiments were performed according to the international and national guidelines for ethical conduct on the care and humane use of animals with approval of the Ethical Committee on Animal Experimentation, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand. Mouse strain ICR was intraperitoneally infected with P. berghei (106 infected (i) RBC), and blood from tail vein was collected for determining parasitaemia.
Transfection of plasmids into
P. berghei was performed according to a previously described protocol[
18]. In brief, 5–10 μg of each construct were linearized by digestion with
Sac II, and transfected into purified schizonts using Basic Parasite Nucleofector Kit 2 (Lonza AG, Cologne, Germany) and Amaxa Nucleofector™ device (Amaxa Biosystems GmbH, Cologne, Germany) according to pre-set U033 program. Transfected parasites then intravenously injected into mice tail vein and selected by providing the mice with 70 μg/ml PYR (Sigma-Aldrich, Missouri, USA) in drinking water.
For localization studies,
P. falciparum strain 3D7 was cultured in human RBC (5% haematocrit) in RPMI-1640 medium (Invitrogen™) supplemented with 0.3 g/l L-glutamine, 5 g/l hypoxanthine and 10% human serum under an atmosphere of 1% O
2 and 5% CO
2[
19]. Transient transfection of
P. falciparum with plasmids was performed by electroporation as previously described[
20]. In short, 5-10% synchronous ring stage parasites were electroporated with 100 μg of plasmid using Gene Pulser Xcell Electroporation System (Bio-Rad Laboratories, California, USA) at 0.310 kV and 950 μF. pRL_
Pf cSHMT and pGL_
Pf mSHMT transfected parasites were cultured in the presence of 2 nM WR99210 and 2 μg/ml blasticidin S (Invitrogen™) respectively.
Molecular characterization of transfected P. berghei
Blood from transfected P. berghei-infected mice was collected by heart puncture. White blood cells were removed by passage through a syringe packed with Whatman® CF11 cellulose powder. gDNA was extracted from intra-erythrocytic transfected parasites using Genomic DNA Mini Kit (Geneaid Biotech, Taiwan), and was used in PCR and Southern blotting to assess integration of plasmid constructs at the desired loci. Diagnostic PCR to amplify endogenous Pbcshmt, 5′ integration fragment, and 3′ integration fragment was performed using primer pairs of Xho I Pbcshmt F and Bam HI Pbcshmt R, 5′UTR int Pbcshmt F and 5′UTR int Pbdhfr-ts R, and 3′intDS F and 3′UTR int Pbcshmt R, respectively. The protocols described above were conducted also with wild type parasites. The putative Pbmshmt was amplified as a control (using Xho I putative Pbmshmt F and Bam HI putative Pbmshmt R primers).
For Southern blot hybridization, approximately 20 μg of gDNA extracted from transgenic and wild type parasites were digested with Eco RV and Bgl II. DNA fragments were separated by 1% agarose gel-electrophoresis and transferred to nylon membrane (Merck Milipore, Massachusetts, USA) for hybridization with digoxigenin-labelled 5′UTR and 3′UTR probes of Pbcshmt according to the manufacturer’s protocol (DIG High Prime DNA Labeling and Detection Kit II; Invitrogen™).
Expression of Pbcshmt, putative Pbmshmt, Pvcshmt, and Pbα-tubulin-2 in wild type and transgenic P. berghei parasites were assessed by RT-PCR. Expression level of putative Pbmshmt was measured by quantitative RT-PCR (qRT-PCR) using CFX96™ Real-Time System and iQ™ SYBR® Green Supermix (Bio-Rad Laboratories) normalized to Pbα-tubulin-2 expression level Relative gene expression using 2-ΔΔCT formula. Total RNA and first strand cDNA were prepared for analysis by RT-PCR and qRT-PCR as described above.
Parasite growth study
Three ICR mice per group were injected intravenously in the tail vein with either wild type or transgenic parasites (106 iRBC/mice). Parasite numbers were counted every day using Giemsa-stained blood smears (from tail vein) under a light microscope.
Fluorescence microscopy
Parasites were stained with Mitotracker™ (Roche, Basel, Switzerland) and Hoechst 33258 (Sigma-Aldrich) dyes according to manufacturer’s protocols. Localization of fluorescent protein-tagged SHMT isoforms in transfected parasites was determined using Zeiss LSM 700 laser scanning confocal microscope (Carl Zeiss Micro-Imaging GmbH, Germany) at excitation and emission wavelengths of 555 nm and 572 nm respectively for DsRed, and at 488 nm and 509 nm respectively for GFP. Images were processed using ZEN 2009 software.
Discussion
SHMT links together several metabolic pathways, including biosynthesis of folate, dTMP, and methionine. The biological necessity of this enzyme in malaria parasites has been proposed as
shmt transcripts are markedly increased during the rapid intra-erythrocytic stage progression[
8]. Similar to other eukaryotes,
Plasmodium spp. has two SHMT isoforms, a functioning
cshmt and a putative
mshmt allele. It is worth noting that, unlike other eukaryotes where c- and m-SHMT isozymes are highly conserved,
Pf cSHMT and
Pf mSHMT share only ~20% similarity with each other[
30].
