Maurer's clefts of Plasmodium falciparum are secretory organelles that concentrate virulence protein reporters for delivery to the host erythrocyte

Abstract

In blood-stage infection by the human malaria parasite Plasmodium falciparum, export of proteins from the intracellular parasite to the erythrocyte is key to virulence. This export is mediated by a host-targeting (HT) signal present on a "secretome" of hundreds of parasite proteins engaged in remodeling the erythrocyte. However, the route of HT-mediated export is poorly understood. Here we show that minimal soluble and membrane protein reporters that contain the HT motif and mimic export of endogenous P falciparum proteins are detected in the lumen of "cleft" structures synthesized by the pathogen. Clefts are efficiently targeted by the HT signal. Furthermore, the HT signal does not directly translocate across the parasitophorous vacuolar membrane (PVM) surrounding the parasite to deliver protein to the erythrocyte cytoplasm, as suggested by current models of parasite protein trafficking to the erythrocyte. Rather, it is a lumenal signal that sorts protein into clefts, which then are exported beyond the PVM. These data suggest that Maurer's clefts, which are unique to the virulent P falciparum species, are pathogen-induced secretory organelles that concentrate HT-containing soluble and membrane parasite proteins in their lumen for delivery to the host erythrocyte.

Figure 1

HT^sol-GFP, a minimal soluble reporter exported to the erythrocyte cytoplasm and detected in clefts. (A) 0° projection of an erythrocyte infected with transgenic parasites expressing HT^sol-GFP. Arrow indicates GFP-labeled intraerythrocytic structure, possibly a cleft. (B) Immunoelectron micrographs of trophozoite parasite (p)-infected cells expressing HT^sol-GFP. Ultrathin sections were probed with antibodies to GFP and secondary antibody gold (10 nm) conjugate. Arrows indicate gold particles at intraerythrocytic Maurer's clefts (MC). No gold labeling was detected in absence of primary antibody or when a nonspecific primary was used (not shown). Bar, 200 nm. (C) Single optical section of an infected erythrocyte expressing HT^sol-GFP. Samples were treated to release soluble GFP, fixed, and probed with antibodies to GFP (green) and P falciparum Skeletal Binding Protein1 (PfSBP1, red). Arrow, GFP labeled cleft structures at the periphery of infected erythrocyte; arrowheads, clefts proximal to the parasite. In fluorescence micrographs, p denotes parasite nucleus stained with Hoechst 33342; bar, 2 μm. Schematic representation of the construct is indicated above with ER-type signal sequence (red), sequence containing HT signal (blue) fused to GFP (green) and myc (orange).

Figure 2

Lumenal association of HT^sol-GFP at Maurer's clefts. (A) Schematic representation of the infected erythrocyte (left) and its permeabilization (dotted lines) after treatment with tetanolysin (top) or saponin (bottom). Panels of fluorescent images show infected erythrocyte expressing HT^sol-GFP, permeabilized with tetanolysin (top) or saponin (bottom), and probed with antibodies to GFP (green) and PfStomatin (red). Respective merged images are also shown. Dotted lines indicate erythrocyte periphery. Arrows show intraerythrocytic clefts. (B) 0° projections of an rHT-GFP–loaded erythrocyte ghost infected with 3D7 P falciparum (top) or a mock-loaded erythrocyte ghost infected with transgenic parasite expressing HT^sol-GFP (bottom). Empty arrowhead, cleft structure not labeled with intraerythrocytic rHT-GFP; solid arrowhead, GFP labeled cleft. (C) Cells in panel B fixed, permeabilized, and probed with antibodies to GFP (green) and resident cleft protein PfSBP1 (red). Arrows show clefts. (D) Immunoelectron microscopy of cells in panel B showing distribution of GFP associated with Maurer's clefts (MC). Bar indicates 500 nm. (E) Bar graph showing the percentage colocalization between GFP and Maurer's cleft in indicated samples by fluorescence microscopy. (F) Quantitation for the number of gold particles (measuring GFP) associated with clefts by immunoelectron microscopy over 20 infected erythrocytes. In all fluorescence micrographs: p, parasite (nucleus stained with Hoechst 33342; blue); ec, erythrocyte cytosol; bar, 2 μm.

