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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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A CD81 mAb binds poorly to the non-functional mutant VVD (135–137)→AAA but does not block infection.A: Hepa 1–6 cells were transfected with the indicated construct in pEGFP-N3 and analyzed for the surface expression and recognition of the transgene by several CD81 mAb using 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). B: HepG2-A16/CD81 cells were infected with P. yoelii sporozoites in the presence of the indicated mAbs at 25 µg/ml except when otherwise indicated. All mAbs are directed to CD81 except TS9 which is a CD9 mAb and does not inhibit P. yoelii infection.
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ppat-1000010-g006: A CD81 mAb binds poorly to the non-functional mutant VVD (135–137)→AAA but does not block infection.A: Hepa 1–6 cells were transfected with the indicated construct in pEGFP-N3 and analyzed for the surface expression and recognition of the transgene by several CD81 mAb using 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). B: HepG2-A16/CD81 cells were infected with P. yoelii sporozoites in the presence of the indicated mAbs at 25 µg/ml except when otherwise indicated. All mAbs are directed to CD81 except TS9 which is a CD9 mAb and does not inhibit P. yoelii infection.

Mentions: The surface expression and the conformation of the CD81 mutants unable to support infection by P. yoelii sporozoites were tested. Hepa1.6 cells were transiently transfected with the different constructs and surface expression was assessed by flow-cytometry, using a panel of 7 CD81 mAbs produced in the mouse (Fig. 6A). Most anti-tetraspanin mAbs if not all do not recognize the denatured (reduced) protein indicating the recognition of conformational epitopes. This is confirmed by our analysis of chimeric CD9/CD81 molecules with swaps in the LEL, which were not recognized (or only at a low level) by all CD9 or CD81 mAbs except by mAb 10B1 to CD9 and 1D6 to CD81, both able to recognize the CDE region of the corresponding tetraspanin (Table I). All mutants were recognized by 6 CD81 mAbs with a staining very similar to the staining of WT CD81, therefore indicating a correct conformation. Interestingly, 5A6 had a ∼70% reduction of binding to the VVD (135–137)→AAA mutant. The fact that 5A6 was the only CD81 mAb to have a reduced ability to bind to this mutant indicates that the epitope recognized by this mAb critically depends on residues located at the A–B junction. This is coherent with the second Val being substituted by a Met in mouse CD81, which is not recognized by the mAb (Fig. 3A).


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)

A CD81 mAb binds poorly to the non-functional mutant VVD (135–137)→AAA but does not block infection.A: Hepa 1–6 cells were transfected with the indicated construct in pEGFP-N3 and analyzed for the surface expression and recognition of the transgene by several CD81 mAb using 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). B: HepG2-A16/CD81 cells were infected with P. yoelii sporozoites in the presence of the indicated mAbs at 25 µg/ml except when otherwise indicated. All mAbs are directed to CD81 except TS9 which is a CD9 mAb and does not inhibit P. yoelii infection.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000010-g006: A CD81 mAb binds poorly to the non-functional mutant VVD (135–137)→AAA but does not block infection.A: Hepa 1–6 cells were transfected with the indicated construct in pEGFP-N3 and analyzed for the surface expression and recognition of the transgene by several CD81 mAb using 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). B: HepG2-A16/CD81 cells were infected with P. yoelii sporozoites in the presence of the indicated mAbs at 25 µg/ml except when otherwise indicated. All mAbs are directed to CD81 except TS9 which is a CD9 mAb and does not inhibit P. yoelii infection.
Mentions: The surface expression and the conformation of the CD81 mutants unable to support infection by P. yoelii sporozoites were tested. Hepa1.6 cells were transiently transfected with the different constructs and surface expression was assessed by flow-cytometry, using a panel of 7 CD81 mAbs produced in the mouse (Fig. 6A). Most anti-tetraspanin mAbs if not all do not recognize the denatured (reduced) protein indicating the recognition of conformational epitopes. This is confirmed by our analysis of chimeric CD9/CD81 molecules with swaps in the LEL, which were not recognized (or only at a low level) by all CD9 or CD81 mAbs except by mAb 10B1 to CD9 and 1D6 to CD81, both able to recognize the CDE region of the corresponding tetraspanin (Table I). All mutants were recognized by 6 CD81 mAbs with a staining very similar to the staining of WT CD81, therefore indicating a correct conformation. Interestingly, 5A6 had a ∼70% reduction of binding to the VVD (135–137)→AAA mutant. The fact that 5A6 was the only CD81 mAb to have a reduced ability to bind to this mutant indicates that the epitope recognized by this mAb critically depends on residues located at the A–B junction. This is coherent with the second Val being substituted by a Met in mouse CD81, which is not recognized by the mAb (Fig. 3A).

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