Here, results show that
Pfmshmt is a functional gene by demonstrating the expression of gene product throughout the asexual stage development. The presence of two isoforms in
Plasmodium spp. raises the possibility of a redundant role and a potential overlap in their functional activity. For instance, in mice, examination of nuclear extracts of
cshmt- knockout mice showed 25% SHMT activity compared to wild type mice; the remaining SHMT activity is due to the presence of mSHMT in the nuclear extract, suggesting a redundant function of the two murine SHMT isoforms[
29]. In the case of
Plasmodium spp., null-mutants of
cshmt-knockout parasite clones could not be recovered from transfected
P. falciparum, even though methionine, folinic acid, or a mixture of these compounds was supplemented at concentrations 10-fold higher than that present in RPMI. However, in
P. berghei,
cshmt-knockout parasite clones could only be recovered when complemented with
cshmt from another
Plasmodium species (in this case
P. vivax). These results provide experimental confirmation of the essentiality of
cshmt in the survival of malaria parasites. In addition, these results suggest that there is functional conservation of cSHMT among
Plasmodium spp., but not between cSHMT and mSHMT of the same species. On-going efforts to express recombinant mSHMT are in progress in order to confirm its role in malaria parasites.
The first 24 N-terminal amino acids of putative plasmodial mSHMT contain several basic amino acids characteristics of mitochondrial targeting sequence[
26,
31]. Previous study observed that the first 100 N-terminus of
Pf mSHMT is sufficient for mitochondria targeting[
27]. In this study, transfection system using GFP reporter gene was taken to examine the cellular localization of
Pf mSHMT and to identify the minimum sequence required for mitochondrial targeting of this enzyme. Contrary to the previous prediction, the removal of putative mitochondria signal sequence (N-terminus amino acids 1–24) of
Pf mSHMT did not affect its localization to the mitochondria, suggesting that that the targeting sequence may be downstream of the putative mitochondria targeting sequence. Systematic deletions of the first 120 amino acids of
Pf mSHMT demonstrated that the minimum leader sequence for mitochondrial targeting lies between amino acids 25–80. However, detection of cytoplasmic/mitochondria fluorescence of N1-80-GFP suggests that a more complex mechanism may be involved, such that a longer signal sequence may provide more specific localization to the mitochondria.
Intracellular localizations of
Pf cSHMT and
Pf mSHMT were addressed in this study by direct observation of SHMTs fusion with reporter protein compared to previously published work using immunofluorescence approach (IFA)[
27]. The IFA with polyclonal antibody suggested a stage dependent localization pattern where
Pf cSHMT appeared in the cytoplasm, and also to apicoplast in the mid/late trophozoite to schizont stage.
Pf mSHMT appeared mainly in the mitochondria with some distribution in the cytoplasm. Multi-organelle localizations observed in these IFA experiments may be in part due to cross-reaction of polyclonal antibodies. Whilst the current work relies upon the intrinsic fluorescence from GFP or DsRed fused to SHMT of interest, with the assumption that the fusion proteins behave the same as native SHMTs. Despite different approaches, these studies are complementary of each other, as both studies revealed distinct compartment localization of
Pf cSHMT and
Pf mSHMT.
Various phenotypic consequences in
shmt-deficient cells have been described. Inactivation of
shmt results in glycine auxotroph phenotype in some organisms, such as
Escherichia coli[
32], while
shmt mutations in
Caenorhabditis elegans lead to maternal-effect lethal phenotype[
33], pointing to the essential role of SHMT. In this study, attempts were made to generate
Pbcshmt null mutant but the gene could not be replaced by a knockout construct. The refractoriness of
Pbcshmt locus was ruled out as our attempts to replace the endogenous gene with
Pvcshmt were successful. Additionally, the redundancy role of SHMTs in malaria parasite can be excluded. Transgenic
P. berghei parasites containing
Pvcshmt were able to infect murine red blood cells and complete their blood stage life cycle, albeit at a lower parasitaemia when compared with that of the wild type parasites. This implies that replacement of
shmt affects fitness of transgenic parasite, which may be due to differences in catalytic efficiency between rodent and human plasmodial enzymes. This could readily be proven by comparing kinetic parameters of recombinant
Pb cSHMT and
Pv cSHMT. It should also be noted that the expression of
Pv cSHMT was regulated by
Pb eef1α promoter, which might have an effect on the growth of mutant parasite.
Plasmodium SHMT has been suggested to be the rate-limiting enzyme in dTMP synthesis pathway[
8], and thus is a potential target for drug development. Various classes of compounds, including 2,4-diaminopyrimidine, have been proposed to be effective inhibitors of
Plasmodium SHMT based on binding affinity obtained from molecular docking calculations[
34,
35]. The recent study has shown that a number of 2,4-diaminopyrimidine compounds can inhibit
Plasmodium SHMT[
21]. Further optimization employing a target-based design approach should allow design of more effective anti-malarial drugs targeting
Plasmodium SHMT.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
WP performed the study and drafted the manuscript. DK participated in the design of the study and drafted the manuscript. CU and YY discussed and commented on the manuscript. UL conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.