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

Bhattacharjee S, van Ooij C, Balu B, Adams JH, Haldar K - Blood (2007)

Bottom Line: 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.Rather, it is a lumenal signal that sorts protein into clefts, which then are exported beyond the PVM.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pathology and Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.

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.

Show MeSH

Related in: MedlinePlus

HTsol-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 HTsol-GFP. Arrow indicates GFP-labeled intraerythrocytic structure, possibly a cleft. (B) Immunoelectron micrographs of trophozoite parasite (p)-infected cells expressing HTsol-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 HTsol-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).
© Copyright Policy - creativecommons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2234068&req=5

Figure 1: HTsol-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 HTsol-GFP. Arrow indicates GFP-labeled intraerythrocytic structure, possibly a cleft. (B) Immunoelectron micrographs of trophozoite parasite (p)-infected cells expressing HTsol-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 HTsol-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).

Mentions: Parasite proteins exported to the erythrocyte cytoplasm via the HT motif vary greatly in size, amino acid composition, and sequence. Thus, to understand the role of HT motif in all soluble proteins, we followed a minimal reporter composed of a cleavable endoplasmic reticulum (ER)-type signal sequence (SS), HT motif, and GFP, known to result in the delivery of green fluorescence in the erythrocyte cytoplasm9 (Figure 1A). In these cells, green fluorescence is also detected in association with the parasite, probably because of constitutive action of the promoter that leads to overproduction of the protein and its accumulation in the parasite during blood stage growth.9 In addition, structures in the erythrocyte cytoplasm also seem to be labeled by the fluorescent reporter (Figure 1A arrows). Immunoelectron microscopy confirmed the presence of gold particles in the erythrocyte cytosol as well as in flattened lamellar structures called Maurer's clefts (Figure 1B arrow) in the host cell. When the infected erythrocyte membrane was permeabilized with tetanolysin and cells were subsequently fixed, permeabilized, and probed with antibodies, GFP was largely associated with punctate structures that colocalized with a resident protein of Maurer's clefts (Figure 1C). This cleft-associated GFP was detected both proximal to the parasite (Figure 1C arrowheads) and in the periphery of the erythrocyte (Figure 1C arrow). Previous studies have shown that full-length parasite proteins exported to the erythrocyte cytoplasm have also been located to the Maurer's clefts,9 suggesting that a minimal secretory reporter mimics this feature of parasite protein export to the erythrocyte.


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

Bhattacharjee S, van Ooij C, Balu B, Adams JH, Haldar K - Blood (2007)

HTsol-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 HTsol-GFP. Arrow indicates GFP-labeled intraerythrocytic structure, possibly a cleft. (B) Immunoelectron micrographs of trophozoite parasite (p)-infected cells expressing HTsol-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 HTsol-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).
© Copyright Policy - creativecommons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2234068&req=5

Figure 1: HTsol-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 HTsol-GFP. Arrow indicates GFP-labeled intraerythrocytic structure, possibly a cleft. (B) Immunoelectron micrographs of trophozoite parasite (p)-infected cells expressing HTsol-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 HTsol-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).
Mentions: Parasite proteins exported to the erythrocyte cytoplasm via the HT motif vary greatly in size, amino acid composition, and sequence. Thus, to understand the role of HT motif in all soluble proteins, we followed a minimal reporter composed of a cleavable endoplasmic reticulum (ER)-type signal sequence (SS), HT motif, and GFP, known to result in the delivery of green fluorescence in the erythrocyte cytoplasm9 (Figure 1A). In these cells, green fluorescence is also detected in association with the parasite, probably because of constitutive action of the promoter that leads to overproduction of the protein and its accumulation in the parasite during blood stage growth.9 In addition, structures in the erythrocyte cytoplasm also seem to be labeled by the fluorescent reporter (Figure 1A arrows). Immunoelectron microscopy confirmed the presence of gold particles in the erythrocyte cytosol as well as in flattened lamellar structures called Maurer's clefts (Figure 1B arrow) in the host cell. When the infected erythrocyte membrane was permeabilized with tetanolysin and cells were subsequently fixed, permeabilized, and probed with antibodies, GFP was largely associated with punctate structures that colocalized with a resident protein of Maurer's clefts (Figure 1C). This cleft-associated GFP was detected both proximal to the parasite (Figure 1C arrowheads) and in the periphery of the erythrocyte (Figure 1C arrow). Previous studies have shown that full-length parasite proteins exported to the erythrocyte cytoplasm have also been located to the Maurer's clefts,9 suggesting that a minimal secretory reporter mimics this feature of parasite protein export to the erythrocyte.

Bottom Line: 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.Rather, it is a lumenal signal that sorts protein into clefts, which then are exported beyond the PVM.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pathology and Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.

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.

Show MeSH
Related in: MedlinePlus