Figure 3

The HT motif targets proteins to Maurer's clefts without translocation into the erythrocyte cytoplasm. (A) Live infected erythrocytes expressing HT-GFP^membmyc (top) and Δ-GFP^membmyc (bottom) viewed under bright-field image, GFP fluorescence, and merged optics. Western blot using antibodies to GFP indicating the detection of 39-kDa fusion product for HT-GFP^membmyc-expressing cells (lane 1), 39/41-kDa doublet fusion product for Δ-GFP^membmyc-expressing cells (lane 2), or no signal for untransfected cells (lane 3) is shown at the right. In lane 2, the 41-kDa band is a precursor that in pulse chase experiments can be chased into the 39-kDa band (data not shown). Vertical lines have been inserted to indicate repositioned gel lanes. (B) Indirect immunofluorescence assay showing distribution of GFP (green), associated with HT-GFP^membmyc (top panel) or Δ-GFP^membmyc (bottom panel) relative to the Maurer's cleft protein PfSBP1 (red). Parasite nucleus (p) is stained with Hoechst 33342. (C) Localization of HT-GFP^membmyc in Maurer's clefts (MC, top) and Δ-GFP^membmyc in parasitophorous vacuolar membrane (PVM, bottom) by immunoelectron microscopy. Empty arrowheads indicate gold particles showing distribution of GFP chimeras. Bar, 500 nm; p, parasite. Micrograph at the bottom has been magnified twice compared with that at the top to distinguish the parasite plasma membrane (PPM) from the PVM. (D) Western blots of tetanolysin-released infected erythrocyte cytoplasm supernatant (S) and pellet (P) fractions of cells expressing HT-GFP^membmyc (left panel), Δ-GFP^membmyc (middle panel), and HT-GFP^membC-term (right panel) probed for GFP, PVM marker PfStomatin, and parasite cytoplasmic protein PfFKBP. Hemoglobin (Hb) released (in S) by tetanolysin is expressed as a percentage of the total Hb detected by hypotonic lysis. (E) Live infected erythrocytes expressing HT-GFP^membCterm viewed under bright-field image, GFP fluorescence, and merged optics. Schematic representation for all constructs are indicated above with ER-type signal sequence (red), sequence containing HT signal (blue) or its replacement (solid black triangle) fused to GFP (green), transmembrane region (black), and myc (orange). C-terminal region (derived from PfEMP1) is depicted in yellow.

Figure 4

The HT motif sorts protein into Maurer's clefts without translocation across the PVM. (A) For both HT-GFP^membmyc and Δ-GFP^membmyc, Western blots show protection of GFP but quantitative degradation of myc and erythrocyte spectrin after addition of trypsin to cells where the infected erythrocyte membrane was permeabilized with tetanolysin (lanes 2 and 6). Saponin (which additionally permeabilizes PVM and clefts, lanes 4 and 8) renders GFP susceptible to protease. *, trypsin digested GFP product of 25-kDa. Molecular mass markers are expressed in kilodaltons (kDa). (B) Single optical sections of ghosts resealed with Alexa Fluor 594 anti-GFP antibodies infected with parasites expressing HT-GFP^membmyc (top panel) or Δ-GFP^membmyc (bottom panel). Cells were viewed live using optics for GFP (green), Alexa Fluor 594/Rhodamine (red), and the merged image is shown in the right panel. Arrows, GFP labeled clefts not labeled with anti-GFP Alexa Fluor 594 conjugate. (C) Immunofluorescence assay of resealed ghosts infected with parasites expressing HT-GFP^membmyc (top panel) or Δ-GFP^membmyc (bottom panel) permeabilized with saponin and treated with anti-GFP Alexa Fluor 594-conjugated antibodies. Images under GFP (green) and Alexa 594 (red) optics and their respective merge are shown. Arrowhead, region of colocalization (in yellow) between GFP and Alexa 594. In all cells, the parasite (p) nuclei were stained with Hoechst 33342 (blue); bar, 2 μm. Schematic representation of the construct is indicated above with ER-type signal sequence (red), sequence containing HT signal (blue) or its replacement (filled triangle in black) fused to GFP (green), transmembrane region (black), and myc (orange).

