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Hepatocyte permissiveness to Plasmodium infection is conveyed by a short and structurally conserved region of the CD81 large extracellular domain.

Yalaoui S, Zougbédé S, Charrin S, Silvie O, Arduise C, Farhati K, Boucheix C, Mazier D, Rubinstein E, Froissard P - PLoS Pathog. (2008)

Bottom Line: Still, the molecular mechanisms underlying sporozoite invasion are largely unknown.By site-directed mutagenesis, we have demonstrated the key role of a solvent-exposed region around residue D137 within this domain.This study has uncovered a new functionally important region of CD81, independent of HCV E2 envelope protein binding domain, and further suggests that CD81 may not interact directly with a parasite ligand during Plasmodium infection, but instead may regulate the function of a yet unknown partner protein.

View Article: PubMed Central - PubMed

Affiliation: Université Pierre et Marie Curie-Paris6, UMR S511, Paris, France.

ABSTRACT
Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a malaria infection, and thus represents an attractive target for anti-malarial interventions. Still, the molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that the tetraspanin CD81, a known receptor for the hepatitis C virus (HCV), is required on hepatocytes for infection by sporozoites of several Plasmodium species. Here we have characterized CD81 molecular determinants required for infection of hepatocytic cells by P. yoelii sporozoites. Using CD9/CD81 chimeras, we have identified in CD81 a 21 amino acid stretch located in a domain structurally conserved in the large extracellular loop of tetraspanins, which is sufficient in an otherwise CD9 background to confer susceptibility to P. yoelii infection. By site-directed mutagenesis, we have demonstrated the key role of a solvent-exposed region around residue D137 within this domain. A mAb that requires this region for optimal binding did not block infection, in contrast to other CD81 mAbs. This study has uncovered a new functionally important region of CD81, independent of HCV E2 envelope protein binding domain, and further suggests that CD81 may not interact directly with a parasite ligand during Plasmodium infection, but instead may regulate the function of a yet unknown partner protein.

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Mutation of the SEL β-sheet abolishes P. yoelii infection and alters the LEL conformation.A: Sequence of CD81 and CD9 SEL, and description of the SEL mutants. The boxes indicate the β-sheet [36]. B: HepG2-A16 cells were transiently transfected with plasmids expressing CD9, CD81, or CD81 molecules with mutations in the SEL, and infected two days later with P. yoelii sporozoites. After two days incubation, the number of EEF-infected cells was determined by immunofluorescence in triplicate wells. Results are expressed as mean±s.d. **, p<0.01 as compared to mock-transfected cells. C: Hepa 1–6 cells were transfected with the indicated constructs and analyzed for the surface expression of the transgene by flow-cytometry analysis. Data are expressed as mean fluorescence intensity. In this experiment, the antibodies were used at 20 µg/ml (JS64, M38, JS81) or at 1/100 ascitic fluid dilution (all other mAbs).
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ppat-1000010-g008: Mutation of the SEL β-sheet abolishes P. yoelii infection and alters the LEL conformation.A: Sequence of CD81 and CD9 SEL, and description of the SEL mutants. The boxes indicate the β-sheet [36]. B: HepG2-A16 cells were transiently transfected with plasmids expressing CD9, CD81, or CD81 molecules with mutations in the SEL, and infected two days later with P. yoelii sporozoites. After two days incubation, the number of EEF-infected cells was determined by immunofluorescence in triplicate wells. Results are expressed as mean±s.d. **, p<0.01 as compared to mock-transfected cells. C: Hepa 1–6 cells were transfected with the indicated constructs and analyzed for the surface expression of the transgene by flow-cytometry analysis. Data are expressed as mean fluorescence intensity. In this experiment, the antibodies were used at 20 µg/ml (JS64, M38, JS81) or at 1/100 ascitic fluid dilution (all other mAbs).

Mentions: Our initial data, using CD9×81 and CD9LEL81 chimeras, indicated that CD81 SEL could be replaced with that of CD9 without affecting the ability to support P. yoelii sporozoites infection (Fig. 1). Both CD9 and CD81 SEL are predicted to contain a β-sheet that was proposed to tightly interact with the LEL [36]. To further investigate the role of the SEL, residues of this β-sheet were mutated to Ala. Two mutants were designed: SEL-CD81, in which NLLYLE (43–48) was mutated to AAAAAA in the hCD81 molecule, and SEL-CD9LEL81, in which TKSIFEQ (41–47) was mutated to AAAAAAA in the CD9LEL81 chimeric molecule (Fig. 8A). None was able to confer cell susceptibility to P. yoelii infection (Fig. 8B). These mutants are normally expressed at the cell surface as shown by the binding of the non-conformational mAb 1D6 (Fig. 8C). Five out of 7 CD81 mAb tested showed a reduced binding to cells expressing this mutant as compared to cells expressing WT CD81, indicating that the conformation of the LEL of these mutants is modified (Fig. 8C).


