The role of KAHRP domains in knob formation and cytoadherence of P falciparum-infected human erythrocytes

Abstract

Surface protrusions of Plasmodium falciparum-infected erythrocytes, called knobs, display focal aggregates of P falciparum erythrocyte membrane protein 1 (PfEMP1), the adhesion ligand binding endothelial-cell receptors. The resulting sequestration of infected erythrocytes in tissues represents an important factor in the course of fatalities in patients with malaria. The main component of knobs is the knob-associated histidine-rich protein (KAHRP), and it contributes to altered mechanical properties of parasite-infected erythrocytes. The role of KAHRP domains in these processes is still elusive. We generated stable transgenic P falciparum-infected erythrocytes expressing mutant versions of KAHRP. Using atomic force and electron microscopy we show that the C-terminal repeat region is critical for the formation of functional knobs. Elasticity of the membrane differs dramatically between cells with different KAHRP mutations. We propose that the 5' repeat region of KAHRP is important in cross-linking to the host-cell cytoskeleton and this is required for knob protrusion and efficient adhesion under physiologic flow conditions.

Figure 1.

Targeted gene truncation of KAHRP. (A) Schematic representations of the plasmid vector pHH1 used for integration, the endogenous KAHRP gene, and the expected integration event. Promoter (arrow) and terminator (T) regions are depicted as shaded, the resistance marker human DHFR (hDHFR) in white, the endogenous KAHRP gene in gray, and the truncated KAHRP gene in black boxes. Relevant restriction enzyme sites are shown and sizes of each expected fragment are shown below in kilobases. (B) Southern blot analysis of Afl II-HindIII-digested gDNA from 1 to 8 of: 3D7-K119, -K245, -K362, -K405, -K530, -K589, -K(re), and 3D7 parental parasite lines. Note that for 3D7-K119, the construct does not have the second AflII site in the truncated KAHRP gene. Therefore, there is no 1.45-kb band present on the blot but a band representing the distance between the first Afl II site and the HindIII site. For 3D7-K245 the band happens to be a very similar size to the 4.088-kb band and therefore only one double band is visible for this mutant cell line.

Figure 2.

Expression of truncated KAHRP. (A) Schematic representation of truncated proteins (K119, etc) expressed in the transfected lines indicating the number of amino acids of each protein. Red boxes represent the signal peptide, yellow boxes the His-rich repeats, blue boxes the 5′, and green boxes the 3′ repeat regions (not drawn to scale). (B) Western blot analysis of all mutant cell lines compared to 3D7 parasite line detected with anti-K1A antisera. (C) Immunofluorescence assay of K119 compared to K(re) probed with 2 antibodies against different regions of KAHRP. The anti-K1A antibody recognizes the His-rich repeat region, whereas the anti-KAHRP (repeats) antibody was raised against the 5′ and 3′ repeat regions. The first panel of each row shows a phase-contrast image of an infected erythrocyte, the second panel shows nuclear DNA of the parasite stained with DAPI, the third panel shows the reaction with the KAHRP-specific antibody, and the fourth panel represents an overlay of the 3 previous panels. The anti-K1A antibody shows no cross-reaction with uninfected erythrocytes as shown by lack of reactivity in contrast to strong rim fluorescence observed in all P falciparum-infected red blood cells.

Figure 3.

Surface characteristics of mutant cell lines expressing truncated versions of KAHRP. Scanning electron micrographs of (A) 3D7-K119, (B) 3D7-K245, (C) 3D7-K362, (D) 3D7-K405, (E) 3D7-K530, (F) 3D7-K589, (G) 3D7-K(re), (H) parental 3D7, and (I) KAHRP KO8 parasite lines. The bar represents 2 μm.

Figure 4.

Ultrastructural features of knobs in KAHRP mutant cell lines. Transmission electron micrographs of erythrocytes infected with (A) 3D7-K119, (B) 3D7-K245, (C) 3D7-K362, (D) 3D7-K405, (E) 3D7-K530, (F) 3D7-K589, (G) 3D7-K(re), and (H) 3D7 (parental strain). Arrowheads indicate knob formation. Insets show a higher magnification of the areas of each cell shown in panels A, D, and E.

Figure 5.

Knob and surface topography of mutant cells lines expressing truncated versions of KAHRP. AFM of (A) uninfected (i), 3D7-K119-infected (ii), 3D7-K405-infected (iii), and 3D7-K(re)-infected (iv) red blood cells. The images show a 3-dimensional reconstruction of an area representative of 1 μm on each studied cell line. (B) Analysis of the frequency of a certain height (ii) and diameter (i) of knobs in uninfected (yellow), K119-infected (green), K405-infected (blue), and K(re)-infected (red) erythrocytes. The pre-knobs of K(re)-infected erythrocytes are shown in orange. (C) Surface profile of K(re)-infected red blood cells. Height of knobs was measured along a distance of 1.2 μm on the surface of the cell shown on the left panel (curved line). The pre-knobs are visible as minor peaks (arrowheads).

Figure 6.

Adhesive and mechanical properties of KAHRP mutant cell lines. (A) Membrane shear elastic modulus of erythrocytes infected with 3D7-K119, 3D7-K530, and 3D7-K(re) compared with uninfected red blood cells (uRBCs). Each point represents an individual erythrocyte. (B) Cells were flowed over monolayers of platelets expressing CD36. A physiologically relevant wall shear stress of 0.05 Pa was applied and error bars represent mean values ± SEM. The percentage represents a comparison of the adherence of each mutant cell line with 3D7-K(re)-infected and 3D7-infected erythrocytes. (C) Cells were flowed over monolayers of platelets expressing CD36 at 0.05 Pa. Adherent cells were then exposed to stepwise increases in wall shear stress. The number of infected cells remaining adherent after exposure to each stress was determined and is shown as the proportion of adherent infected cells relative to the number adhering at 0.05 Pa. Each value represents the mean ± SEM for 3 experiments for each parasite line. • indicates 3D7-K119-infected; □, 3D7-K530-infected; and ▪, 3D7-K(re)-infected erythrocytes.

Figure 7.

PfEMP1 trafficking and surface exposure on KAHRP mutant cell lines. (A) Immunofluorescence assay on 3D7-K119- and 3D7-K(re)-infected erythrocytes with anti-ATS antibody detecting PfEMP1 and anti-MAHRP, a Maurer clefts marker. The first panel in each row shows a phase-contrast image, the second panel is nuclear DNA stained with DAPI, the third panel is the reaction with the anti-ATS antibody for PfEMP1 detection, the fourth panel is the reaction with anti-MAHRP antibody, and the fifth panel illustrates an overlay of the previous 4 panels with colocalization shown in yellow. (B) Western blot analysis of trypsin treated (+) or untreated (-) intact erythrocytes infected with all mutant cell lines. The anti-ATS antibody detects cleaved PfEMP1 in case of presence of PfEMP1 on the surface in trypsin-treated samples (arrowhead). The full-length PfEMP1 runs at the same molecular weight as cross-reactive uRBC proteins.