Cooperative domains define a unique host cell-targeting signal in Plasmodium falciparum-infected erythrocytes

Abstract

When the malaria parasite Plasmodium falciparum infects an erythrocyte, it resides in a parasitophorous vacuole and remarkably exports proteins into the periphery of its host cell. Two of these proteins, the histidine-rich proteins I and II (PfHRPI and PfHRPII), are exported to the erythrocyte cytoplasm. PfHRPI has been linked to cell-surface "knobby" protrusions that mediate cerebral malaria and are a frequent cause of death. PfHRPII has been implicated in (i) the production of hemozoin, the black pigment associated with disease, as well as (ii) interactions with the erythrocyte cytoskeleton. Here we show that a tripartite signal that is comprised of an endoplasmic reticulum-type signal sequence followed by a bipartite vacuolar translocation signal derived from HRPII and HRPI exports GFP from the parasitophorous vacuole to the host cytoplasm. The bipartite vacuolar translocation signal is comprised of unique, peptidic (approximately equal to 40-aa) sequences. A domain within it contains the signal for export to "cleft" transport intermediates in the host erythrocyte and may thereby regulate the pathway of export to the host cytoplasm. A signal for posttranslational, vacuolar exit of proteins has hitherto not been described in eukaryotic secretion.

Fig. 1.

Structure of PfHRPII and PfHRPI and expression of transgenes. (Ai) PfHRPII protein sequence (44) contains a cleavable, ER-type signal peptide (shown in orange) as predicted by Signal P. An asparagine-rich region called domain I (shown in black) is followed by a histidine-rich C-terminal domain (II, shown in pink). Subscript numbers indicate truncations used in GFP constructs. (Aii) For PfHRPI (9), sequences for the ER-type signal (lowercase), domain I (gray), and the first histidine region (II, amino acids 61-106, in blue) are shown. (B) Western blot analysis of total infected cell lysates containing fusion proteins PfHRPIIGFP (lane i), SSHRPIIDomainIGFP (lane ii), SSGFP (lane iii), SSHRPIIDomainIHis.reg._57-124 GFP (lane iv), SSHRPIIDomainIHis.minGFP (lane v), GFPHis.reg._154-327 (lane vi), and SSGFPHis.reg._154-327 (lane vii) and probed with antibodies to GFP. Arrowheads indicate the expected fusion protein. Arrows indicate predicted unprocessed secretory precursors. Some constructs showed low levels of degradation product that migrated at the predicted size of GFP (asterisk). Molecular mass (in kDa) is shown on the left side of each blot.

Fig. 2.

Identification and characterization of a VTS in P. falciparum-infected erythrocytes. (A) Projections (0°) of live cells expressing PfHRPIIGFP (i), SSHRPIIDomainIGFP (ii), SSGFP (iii), SSHRPIIDomainIHis.reg._57-124GFP (iv), SSHRPIIDomainIHis.min.GFP (v), GFPHis.Reg._154-327 (vi), and SSGFPHis.reg._154-327 (vii). Green, GFP; blue, nuclei; p, parasite; arrow, vacuolar tubules and loops; arrowhead, punctate spot; e, erythrocyte. In GFP fusions: orange, the SS; black, domain I; hatched region, deleted domain I (ΔDomainI) construct (see Materials and Methods); pink, histidine-rich regions (of differing lengths; domain II); green, GFP. A minimal (9-aa) histidine-rich sequence (containing four histidines) from the histidine domain II downstream of domain I is essential for vacuolar export of GFP to the erythrocyte cytoplasm. (B) Western blots of supernatant (S) and pellet (P) fractions after tetanolysin treatment of cells (see Materials and Methods) expressing PfHRPIIGFP (lanes 1 and 2) and SSDomain-IGFP (lanes 3 and 4). PfFKBP in the plasmodial cytoplasm was used to confirm parasite integrity (the antibody was a kind gift of T. Wandless, (Stanford University, Stanford, CA). (C) Live cells expressing SSHRPIIΔDomainIHis. reg._57-124 GFP (i), SSGFPHRPIIDomainIHis.min (ii), and SSHRPIIDomainIGFPHis. reg._154-327 (iii). Domain I seems to be required along with the minimum histidine sequence to form a VTS. Further, DomainIHis.min must be contiguous and immediately downstream of SS to mediate protein export from the PV to the erythrocyte cytoplasm. (Scale bar, 5 μm.)

Fig. 3.

The Maurer's clefts are intermediates in PfHRPII transport and are targeted by its histidine-rich sequences. Single optical sections of trophozoite-infected cells expressing SSGFPHis_154-327 (A-C) or PfHRPIIGFP (D-F) are shown. Cells were fixed, permeabilized, subjected to indirect immunofluorescence assays (see Materials and Methods), and probed with antibodies to GFP (green) and a Maurer's cleft resident protein PfSBP (red). Magnification of the boxes in C and F are shown as C′ and F′, respectively. The extent of colocalization is shown in yellow. In C′ and F′, arrows indicate GFP-labeled tubules or vesicles connecting clefts (arrowheads) to each other or the vacuolar parasite (P). Nuclei were stained with Hoechst (blue). (Scale bar, 5 μm.)

Fig. 4.

Functionally equivalent bipartite VTS from PfHRPI/PfHRPII and histidine-to-arginine substitutions. Note that, in chimeric VTS, HRPI sequences are shown in bold type, and HRPII sequences are shown in bold and italic type. Live cells expressing SSHRPIDomainIGFP (A), SSHRPIDomainIHRPIIHis.min.GFP (B), SSHRPIIDomainIHRPIHis.min.GFP (C), and SSHRPIIDomainIArg.min.GFP (D) are shown. DNA was visualized by Hoechst stain (blue). e, erythrocyte; p, parasite. The color code shows indicated regions in each chimera. Domain I and minimal histidine sequences from domain II of HRPI and HRPII are interchangeable in a VTS. Arginine can substitute for histidine in a VTS. (Scale bar, 5 μm.)