Fig 1.
Comparison of Reticulocyte enriched Erythrocyte Population (REP) and wild type Erythrocyte Population (wtEP) reveals metabolite enrichment in rodent and human reticulocytes.
A. Dynamics of reticulocyte enrichment in peripheral blood in vivo followed by Phenylhydrazine-HCl (phz) treatment of mice. Reticulocytes were harvested at day 5 post phz treatment. The error is given as the standard deviation (S.D.) of 3 independent biological replicates. B. Volcano plot showing distribution of putative metabolites according to their fold change in abundance in REP vs wtEP in rodent blood. All significant changes are represented above the broken horizontal line. Coloured dots indicate metabolites which are: Blue- significantly up-regulated, Red- significantly down-regulated, Yellow- significant but little change, Brown- non-significant. n = 3 independent biological replicates (with four internal technical replicates each). Significance tested by Welch’s T-test (α < 0.05). See Fig A-C in S1 Text and S1 Table in for the complete list of detected metabolites and their respective abundance fold changes. C. Representative metabolites up-regulated in reticulocytes compared to mature erythrocytes in human and rodent erythrocytes. Relative levels (peak intensities) are expressed as fold change observed in reticulocyte vs mature erythrocytes. Dotted line indicates no change and error bars indicate R.S.D. (Relative Standard Deviation) of peak intensities from reticulocyte samples multiplied to the fold change values from n = 3 independent biological replicates.
Fig 2.
Metabolites of intermediary carbon metabolism (ICM) and pyrimidine biosynthesis are up-regulated in reticulocytes.
A. Top panel: Fold change of relative levels (peak intensities) of metabolites of carbon metabolism in rodent REP compared to wtEP. Dotted line indicates no change and error bars indicate R.S.D. (Relative Standard Deviation) of peak intensities from reticulocyte samples multiplied to the fold change values from n = 3 independent biological replicates. Bottom panel: Schematic representation of intermediary carbon metabolism (ICM) in Plasmodium cytosol. Genes marked with (✓) were deleted in P. berghei blood stages and the ones marked with (✕) could not be deleted even after repeated attempts. pepc: Phosphoenolpyruvate Carboxylase (PBANKA_101790), mdh: Malate Dehydrogenase (PBANKA_111770), aat: Aspartate Amino Transferase (PBANKA_030230). B. Top panel: Fold change of relative levels (peak intensities) of metabolites of pyrimidine biosynthesis in rodent REP compared to wtEP. Dotted line indicates no change and error bars indicate R.S.D. (Relative Standard Deviation) of peak intensities from reticulocyte samples multiplied to the fold change values from n = 3 independent biological replicates. Bottom panel: Schematic representation of pyrimidine biosynthesis pathway in Plasmodium cytosol. Genes marked with (✓) were deleted in P. berghei blood stages and the ones marked with (✕) could not be deleted even after repeated attempts. cpsII: Carbamoyl phosphate synthetase II (PBANKA_140670), act: Aspartate carbamoyltransferase (PBANKA_135770), dhoase: Dihydroorotase (PBANKA_133610), dhodh: Dihydroorotate dehydrogenase (PBANKA_010210), oprt: Orotate phosphoribosyltransferase (PBANKA_111240), ompdc: Orotidine 5′-monophosphate decarboxylase (PBANKA_050740). (Also see Fig B in S1 Text for gene deletion strategy and confirmation)
Fig 3.
Phenotypic analyses of blood stage mutant P. berghei parasites.
A. in vivo growth assay of mutants in mixed infections in competition with wild type parasites over 12 days. Coloured lines represent non-linear fit of percentage of mutant parasites in total parasite population. Data representative of n = 3 independent biological replicates. (Also see Fig C- A, B and C in S1 Text.) B. Lethality experiment in C57/B6 mice by wt and mutant P. berghei parasites. 104 parasites were injected intra-peritoneally in mice (n = 5) on day 0 and they were monitored for 21 days. The mice were culled humanely when they showed severe malaria pathology. All mutant parasites were found to be lethal to mice. C. Gametocyte conversions during blood stages in mutant P. berghei parasites over 5 days post infection. Data from 2 independent observed gametocyte conversion experiments are shown ± S.D. Gametocyte conversion was observed using a wt parent line which expresses GFP in male gametocytes and RFP in female gametocytes (RMgm-164). P. berghei mutants were generated in the same genetic background and analysed using FACS determining the number of gametocytes in infected blood. P-values: *p<0.05, **p<0.005, ***p<0.0005, paired two tailed t-test. D. Exflagellation (male gamete formation) in mutant P. berghei parasites normalised to wt in in vitro activation assay. The error is given as the SD of n = 3 independent biological replicates. P-values: **p<0.005, ***p<0.0005, paired two tailed t-test. E. Determination of DNA content of male gametocytes over 20 minutes post activation by FACS analysis in mutant P. berghei parasites normalised to wt. DNA content was determined in Hoechst-33258-stained MACS purified gametocytes. Before activation (0minutes) males show low DNA content with increasing amounts post activation reaching maximum levels between 8 to 12 minutes in wt. Data from 3 independent biological replicates are given ± S.D. P-values: **p<0.005, ***p<0.0005, unpaired two tailed t-test (also see Fig C- D in S1 Text).
Fig 4.
Mosquito stage development of P. berghei mutant parasites (also see Fig D in S1 Text).
A. in vitro ookinete conversion of mutant P. berghei parasites as compared to wt. The error is given as the S.D. of n = 3 independent biological replicates. P-values: **p<0.005, ***p<0.0005, unpaired two tailed t-test. B. in vitro ookinete conversion assay to measure fertility of mutant P. berghei gametocytes. Fertility of mutant P. berghei gametocytes was analysed by their capacity to form ookinetes by crossing gametes with RMgm-348 (Pb270, p47-) which produces viable male gametes but non-viable female gametes and RMgm-15 (Pb137, p48/45-) which produces viable female gametes but non-viable male gametes. The error is given as the S.D. of n = 2 independent biological replicates. P-values: *p<0.05, **p<0.005, unpaired two tailed t-test. C. Number of mature oocysts at day 14 post infected blood feed in mosquito mid guts. n = 40 mosquitoes cumulative of two independent biological replicates. ***p<0.0005, unpaired two tailed t-test. D. Infection prevalence (percentage of observed mosquitoes found to be infected) and infection load (median of number of oocysts found per mosquito) in mutant P.berghei parasites compared to wt.
Fig 5.
P. berghei and P. falciparum inhibition by dihydroartemisinin (DHA) and 5-fluoroorotic acid (5FOA) in vitro.
Error bars indicate S.D. from n = 3 biological replicates.