Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes

Abstract

After invading human erythrocytes, the malarial parasite Plasmodium falciparum, initiates a remarkable process of secreting proteins into the surrounding erythrocyte cytoplasm and plasma membrane. One of these exported proteins, the knob-associated histidine-rich protein (KAHRP), is essential for microvascular sequestration, a strategy whereby infected red cells adhere via knob structures to capillary walls and thus avoid being eliminated by the spleen. This cytoadherence is an important factor in many of the deaths caused by malaria. Green fluorescent protein fusions and fluorescence recovery after photobleaching were used to follow the pathway of KAHRP deployment from the parasite endomembrane system into an intermediate depot between parasite and host, then onwards to the erythrocyte cytoplasm and eventually into knobs. Sequence elements essential to individual steps in the pathway are defined and we show that parasite-derived structures, known as Maurer's clefts, are an elaboration of the canonical secretory pathway that is transposed outside the parasite into the host cell, the first example of its kind in eukaryotic biology.

Fig. 1. Structure of the KAHRP–GFP chimeric proteins expressed in transgenic P.falciparum. (A) The domain structure of the endogenous KAHRP protein is shown in (i), where (S) depicts the hydrophobic core of the putative signal sequence, (His) is the histidine-rich region, and (5′) and (3′) are the 5′ repeat and the 3′ repeat regions. The KAHRP(–His)–GFP chimeric protein expressed from the transfection plasmid pHH2-KAHRP(–His)–GFP is shown in (ii). This chimeric protein contains the putative hydrophobic signal (S) within the first 60 amino acids of the KAHRP protein fused to GFP. The KAHRP(+His)–GFP chimeric protein expressed from transfection plasmid pHH2-KAHRP(+His)–GFP is shown in (iii). This chimeric protein contains the putative signal sequence (S) and the histidine-rich region (His) fused to GFP. (B) The sequences of the KAHRP–GFP fusions expressed in the 3D7–His (i) and 3D7+His (ii) transgenic parasites. N-terminal lower-case sequence represents the putative signal peptide. The cleavage point predicted by SignalP (http://www.cbs.dtu.dk/services/SignalP/) is indicated by (\). Upper-case sequence represents the mature peptide fused with GFP.

Fig. 2. Expression of GFP fusion proteins in transgenic P.falciparum transfected with pHH2-KAHRP(–His)–GFP and pHH2-KAHRP(+His)–GFP. (A) Western blot analysis of GFP fusion protein expression in Percoll-purified preparations of erythrocytes infected with the untransfected parent line 3D7 and the 3D7 lines stably transfected with pHH2-KAHRP(–His)–GFP (3D7–His) and pHH2-KAHRP(+His)–GFP (3D7+His). (B) Time-course of KAHRP–GFP fusion expression in the KAHRP(–His) and KAHRP(+His) transgenic parasite transfectants over 40 h. Parasites were synchronized by two consecutive sorbitol treatments 4 h apart (time zero corresponds to the second sorbitol lysis), aliquots taken 8-hourly and saponin lysed. Saponin lyses the erythrocyte plasma and parasitophorous vacuole membranes, hence the depletion of undegraded GFP products. As a control, the levels of expression of the housekeeping protein HSP70 were also examined.

Fig. 3. Expression of KAHRP(–His)–GFP and KAHRP(+His)–GFP chimeric proteins at different stages of the intra-erythrocytic life cycle of P.falciparum. (A–E) Expression of KAHRP(–His)–GFP in 3D7–His and (F–J) expression of KAHRP(+His)–GFP in 3D7+His. The first image in each set represents the fluorescence signal from the GFP chimeric protein, the second is the bright field image, with an overlay of these images in the third panel. The KAHRP(–His)–GFP-expressing parasites, 3D7–His, traffic GFP to the parasitophorous vacuole, showing a ‘necklace of beads’ pattern in ring stages (A). In trophozoite stages, distortions and evaginations of the parasitophorous vacuole can be observed (B and C). In late trophozoites, GFP can be seen in association with the parasitophorous vacuole and the food vacuole (arrow) (D). Schizonts show a ‘bunch of grapes’ pattern (E). KAHRP(+His)–GFP-expressing parasites, 3D7+His, traffic GFP to the parasitophorous vacuole early in development (i.e. early ring stages, F). In later stages, the chimeric protein is located in the host cell cytoplasm (G–J). The trafficking appeared to involve transit through the parasitophorous vacuole to foci in the erythrocyte cytosol (G and H). The foci then appeared to dissipate, and the GFP chimera became concentrated at the cytoplasmic side of the erythrocyte plasma membrane (I and J).

