DPAP3 expression in <i>P</i>. <i>falciparum</i> can be conditionally regulated.

<p><b>A.</b> Western blot analysis showing dose-dependent PfDPAP3-HA protein expression in two independent clones after treatment with glucosamine (GlcN) for one (upper panel) or two (lower panel) cell cycles (Cyc1 and Cyc2, respectively). EXP2 serves as the loading control. <b>B.</b> Densitometry of PfDPAP3 expression levels in PfDPAP3-HAglmS Clone 1 parasites grown in the presence or absence of glucosamine for one cycle revealed DPAP3 expression levels were knocked down by 91%. Error bars represent ± SEM from three independent experiments. <b>C.</b> IFA showing DPAP3-HA expression is significantly reduced within 24 hrs after addition of 2.5 mM GlcN when the parasites are at schizont stage. Scale bars represent 5 μm.</p

The localization of DPAP3 is distinct from proteins that localize to the apical organelles.

<p><b>A.</b> IFA on RBCs infected with PfDPAP3-HAglmS parasites and fixed with acetone/methanol. DPAP3 is labeled with the anti-HA antibody. The scale bars represent 5 μm. <b>B.</b> Immuno-electron microscopy of PfDPAP3-HAglmS parasites with anti-HA antibody shows labelling for DPAP3 (as indicated by the arrowheads) in a mature schizont at the periphery of the rhoptry bulb (RB), rhoptry neck (RN) as well as at the PV/parasitophorous vacuole membrane (PVM).</p

Knockdown of DPAP3 does not affect maturation of SUB1 substrates.

<p>Representative western blots (n = 3–5) of schizont (Sch) and merozoite (M) lysates and culture supernatant (CS) collected after egress (SERA5) or schizont lysates (RAP1, RAMA) made from infected RBCs grown in the presence (+) or absence (-) of 2.5 mM GlcN. Densitometry of bands relative to the EXP2 loading control and between treatment groups revealed no significant difference in the processing of SERA5, RAP1 and RAMA as a result of DPAP3 knockdown.</p

Generation of an inducible PTEX88 knockdown line in <i>P</i>. <i>berghei</i>.

<p><b>A</b>: Schematic representation of the targeting construct used to generate the PbPTEX88 iKD line. Successful integration results in the full <i>ptex88</i> gene being under control of a minimal promoter (solid black arrow) and the TRAD transactivator under the <i>ptex88</i> 5' UTR. Arrows indicate binding sites for diagnostic PCR primers. <b>B</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. <b>C</b>: Quantitative RT-PCR on parasite material isolated from PbPTEX88 iKD parasites shows a significant knockdown of <i>ptex88</i> transcript in parasites exposed to ATc.</p

PTEX88-glmS parasites exposed to glucosamine export PfEMP1 but are less cytoadherent to CD36.

<p><b>A</b>: 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>B</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.</p

Effect of PTEX88 knockdown on <i>P</i>. <i>falciparum</i> protein export.

<p>Immunofluorescence assays showing that exported proteins <b>A</b>: RESA, <b>B</b>: STEVOR, <b>C</b>: SBP1, <b>D</b>: KAHRP, <b>E</b>: RIF50, <b>F</b>: 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 25–75^th and 10–90 percentiles, respectively.</p

Knockdown of PTEX88 in <i>P</i>. <i>berghei</i> impacts growth of <i>P</i>. <i>berghei in vivo</i>.

<p><b>A</b>: 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 <i>t</i>-test. <b>B</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. <b>C</b>: Conditional depletion of PTEX88 does not impact on merozoite formation within schizonts (n = 25).</p

Generation of a PTEX88 knockdown line in <i>Plasmodium falciparum</i>.

<p><b>A</b>: Schematic of targeting construct designed to integrate into the endogenous <i>ptex88</i> locus by single crossover recombination. Arrows indicate binding sites for diagnostic PCR primers. SM, selectable marker; HA, haemaglutinin epitope tag; glmS, glmS ribozyme. <b>B</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. <b>C</b>: 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. <b>D</b>: 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).</p

Conditional depletion of PTEX88 affects parasite burden and virulence.

<p><b>A, C</b>: Parasitemia and <b>B</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 <i>t</i>-test for parasitemias or by log-rank test for survival curves, which plots the % of mice that have not succumbed to cerebral malaria. <b>D</b>: The parasite load in tissues of mice from <b>C</b> was determined by normalizing the expression levels of parasite <i>18S ribosomal RNA</i> against the mouse <i>hrpt</i> house-keeping gene.</p