Fig 1.
Generation of a PTEX88 knockdown line in Plasmodium falciparum.
A: Schematic of targeting construct designed to integrate into the endogenous ptex88 locus by single crossover recombination. Arrows indicate binding sites for diagnostic PCR primers. SM, selectable marker; HA, haemaglutinin epitope tag; glmS, glmS ribozyme. B: Diagnostic PCR on wildtype (WT) and transgenic genomic DNA using the indicated primer combinations to test for integration of the targeting construct. Absence of a product using primer combination A/B in PTEX88-HA and PTEX88-glmS gDNA indicates these parasite lines are clonal. C: Representative Western blot showing levels of tagged PTEX88 in PTEX88-HA control parasites and PTEX88-glmS parasites after incubation with the indicated concentration of glucosamine. EXP2 was used as a loading control. GlcN, glucosamine. D: Densitometry performed on Western blots to quantify the protein levels of tagged PTEX88 in the control PTEX88-HA line and the PTEX88-glmS line. The protein level of PTEX88 does not decrease in the control PTEX88-HA line but decreases in the PTEX88-glmS with increasing GlcN concentrations by up to 90% when compared to the sample without GlcN (n = 4).
Fig 2.
Effect of PTEX88 knockdown on P. falciparum protein export.
Immunofluorescence assays showing that exported proteins A: RESA, B: STEVOR, C: SBP1, D: KAHRP, E: RIF50, F: PfEMP1 are still exported after knockdown of PTEX88. Mean fluorescence intensity was calculated for RESA, STEVOR and KAHRP, whilst the number of Maurer’s clefts was calculated for SBP1. Boxes and whiskers delineate 25–75th and 10–90 percentiles, respectively.
Fig 3.
Knockdown of PTEX88 does not affect the growth of P. falciparum.
A: Giemsa stained blood smears of P. falciparum-infected erythrocytes shows no effect on parasite growth in the PTEX88-glmS line when treated with 2.5mM glucosamine relative to the untreated parasites. B: Lactate dehydrogenase (LDH) activity assay of infected erythrocytes assayed approximately 24 hours after parasite invasion indicate that growth of control parasites and PTEX88-glmS parasites is unaffected upon glucosamine treatment. (n = 4). C: Reduction of PTEX88 protein levels with 2.5 mM glucosamine does not affect the merozoite formation with a schizont (n = 15 per group).
Fig 4.
Generation of an inducible PTEX88 knockdown line in P. berghei.
A: Schematic representation of the targeting construct used to generate the PbPTEX88 iKD line. Successful integration results in the full ptex88 gene being under control of a minimal promoter (solid black arrow) and the TRAD transactivator under the ptex88 5' UTR. Arrows indicate binding sites for diagnostic PCR primers. B: Diagnostic PCR on parasite genomic DNA using indicated primer combinations demonstrate correct integration of the targeting construct. Absence of a product using primer combination A/B in PbPTEX88 iKD gDNA indicates this line is clonal. C: Quantitative RT-PCR on parasite material isolated from PbPTEX88 iKD parasites shows a significant knockdown of ptex88 transcript in parasites exposed to ATc.
Fig 5.
Effect of PTEX88 knockdown on protein export in P. berghei.
A: Immunofluorescence assays showing the P. berghei proteins PBANKA_114540 and PBANKA_122900 are still exported after conditional depletion of PTEX88 in PbPTEX88iKD parasites using ATc. Export was measured by calculating MFI, shown as the mean + SEM. B: Surface labelling of parasite antigens on PbPTEX88 iKD parasites harvested between 4 or 5 days post infection compared with infected erythrocytes not exposed to ATc as measured by FACS (n = 6). A mixed ANOVA was used to test for statistical significance.
Fig 6.
Knockdown of PTEX88 in P. berghei impacts growth of P. berghei in vivo.
A: Parasitemia of Balb/c mice administered either ATc (dashed line) or vehicle control (solid line) after intraperitoneal administration of asynchronous PbANKA wildtype (WT) parasites (left panel) or PbPTEX88 iKD parasites (right panel). Each data point represents the mean ± SEM, n = 6 mice per group. ***P<0.001 as determined by unpaired t-test. B: Conditional depletion of PTEX88 leads to a lower fold-increase in parasitemia (left panel) and increase in circulating schizonts (right panel) in synchronous infections initiated by intravenous injection of merozoites. C: Conditional depletion of PTEX88 does not impact on merozoite formation within schizonts (n = 25).
Fig 7.
Conditional depletion of PTEX88 affects parasite burden and virulence.
A, C: Parasitemia and B: Survival curves of C57/Bl6 mice administered either ATc (dashed lines) or vehicle control (solid lines) after intraperitoneal administration of 1x106 PbPTEX88 iKD parasites. Crosses represent the number of deaths. *P<0.05, **P<0.01, ***P<0.001 as determined by unpaired t-test for parasitemias or by log-rank test for survival curves, which plots the % of mice that have not succumbed to cerebral malaria. D: The parasite load in tissues of mice from C was determined by normalizing the expression levels of parasite 18S ribosomal RNA against the mouse hrpt house-keeping gene.
Fig 8.
PTEX88-glmS parasites exposed to glucosamine export PfEMP1 but are less cytoadherent to CD36.
A: Adherence of trophozoite-stage infected erythrocytes to recombinant CD36 under flow conditions (0.1 Pa) ± SEM, n = 30 (***, P<0.001), unpaired t-test. B: Trypsin digestion of surface exposed PfEMP1 in erythrocytes infected with PTEX88-glmS parasites grown in the presence and absence of glucosamine (GlcN). P: pre-treated, T: trypsin treated. Full-length PfEMP1 (~270 kDa, black arrowhead) and a cross-reactive spectrin band (~240 kDa, asterisks) are indicated. Trypsin cleavage products (75 kDa) are indicated with an empty arrowhead.