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Identification of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) as the rosetting ligand of the malaria parasite P. falciparum.

Chen Q, Barragan A, Fernandez V, Sundström A, Schlichtherle M, Sahlén A, Carlson J, Datta S, Wahlgren M - J. Exp. Med. (1998)

Bottom Line: Rosetting PfEMP1 contains clusters of glycosaminoglycan-binding motifs.A recombinant fusion protein (Duffy binding-like 1-glutathione S transferase; Duffy binding-like-1-GST) was found to adhere directly to normal erythrocytes, disrupt naturally formed rosettes, block rosette reformation, and bind to a heparin-Sepharose matrix.The adhesive interactions could be inhibited with heparan sulfate or enzymes that remove heparan sulfate from the cell surface whereas other enzymes or similar glycosaminoglycans of a like negative charge did not affect the binding.

View Article: PubMed Central - PubMed

Affiliation: Microbiology and Tumor Biology Center, Karolinska Institutet, the Swedish Institute for Infectious Disease Control, S-171 77 Stockholm, Sweden.

ABSTRACT
Severe Plasmodium falciparum malaria is characterized by excessive sequestration of infected and uninfected erythrocytes in the microvasculature of the affected organ. Rosetting, the adhesion of P. falciparum-infected erythrocytes to uninfected erythrocytes is a virulent parasite phenotype associated with the occurrence of severe malaria. Here we report on the identification by single-cell reverse transcriptase PCR and cDNA cloning of the adhesive ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1). Rosetting PfEMP1 contains clusters of glycosaminoglycan-binding motifs. A recombinant fusion protein (Duffy binding-like 1-glutathione S transferase; Duffy binding-like-1-GST) was found to adhere directly to normal erythrocytes, disrupt naturally formed rosettes, block rosette reformation, and bind to a heparin-Sepharose matrix. The adhesive interactions could be inhibited with heparan sulfate or enzymes that remove heparan sulfate from the cell surface whereas other enzymes or similar glycosaminoglycans of a like negative charge did not affect the binding. PfEMP1 is suggested to be the rosetting ligand and heparan sulfate, or a heparan sulfate-like molecule, the receptor both for PfEMP1 binding and naturally formed erythrocyte rosettes.

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Identification of rosetting PfEMP1. (A) Rosetting, single P. falciparum–infected erythrocyte is seen by light microscopy held by a 5-μm micropipette (A, 1). The uninfected erythrocytes are stripped of the infected  cell and careful examination confirms that it indeed is infected by a single parasite (A, 2–3). B shows the amplification of a 434-bp band in four (from reactions 3, 4, 5, and 7) out of eight single-infected, rosetting erythrocytes using  degenerate primers generated from the primary sequence of the DBL-1 domain of PfEMP1. C shows the amplification pattern with the same primers as  in B of bulk cultures of rosetting (R+) FCR3S1.2 cultures and the R−  FCR3s/a parasites. Note that the 434-bp product is only seen with the R+  parasites. D shows the hybridization pattern in Northern blotting of the 434-bp sequence to mRNA extracted from the highly rosetting parasite  FCR3S1.2 (84% R+) and the weak hybridization to the R− FCR3S/a parasite (9% R+). E shows the autoradiogarph of a Triton X-100 insoluble, SDS-soluble extract of FCR3S1.2-infected erythrocytes after radio-iodination labeling. PfEMP1 (arrow) is labeled on FCR3S1.2-infected erythrocytes and is  cleaved by low concentrations of trypsin.
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Figure 1: Identification of rosetting PfEMP1. (A) Rosetting, single P. falciparum–infected erythrocyte is seen by light microscopy held by a 5-μm micropipette (A, 1). The uninfected erythrocytes are stripped of the infected cell and careful examination confirms that it indeed is infected by a single parasite (A, 2–3). B shows the amplification of a 434-bp band in four (from reactions 3, 4, 5, and 7) out of eight single-infected, rosetting erythrocytes using degenerate primers generated from the primary sequence of the DBL-1 domain of PfEMP1. C shows the amplification pattern with the same primers as in B of bulk cultures of rosetting (R+) FCR3S1.2 cultures and the R− FCR3s/a parasites. Note that the 434-bp product is only seen with the R+ parasites. D shows the hybridization pattern in Northern blotting of the 434-bp sequence to mRNA extracted from the highly rosetting parasite FCR3S1.2 (84% R+) and the weak hybridization to the R− FCR3S/a parasite (9% R+). E shows the autoradiogarph of a Triton X-100 insoluble, SDS-soluble extract of FCR3S1.2-infected erythrocytes after radio-iodination labeling. PfEMP1 (arrow) is labeled on FCR3S1.2-infected erythrocytes and is cleaved by low concentrations of trypsin.