Fig. 4. Detection of GFP by immunogold labelling with anti-GFP of ultra-thin sections of 3D7–His (A and B) and 3D7+His (C and D) parasites. In KAHRP(–His)–GFP expressing parasites GFP localizes to the parasitophorous vacuole (A and B) and can also be seen in the food vacuole (A). In KAHRP(+His)–GFP-expressing parasites, GFP localizes to the erythrocyte cytoplasm (C and D) and can be seen associated with the knob structure (D). Scale bars are 400 nm in (A–C) and 250 nm in (D).

Fig. 5. Confocal fluorescence microscopy and selective FRAP of P.falciparum-infected erythrocytes expressing KAHRP(–His)–GFP. In each row, the left-hand panels represent pre-bleach images collected at low laser power. The second panels represent images collected 1 s after exposing a defined area (circle, first panels) to a high intensity laser pulse (100% of the laser power). The third panels represent images collected 0.5–1 min after the bleach pulse and the right-hand panels show differential interference contrast images of the cells. (A–D) A young trophozoite stage-infected erythrocyte showing a ‘necklace of beads’ fluorescence pattern. A region of the parasitophorous vacuole was subjected to a 5 s bleach pulse (A and B) and re-imaged after 1 min (C). The bleach site is indicated with an arrow. (E–H) A trophozoite stage parasite showing a more homogeneous parasitophorous vacuole fluorescence pattern. A region of the parasitophorous vacuole was subjected to a 3 s bleach pulse (E and F) and re-imaged after 30 s (G). Partial recovery of fluorescence into the bleached area was observed (arrow). (I–L) A trophozoite stage-infected erythrocyte showing a parasitophorous vacuole evagination was subjected to a 10 s bleach (I–L) and re-imaged after 1 min (K). Pixel values in (J) and (K) have been multiplied by four for the image to be visible. (M–P) A trophozoite stage-infected erythrocyte showing a parasitophorous vacuole evagination was subjected to a 3 s bleach pulse (M and N) and re-imaged after 1 min (O). No recovery of fluorescence into the bleached area was observed (arrow). (Q–T) A late trophozoite stage-infected erythrocyte showing a fluorescent compartment within the parasite was subjected to a 3 s bleach (Q and R) and re-imaged after 1 min (S). The parasitophorous vacuole did not undergo bleaching and there was no recovery of fluorescence into the internal compartment (arrow). The bar in (A) represents 5 µm.

Fig. 6. Confocal fluorescence microscopy and selective photobleaching of different intra-erythrocytic stages of P.falciparum-infected erythrocytes expressing the KAHRP(+His)–GFP. (A–D) A ring stage-infected erythrocyte showing bright fluorescence within the parasite and weak diffuse fluorescence within the erythrocyte cytosol. A region within the erythrocyte cytosol was subjected to a 3 s bleach pulse (A and B) and re-imaged after 1 min (C). A diffuse pattern was observed after bleaching with no recovery within the time-frame examined (arrow). (E–H) The same ring-stage parasite was subjected to a 3 s laser pulse targeting the parasite (E and F) and re-imaged after 1 min (G). The parasite-associated fluorescence was completely ablated (arrow). (I–L) A trophozoite stage-infected erythrocyte showing a fluorescent compartment within the parasite was subjected to a 3 s bleach (I and J) and re-imaged after 2 min (K). No recovery of fluorescence into the bleached area was observed (arrow). (M–P) A trophozoite stage-infected erythrocyte showing punctate fluorescence within the erythrocyte cytosol. Two foci of fluorescence were subjected to a 3 s bleach pulse (M and N) and re-imaged after 2 min (O). Some recovery of the fluorescence associated with the vesicular-like compartments was observed (arrows). (Q–T) A trophozoite stage-infected erythrocyte showing rim-like fluorescence associated with the erythrocyte membrane. A region of membrane-associated fluorescence was subjected to a 3 s bleach (Q and R) and re-imaged after 1 min (S). Partial recovery of fluorescence into the bleached area was observed (arrow). The bar in (A) represents 5 µm.