Hepatocyte permissiveness to Plasmodium infection is conveyed by a short and structurally conserved region of the CD81 large extracellular domain.

Yalaoui S, Zougbédé S, Charrin S, Silvie O, Arduise C, Farhati K, Boucheix C, Mazier D, Rubinstein E, Froissard P - PLoS Pathog. (2008)

Mutation of the SEL β-sheet abolishes P. yoelii infection and alters the LEL conformation.A: Sequence of CD81 and CD9 SEL, and description of the SEL mutants. The boxes indicate the β-sheet [36]. B: HepG2-A16 cells were transiently transfected with plasmids expressing CD9, CD81, or CD81 molecules with mutations in the SEL, and infected two days later with P. yoelii sporozoites. After two days incubation, the number of EEF-infected cells was determined by immunofluorescence in triplicate wells. Results are expressed as mean±s.d. **, p<0.01 as compared to mock-transfected cells. C: Hepa 1–6 cells were transfected with the indicated constructs and analyzed for the surface expression of the transgene by flow-cytometry analysis. Data are expressed as mean fluorescence intensity. In this experiment, the antibodies were used at 20 µg/ml (JS64, M38, JS81) or at 1/100 ascitic fluid dilution (all other mAbs).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2279262&req=5

ppat-1000010-g008: Mutation of the SEL β-sheet abolishes P. yoelii infection and alters the LEL conformation.A: Sequence of CD81 and CD9 SEL, and description of the SEL mutants. The boxes indicate the β-sheet [36]. B: HepG2-A16 cells were transiently transfected with plasmids expressing CD9, CD81, or CD81 molecules with mutations in the SEL, and infected two days later with P. yoelii sporozoites. After two days incubation, the number of EEF-infected cells was determined by immunofluorescence in triplicate wells. Results are expressed as mean±s.d. **, p<0.01 as compared to mock-transfected cells. C: Hepa 1–6 cells were transfected with the indicated constructs and analyzed for the surface expression of the transgene by flow-cytometry analysis. Data are expressed as mean fluorescence intensity. In this experiment, the antibodies were used at 20 µg/ml (JS64, M38, JS81) or at 1/100 ascitic fluid dilution (all other mAbs).
Mentions: Our initial data, using CD9×81 and CD9LEL81 chimeras, indicated that CD81 SEL could be replaced with that of CD9 without affecting the ability to support P. yoelii sporozoites infection (Fig. 1). Both CD9 and CD81 SEL are predicted to contain a β-sheet that was proposed to tightly interact with the LEL [36]. To further investigate the role of the SEL, residues of this β-sheet were mutated to Ala. Two mutants were designed: SEL-CD81, in which NLLYLE (43–48) was mutated to AAAAAA in the hCD81 molecule, and SEL-CD9LEL81, in which TKSIFEQ (41–47) was mutated to AAAAAAA in the CD9LEL81 chimeric molecule (Fig. 8A). None was able to confer cell susceptibility to P. yoelii infection (Fig. 8B). These mutants are normally expressed at the cell surface as shown by the binding of the non-conformational mAb 1D6 (Fig. 8C). Five out of 7 CD81 mAb tested showed a reduced binding to cells expressing this mutant as compared to cells expressing WT CD81, indicating that the conformation of the LEL of these mutants is modified (Fig. 8C).

Bottom Line: Still, the molecular mechanisms underlying sporozoite invasion are largely unknown.By site-directed mutagenesis, we have demonstrated the key role of a solvent-exposed region around residue D137 within this domain.This study has uncovered a new functionally important region of CD81, independent of HCV E2 envelope protein binding domain, and further suggests that CD81 may not interact directly with a parasite ligand during Plasmodium infection, but instead may regulate the function of a yet unknown partner protein.

View Article: PubMed Central - PubMed

Affiliation: Université Pierre et Marie Curie-Paris6, UMR S511, Paris, France.

ABSTRACT
Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a malaria infection, and thus represents an attractive target for anti-malarial interventions. Still, the molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that the tetraspanin CD81, a known receptor for the hepatitis C virus (HCV), is required on hepatocytes for infection by sporozoites of several Plasmodium species. Here we have characterized CD81 molecular determinants required for infection of hepatocytic cells by P. yoelii sporozoites. Using CD9/CD81 chimeras, we have identified in CD81 a 21 amino acid stretch located in a domain structurally conserved in the large extracellular loop of tetraspanins, which is sufficient in an otherwise CD9 background to confer susceptibility to P. yoelii infection. By site-directed mutagenesis, we have demonstrated the key role of a solvent-exposed region around residue D137 within this domain. A mAb that requires this region for optimal binding did not block infection, in contrast to other CD81 mAbs. This study has uncovered a new functionally important region of CD81, independent of HCV E2 envelope protein binding domain, and further suggests that CD81 may not interact directly with a parasite ligand during Plasmodium infection, but instead may regulate the function of a yet unknown partner protein.

Show MeSH
Related in: MedlinePlus