Mentions: Two degenerate primers (Duffy binding-like [DBL]-1.1, 5′-GG[A/T] GC[A/T] TG[TC] GC[A/T] CC[A/T] T[A/T][T/C] [A/C]G-3′; DBL-1.2, 5′-A[A/G][A/G]T A[T/C]TG [T/A]GG [A/T]AC [A/G]TA [A/G]TC-3′) that mapped to the conserved region of all PfEMP1 DBL-1 were modified from the sequences of Su et al. The amplification parameters were first optimized so that the amplified products were visible with normal ethidium bromide staining (25). In brief, one to five parasites, obtained by limiting dilution, were directly emerged in the RT-PCR buffer (Stratagene Corp., La Jolla, CA) with different concentration of primers, MgCl2, KCl, and Tris-HCl. Both DNA and RNA were released from the parasites by heating at 93°C for 3 min. The DNA was degraded by addition of 10 U DNase (Stratagene Corp.). Reverse transcription was carried out immediately after addition of random primers and reverse transcriptase (Perkin-Elmer Corp., Norwalk, CT). The PCR reaction was subsequently performed in the same tube. Through comparison of the amplification efficiency from different reactions, the optimized parameters for single-cell RT-PCR were found to be as follows: 100 mM Tris-Cl, pH 8.3, 3.5 mM MgCl2, 500 mM KCl, and the final concentration of primers was 1 μM. In the subsequent experiments, individual trophozoite-infected rosetting erythrocytes were isolated with a 5-μm glass pipette using an inverted microscope. The selected pRBC was stripped of uninfected RBCs and repeatedly grabbed, ejected, and turned to conclusively ensure that it had pigment and that the selected cell was a single trophozoite-infected RBC (see Fig. 1 A). 50 cycles of amplification at 93°C for 20 s, 55°C for 30 s, and 72°C for 1 min were needed for product detection. Several controls were included in each experiment; one blank control (without parasite[s]) and one without reverse transcriptase to rule out the possibility of contamination and amplification due to the presence of genomic DNA.


Identification of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) as the rosetting ligand of the malaria parasite P. falciparum.

Chen Q, Barragan A, Fernandez V, Sundström A, Schlichtherle M, Sahlén A, Carlson J, Datta S, Wahlgren M - J. Exp. Med. (1998)

Identification of rosetting PfEMP1. (A) Rosetting, single P. falciparum–infected erythrocyte is seen by light microscopy held by a 5-μm micropipette (A, 1). The uninfected erythrocytes are stripped of the infected  cell and careful examination confirms that it indeed is infected by a single parasite (A, 2–3). B shows the amplification of a 434-bp band in four (from reactions 3, 4, 5, and 7) out of eight single-infected, rosetting erythrocytes using  degenerate primers generated from the primary sequence of the DBL-1 domain of PfEMP1. C shows the amplification pattern with the same primers as  in B of bulk cultures of rosetting (R+) FCR3S1.2 cultures and the R−  FCR3s/a parasites. Note that the 434-bp product is only seen with the R+  parasites. D shows the hybridization pattern in Northern blotting of the 434-bp sequence to mRNA extracted from the highly rosetting parasite  FCR3S1.2 (84% R+) and the weak hybridization to the R− FCR3S/a parasite (9% R+). E shows the autoradiogarph of a Triton X-100 insoluble, SDS-soluble extract of FCR3S1.2-infected erythrocytes after radio-iodination labeling. PfEMP1 (arrow) is labeled on FCR3S1.2-infected erythrocytes and is  cleaved by low concentrations of trypsin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199182&req=5