Fig. 7. Brefeldin A inhibition of protein trafficking in P.falciparum-infected erythrocytes. (A) Brefeldin A inhibition of KAHRP(–His)–GFP trafficking (a) and KAHRP(+His)–GFP (b) visualized in live parasites. Control cultures were incubated in the presence of equivalent amounts of ethanol to ensure that it had no effect on growth and cell morphology (c and d). (B) Brefeldin A inhibition of trafficking of KAHRP(+His)–GFP and endogenous KAHRP, PfEMP1 and PfEMP3 and co-localization of these antigens with the ER marker, PfERC (La Greca et al., 1997). First row: co-localization of GFP (green) with the ER marker PfERC (red) in brefeldin A-treated parasites. Second row: co-localization of KAHRP (green) with PfERC (red). Third row: co-localization of PfEMP1 (green) with PfERC (red). Fourth row: co-localization of PfEMP3 (green) with PfERC (red). The third window in each panel represents the merging of the red and green channels. Yellow areas represent regions of co-localization.

Fig. 8. Transient co-localization of KAHRP, PfSBP1, PfEMP1, PfEMP3 and PfSAR1p to Maurer’s clefts in ring stage P.falciparum-infected erythrocytes. First row: co-localization of KAHRP (green) with PfSBP1 (red). Second row: co-localization of KAHRP (green) with PfEMP1 (red). Third row: co-localization of PfSAR1p (green) with PfEMP3 (red). Fourth row: co-localization of PfSAR1p (green) with PfSBP1 (red). The third window in each row represents the merging of the red and green channels. Yellow areas represent regions of co-localization.

Fig. 9. A proposed model of the trafficking of KAHRP to the knobs of P.falciparum-infected erythrocytes. Proteins destined for export are routed through the ER to the parasitophorous vacuole (PV), which presumably involves cleavage of the signal peptide upon translocation into the ER and trafficking through the classical secretory pathway. Recognition of a ‘translocation motif’ in the histidine-rich region of KAHRP (green circles) would result in translocation of this protein across the parasitophorous vacuolar membrane, via a putative ATP-dependent transporter. Freely diffusable KAHRP located in the parasite-infected erythrocyte cytoplasm would be recruited on to Maurer’s clefts (which contain PfSBP) most likely by interaction of the histidine-rich region with the cytoplasmic tail of PfEMP1 (blue rectangles), which may be exposed on the cytoplasmic surface of Maurer’s clefts (MC) in ring stages. Maurer’s clefts, which co-localize with homologues of components of the canonical secretory pathway such as Sar1p and Sec31p (Adisa et al., 2001), and possibly proteins involved in SNARE-mediated membrane fusion such as NSF (Hayashi et al., 2001), are important structures involved in protein traffic and knob complex formation, and their interaction with the erythrocyte plasma membrane appears to involve PfEMP3 (pink triangles), which has structural similarity to Uso1p, a yeast tethering protein involved in ER to Golgi traffic. Our evidence suggests that Maurer’s clefts are involved directly in the deposition and assembly of parasite proteins and come into close proximity with the cytosolic face of the parasite-infected erythrocyte membrane. P.falciparum homologues of proteins associated with the ER (PfSEC61 α and γ, GRP90/BIP, PfERC), Golgi [sphingomyelin synthase (SS), PfERD2], the trans-Golgi network (TGN) (PfRAB6) and those transposed beyond the parasite into the host cell (PfSAR1p, PfSEC31 and PfNSF) that have been localized in infected erythrocytes are marked. TVN refers to tubovesicular network.