Plasmodium serine hydroxymethyltransferase: indispensability and display of distinct localization

Abstract

BackgroundSerine hydroxymethyltransferase (SHMT), a pyridoxal phosphate-dependent enzyme, plays a vital role in the de novo pyrimidine biosynthesis pathway in malaria parasites. Two genes have been identified in Plasmodium spp. encoding a cytosolic SHMT (cSHMT) and putative mitochondria SHMT (mSHMT), but their roles have not been fully investigated.MethodsThe presence of Plasmodium SHMT isoforms in the intra-erythrocytic stage was assessed based on their gene expression using reverse transcription PCR (RT-PCR). Localization studies of Plasmodium SHMT isoforms were performed by transfection of fluorescent-tagged gene constructs into P. falciparum and expressions of fluorescent fusion proteins in parasites were observed using a laser scanning confocal microscope. Genetic targeting through homologous recombination was used to study the essentiality of SHMT in Plasmodium spp.ResultsSemi-quantitative RT-PCR revealed the expression of these two genes throughout intra-erythrocytic development. Localization studies using P. falciparum expressing fluorescent-tagged SHMT showed that PfcSHMT-red fluorescent fusion protein (PfcSHMT-DsRed) is localized in the cytoplasm, while PfmSHMT-green fluorescent fusion protein (PfmSHMT-GFP) co-localized with Mitotracker™-labelled mitochondria as predicted. The essentiality of plasmodial cSHMT was inferred from transfection experiments where recovery of viable knock-out parasites was not achieved, unless complemented with a functional equivalent copy of shmt.ConclusionsDistinct compartment localizations of PfSHMT were observed between cytoplasmic and mitochondrial isoforms, and evidence was provided for the indispensable role of plasmodial cSHMT indicating it as a valid target for development of novel anti-malarials.

Novel Plasmodium falciparum metabolic network reconstruction identifies shifts associated with clinical antimalarial resistance

“…Therefore, in our sensitive models, neither SHMT nor the glycine cleavage system is essential when knocked out individually. This observation conflicts with the literature, as SHMT is essential in cultured parasites [118, 120, 121]. Thus, iPfal17 is unable to predict this intricacy of parasite metabolism, revealing interesting regulatory effects, an uncharacterized location dependency for metabolite generation, or in vivo/in vitro differences in enzyme reversibility (Additional file 6).…”

Inducible Knockdown of Plasmodium Gene Expression Using the glmS Ribozyme

“…The pSSPF2/ Pf Hsp60-GFP-Link plasmid described in [23] was used to construct transfection plasmids. Overlapping synthetic oligonucleotides corresponding to the wild-type and “M9” mutated B. subtilis glmS ribozyme sequences reported in [21] were combined in gene synthesis-PCR and the resulting 166 bp ribozyme element cloned at the 3′-UTR position downstream of the GFP gene in pSSPF2/ Pf Hsp60-GFP-Link via the Xho I and Pst I sites.…”

Kinetic Mechanism and the Rate-limiting Step of Plasmodium vivax Serine Hydroxymethyltransferase

“…Plasmodium SHMT has been shown to be essential for parasite growth and development, making it a prime target for antimalarial drug chemotherapy development (11). The recombinant expression and purification of Plasmodium falciparum (PfSHMT) and Plasmodium vivax SHMT (PvSHMT) as well as their biochemical characterizations have been reported (5,(12)(13)(14).…”