Plasmodium serine hydroxymethyltransferase: indispensability and display of distinct localization

Abstract

Background: Serine 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.

Methods: The 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.

Results: Semi-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.

Conclusions: Distinct 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.

Figure 1

Expression of Pfcshmt and Pfmshmt at different intra-erythrocytic developmental stages. Expressions levels of Pfcshmt and Pfmshmt transcripts were assessed by semi-quantitative RT-PCR of cDNAs prepared from sorbitol-synchronized P. falciparum 3D7 strain. Results are reported as relative values normalized to Pfα-tubulin-2 transcripts.

Figure 2

Localization of P. falciparum SHMT isoforms. Parasites were transfected with pGFP (A), pRL_PfcSHMT (B), pGL_PfmSHMT (C), and pGL_N24del PfmSHMT (D) plasmids expressing fluorescent signals from GFP or DsRed. Schematic diagrams of the recombinant plasmids used are shown alongside the confocal micrographs. Mitochondrion and nucleus is stained with Mitotracker™ (red) and Hoechst 33258 (blue) dye respectively. DIC, differential interference contrast image; rep20, rep20 sequence; bsd, blasticidin S deaminase; hDHFR, human dihydrofolate reductase; gfp, green fluorescence protein; DsRed, red fluorescent protein.

Figure 3

Mitochondrial signal peptide mapping of PfmSHMT. Schematic diagrams depict localization of GFP fusion protein with (i) full-length PfmSHMT, (ii) N-terminus truncated PfmSHMT, and (iii) a series of truncated N-terminal 1–120 amino acid fragment of PfmSHMT. N24del and N80del refer to deletion of N-terminal amino acids at positions 1–24 and 1–80 of PfmSHMT respectively. The numbers in N1-80, N1-40, N21-60, N41-8 0, N61-100 and N81-120 refer to amino acid positions at N-terminus of PfmSHMT.

Figure 4

Targeted deletion of Pbcshmt. (A) Schematic diagram depicting the genomic organization of Pbcshmt locus following disruption or allelic replacement with Pvshmt coding sequence. Enzyme restriction sites, along with fragment sizes and their specific probes are indicated. (B) PCR diagram of molecular characterization of transfected parasites. Lanes 1–5: (1) water control; (2) gDNA of P. berghei wild type; (3) pL0017_(Pv)Δshmt; (4) and (5) gDNA of P. berghei transfected with pL0017_Δshmt and pL0017_(Pv)Δshmt, respectively. Primer pairs a & b, c & d, and e & f (sequences reported in Additional file 1) are used to amplify (i) endogenous Pbcshmt, (iii) 5^′ integrated fragment, and (iv) 3^′ integrated fragment, respectively. Amplification of putative Pbmshmt (ii) was performed as a control. (C) Southern blot hybridized with 5^′ or 3^′UTR probe to confirm Pvcshmt allelic replacement at Pbcshmt locus. DNA was digested with EcoRV and BglII. Lanes are: (1) pL0017_(Pv)Δshmt plasmid, (2) gDNA of P. berghei wild type, and (3) gDNA of transgenic ΔPbPvcshmt P. berghei, respectively. P, I, and WT indicate expected band size for pL0017_(Pv)Δshmt plasmid, integrated Pvcshmt, and endogenous Pbcshmt, respectively. (D) RT-PCR diagram for detection of shmt transcript. Lanes are: (M) 1kb ladder, (−) water control, (RT-) no RT control, (RT+) cDNA, (W) P. berghei wild type, and (V) transgenic P. berghei harbouring Pvcshmt. Pbmshmt and Pbα-tubulin-2 were amplified as control genes.

Figure 5

Morphology and parasite count. Morphology (A) and parasite count (B) of wild type and ΔPbPvcshmt (allelic replacement with Pvshmt coding sequence) transgenic P. berghei. Thin blood smear and parasitaemia determination were performed every day post infection for 9 days.