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
. 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, PfcSHMT and PfmSHMT share only ~20% similarity with each other
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
. 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 PfmSHMT is sufficient for mitochondria targeting
. In this study, transfection system using GFP reporter gene was taken to examine the cellular localization of PfmSHMT 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 PfmSHMT 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 PfmSHMT 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 PfcSHMT and PfmSHMT were addressed in this study by direct observation of SHMTs fusion with reporter protein compared to previously published work using immunofluorescence approach (IFA)
. The IFA with polyclonal antibody suggested a stage dependent localization pattern where PfcSHMT appeared in the cytoplasm, and also to apicoplast in the mid/late trophozoite to schizont stage. PfmSHMT 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 PfcSHMT and PfmSHMT.
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, while shmt mutations in Caenorhabditis elegans lead to maternal-effect lethal phenotype
, 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 PbcSHMT and PvcSHMT. It should also be noted that the expression of PvcSHMT was regulated by Pbeef1α 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
, 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
. Further optimization employing a target-based design approach should allow design of more effective anti-malarial drugs targeting Plasmodium SHMT.