Figure 1: Identification of rosetting PfEMP1. (A) Rosetting, single P. falciparum–infected erythrocyte is seen by light microscopy held by a 5-μm micropipette (A, 1). The uninfected erythrocytes are stripped of the infected cell and careful examination confirms that it indeed is infected by a single parasite (A, 2–3). B shows the amplification of a 434-bp band in four (from reactions 3, 4, 5, and 7) out of eight single-infected, rosetting erythrocytes using degenerate primers generated from the primary sequence of the DBL-1 domain of PfEMP1. C shows the amplification pattern with the same primers as in B of bulk cultures of rosetting (R+) FCR3S1.2 cultures and the R− FCR3s/a parasites. Note that the 434-bp product is only seen with the R+ parasites. D shows the hybridization pattern in Northern blotting of the 434-bp sequence to mRNA extracted from the highly rosetting parasite FCR3S1.2 (84% R+) and the weak hybridization to the R− FCR3S/a parasite (9% R+). E shows the autoradiogarph of a Triton X-100 insoluble, SDS-soluble extract of FCR3S1.2-infected erythrocytes after radio-iodination labeling. PfEMP1 (arrow) is labeled on FCR3S1.2-infected erythrocytes and is cleaved by low concentrations of trypsin.
Mentions: Two degenerate primers (Duffy binding-like [DBL]-1.1, 5′-GG[A/T] GC[A/T] TG[TC] GC[A/T] CC[A/T] T[A/T][T/C] [A/C]G-3′; DBL-1.2, 5′-A[A/G][A/G]T A[T/C]TG [T/A]GG [A/T]AC [A/G]TA [A/G]TC-3′) that mapped to the conserved region of all PfEMP1 DBL-1 were modified from the sequences of Su et al. The amplification parameters were first optimized so that the amplified products were visible with normal ethidium bromide staining (25). In brief, one to five parasites, obtained by limiting dilution, were directly emerged in the RT-PCR buffer (Stratagene Corp., La Jolla, CA) with different concentration of primers, MgCl2, KCl, and Tris-HCl. Both DNA and RNA were released from the parasites by heating at 93°C for 3 min. The DNA was degraded by addition of 10 U DNase (Stratagene Corp.). Reverse transcription was carried out immediately after addition of random primers and reverse transcriptase (Perkin-Elmer Corp., Norwalk, CT). The PCR reaction was subsequently performed in the same tube. Through comparison of the amplification efficiency from different reactions, the optimized parameters for single-cell RT-PCR were found to be as follows: 100 mM Tris-Cl, pH 8.3, 3.5 mM MgCl2, 500 mM KCl, and the final concentration of primers was 1 μM. In the subsequent experiments, individual trophozoite-infected rosetting erythrocytes were isolated with a 5-μm glass pipette using an inverted microscope. The selected pRBC was stripped of uninfected RBCs and repeatedly grabbed, ejected, and turned to conclusively ensure that it had pigment and that the selected cell was a single trophozoite-infected RBC (see Fig. 1 A). 50 cycles of amplification at 93°C for 20 s, 55°C for 30 s, and 72°C for 1 min were needed for product detection. Several controls were included in each experiment; one blank control (without parasite[s]) and one without reverse transcriptase to rule out the possibility of contamination and amplification due to the presence of genomic DNA.

Bottom Line: Rosetting PfEMP1 contains clusters of glycosaminoglycan-binding motifs.A recombinant fusion protein (Duffy binding-like 1-glutathione S transferase; Duffy binding-like-1-GST) was found to adhere directly to normal erythrocytes, disrupt naturally formed rosettes, block rosette reformation, and bind to a heparin-Sepharose matrix.The adhesive interactions could be inhibited with heparan sulfate or enzymes that remove heparan sulfate from the cell surface whereas other enzymes or similar glycosaminoglycans of a like negative charge did not affect the binding.

View Article: PubMed Central - PubMed

Affiliation: Microbiology and Tumor Biology Center, Karolinska Institutet, the Swedish Institute for Infectious Disease Control, S-171 77 Stockholm, Sweden.

ABSTRACT
Severe Plasmodium falciparum malaria is characterized by excessive sequestration of infected and uninfected erythrocytes in the microvasculature of the affected organ. Rosetting, the adhesion of P. falciparum-infected erythrocytes to uninfected erythrocytes is a virulent parasite phenotype associated with the occurrence of severe malaria. Here we report on the identification by single-cell reverse transcriptase PCR and cDNA cloning of the adhesive ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1). Rosetting PfEMP1 contains clusters of glycosaminoglycan-binding motifs. A recombinant fusion protein (Duffy binding-like 1-glutathione S transferase; Duffy binding-like-1-GST) was found to adhere directly to normal erythrocytes, disrupt naturally formed rosettes, block rosette reformation, and bind to a heparin-Sepharose matrix. The adhesive interactions could be inhibited with heparan sulfate or enzymes that remove heparan sulfate from the cell surface whereas other enzymes or similar glycosaminoglycans of a like negative charge did not affect the binding. PfEMP1 is suggested to be the rosetting ligand and heparan sulfate, or a heparan sulfate-like molecule, the receptor both for PfEMP1 binding and naturally formed erythrocyte rosettes.

Show MeSH
Related in: MedlinePlus