Figure 5

HT-dependent protein sorting into clefts may occur at parasite periphery and is not influenced by deletion of the C-terminal domain of PfSBP1. (A) Three-dimensional projections of a live infected erythrocyte expressing HT-GFP^membmyc and stained with TR-ceramide. Clefts at the periphery of the infected erythrocyte (arrows) as well as at or within the perimeter of the vacuolar parasite (empty arrowheads) are visible. (B) 0° projection of live infected erythrocyte expressing HT-GFP^membmyc in 3D7 strains with parental (top) or chromosomal deletion of pfsbp1 (bottom), viewed under GFP optics and merged with bright field. Arrows indicate that the export of HT-GFP^membmyc to cleft structures in parental 3D7 strain is not altered in parasite line with a C-terminal deletion in PfSBP1. Parasite (p) nucleus is stained with Hoechst 33342. Bar, 2 μm.

Figure 6

HT-dependent protein sorting into clefts was not influenced by deletion of putative substrate binding domain in PFE0055c. (A) Deduced amino acid sequence of PFE0055c with an N-terminal ER-type signal sequence (brown), HT motif (bold), followed by sequences containing DnaJ region (orange) with the characteristic HPD (green) motif. Further downstream region include a glycine/phenylalanine-rich stretch and C-terminal substrate-binding domain (underlined). Sequences in blue indicate region deleted in parasite line generated by single crossover recombination as shown in panels D-F. (B) Western blot, using anti-PFE0055c antibodies, detecting the presence of a 42-kDa protein in infected erythrocyte (arrowhead, lane 2) but not uninfected erythrocyte (lane 1). (C) Single optical section of a trophozoite-infected erythrocyte fixed and probed with peptide antibodies to PFE0055c (green) and the cleft protein SBP1 (red). Arrow in merge image shows proximal location of PFE0055c to clefts. (D) Strategy for deletion in the C-terminal substrate-binding region of PFE0055c by single crossover recombination with the chromosomal copy of pfe0055c. P falciparum parasites were transfected with plasmids containing an in-frame fusion of the neomycin resistance gene (npt, green) to an internal fragment of pfe0055c (orange) without sequences encoding for C-terminal substrate-binding domain. Only chromosomal integration of the vector by single crossover with the native pfe0055c (pink) drives npt expression under the control of pfe0055c promoter (Ppfe0055c), thus conferring resistance of antibiotic G418. (E) PCR-based detection for the loss of chromosomal copy of pfe0055c. Positions for primer pairs used for amplification analyses of single crossover recombination are highlighted in panel D. (F) Western blot analysis showing the detection of PFE0055c-NPT fusion protein of 45-kDa in transfected line (arrowhead, lane 1) but in not parental line (lane 2) using antibodies to NPT (top). Parasite protein PfFKBP serves as a loading control (bottom). (G) 0° projection of live infected erythrocyte expressing HT-GFP^membmyc in 3D7 strain with chromosomal deletion of pfe0055c viewed under GFP optics and merged with bright field. Arrow indicates that the export of HT-GFP^membmyc to cleft is not altered by truncation in PFE0055c. Parasite nucleus (p) in all cases is stained with Hoechst 33342 (blue). Bar represents 2 μm.

Figure 7

Schematic for HT-mediated cleft targeting events in erythrocyte infected with the malaria parasite P falciparum. In secretory proteins, a cleavable N-terminal ER-type signal sequence (SS) delivers proteins to the PV (step 1). The HT motif enables protein accumulation in the Maurer's clefts (MC, step 2). Clefts can either bud from the PVM (also step 2) packed with proteins exported to the red cell and function as protein reservoirs underneath the erythrocyte membrane (step 3). The HT motif may also move protein from the lumen of the PVM to lumen of clefts either within the parasite (step 2′) or at the proximity to the parasite plasma membrane (PPM, step 3′). Both steps 2 and 2′ implicate recognition of the HT motif by a putative receptor located at the Maurer's clefts (not shown).