Fig 1.
Generation and verification of the ΔPFA cell line.
A) Strategy for inactivation of PFA0660w via selection-linked integration. Expression of a neomycin resistance marker (NEO) is coupled to integration of the plasmid pSLITGD_PFA into the genomic PFA0660w locus, leading to expression of a truncated (likely inactive) PFA66 missing its substrate binding domain (SBD). Production of PFA and NEO as separate proteins is mediated with a SKIP peptide. B) Integration PCR using gDNA extracts from the cell line ΔPFA and the parental cell line CS2 verifies integration of the plasmid pSLITGD_PFA into the PFA0660w gene in the cell line ΔPFA. Amplification of the wild type PFA0660w locus with the primers C and D is only successful in the parental strain CS2 since integration of the plasmid dramatically increases product size. PCRs using primers spanning the junctions of the integration sites C and B for the 5’ region and A and D for the 3’ region) demonstrate disruption of PFA0660w. C) Western blot verifies truncation of PFA0660w in ΔPFA. The truncated fusion protein was detected using an α-GFP antibody, while the parasite protein SERP served as a loading control. +unskipped product, *GFP degradation product. D) Immunodetection verifies expression of HA-tagged PFA66 in the complementation cell line ΔPFA[PFA::HA].
Fig 2.
Electron microscopy reveals deformed knob morphologies of ΔPFA iRBCs.
A) Scanning electron microscopy shows knobs on the surface of iRBCs. The mutant phenotype of ΔPFA is alleviated upon reintroduction of episomally expressed PFA66 in ΔPFA[PFA::HA]. More pictures can be found in S2 Fig. B) Quantification of knob density via ImageJ in SEM pictures (n = 20) shows significantly fewer knobs on ΔPFA iRBCs. Knob density is restored in the complementation cell line CS2ΔPFA[PFA::HA]. C) Quantification of knob morphology across all iRBCs. Knobs were grouped into three categories: small knobs, enlarged knobs, and elongated knobs. Then every knob on 20 SEM pictures of the three strains was assigned to one of these categories. Each bar represents the distribution of these knobs in the three categories across all pictures of a strain. The ΔPFA strains display an increase in deformed and enlarged knob morphologies compared to CS2 and ΔPFA[PFA::HA]. D) Internal view of the deformed knobs/eKnob via transmission electron microscopy of thin slices. More pictures can be found in S3E Fig) Electron tomography reveals electron-dense material at the base and interior of deformed knobs/eKnobs. The marked area denotes the structure shown in F. F) 3D segmentation of discrete densities within a deformed knob/eKnob depicted with electron tomography. The example shows a severely deformed knob/eKnob. Additionally, electron dense material was detected at the base and inside of these structures.
Fig 3.
ΔPFA iRBCs display altered KAHRP distribution.
A) IFA assay of MeOH-Ac-fixed CS2 and ΔPFA using α-KAHRP antibodies reveals punctate patterns. A trend was noticed towards bigger spots in the truncation strain and verified using automated measuring via an ImageJ algorithm (See Fig 3 B, C). D) Live cell imaging of DAPI stained CS2[KAHRP::mCherry] and ΔPFA[KAHRP::mCherry]. KAHRP::mCherry can be seen in both cell lines as punctate patterns; however, CS2 displays smaller and more dots. E) Immunogold labelling of iRBC sections in TEM using α-KAHRP antibodies. Images demonstrate label associated with normal knobs and deformed knobs in CS2 and ΔPFA, respectively. Framed areas can be seen enlarged below. F) Analysis of label density associated with the cytoplasm and area surrounding knobs. Label density is significantly higher in the area surrounding knobs than the cytoplasm for both strains. G) STED imaging of the KAHRP associated with the internal RBC cytoskeleton. For this analysis CS2 and ΔPFA iRBCs were bound to a dish and then lysed hypotonically. The cell body was then washed away, and the remaining cytoskeleton remained as it would be seen from the inside of the iRBC. These samples were then interrogated with an α-KAHRP antibody and STED imaging. G) Representative images of the KAHRP patterns observed in STED from the CS2 and ΔPFA cell line. KAHRP signals were often found to be bigger in the truncation cell line. H) Computational analysis of KAHRP signals through a self-made ImageJ tool revealed no difference in KAHRP spot numbers between both cell lines. I) Investigation of mean object size demonstrated a slight increase of KAHRP spot size in ΔPFA.
Fig 4.
Investigation of the subcellular composition of deformed knobs in ΔPFA[KAHRP::mCherry] via rSTED imaging in an IFA assay.
A, C) DNA was stained using DAPI; WGA was used to stain the RBC glycocalyx; phalloidin was used to stain actin; and RFP booster was used to label KAHRP::mCherry. B, D) Line scan profiles of fluorescence intensity (arbitrary units) along the yellow arrows shown in the fluorescent overlay. The arrows bisect KAHRP_mCherry-rich structures (likely representing knobs). These profiles demonstrate that in the vertical view, phalloidin (i.e., actin) is localised toward the cytosol from the KAHRP structures. The horizontal view shows that the KAHRP-containing structures form a ring structure. These might contain low amounts of actin but are likely filled with other material(s).
Fig 5.
ΔPFA iRBCs display negligible cytoadherence and lower surface exposed PfEMP1.
A) ΔPFA displays negligible cytoadherence and lower PfEMP1 surface exposure than CS2. CS2 and ΔPFA were assayed to test their ability to adhere to immobilised CSA in Petri dishes using microscopic counting of the cells. Cytoadhesion strength is expressed relative to CS2. Results are shown for six binding assays. B) Analysis of PfEMP1 surface exposure via flow cytometry. IRBCs were stained with DAPI and αVAR2CSA antiserum followed by a Cy3-coupled secondary antibody. ΔPFAs have lower PfEMP1 surface exposure than CS2 in six independent experiments. C, D) Expression of a PFA variant featuring a mutated HPD motif in the cell line ΔPFA[QPD::HA] does not complement reduction in knob abundance (C) and knob deformation (D) observed in ΔPFA. IRBC were purified and imaged via SEM. Knobs were then counted and grouped into three categories.
Fig 6.
The J-domain from PFA66 stimulates the ATPase activity of HsHSP70, HsHSC70 and PfHSP70-X.
A) Single turn-over ATP hydrolysis of HsHSP70 in the presence of a peptide substrate (red circles) or PFAJDS (blue circles). Note that in contrast to Daniyan et al. 2016 [39], we clearly observe a stimulation of the hydrolysis of ATP by HsHsp70 (HSPA1A) in the presence of PFAJDS B) Single turn-over ATPase rates for HsHSC70, HsHsp70 and PfHSP70-X in the absence of any substrates or JDPs (black circles), in the presence of the substrate peptide (red circles) or in the presence of PFAJDH (blue circles). Shown are individual data points and standard deviation of independent experiments (n = 3 to 8). *, p < 0.05; **, p<0.001 (ANOVA with Holm-Sidak’s multiple comparison test).
Fig 7.
Proposed model for eKnob formation and structure.
In opposition to normal knob formation in the CS2 cell line (left) runaway extension of the spiral underlying eKnobs in ΔPFA could drive their elongation (right). KAHRP is still present and associated with the inner lumen of eKnobs, PfEMP1 anchored in the eKnobs is incorrectly presented and has thus a reduced cytoadherence capacity.