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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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Mutations of residues at the A-B helix junction impair the ability of CD81 to support infection by P. yoelii sporozoites.A: Alignment of human CD81, mouse CD81 and human CD9. Residues that contribute to the stability of the subunit fold as reported by Kitadokoro et al. [30] are boxed in red. Additional residues that are buried according to the modelling of Seigneuret et al. [31] are boxed in pink. Some of the buried residues are also involved in the stabilization of the molecule and are therefore boxed in red. Residues potentially in contact with the small extracellular domain, according to the modelling of Seigneuret [36] and not fitting in the previous categories, are in blue. Finally, additional residues identical in both CD9 and CD81 sequences are in parma. The different mutants are also indicated on this alignment. The multiple mutants are designed as follow: VVD: VVD (135–137)→AAA; DDD: DDD (137–139)→AAA; NN: NN (141–142)→AA; EEE: DDD (137–139)→EEE; KDE: DDD (137–139)→KDE B and C: HepG2-A16 cells were transfected with the indicated mutants 48 hours before infection with P. yoelii sporozoites. After two days incubation, the number of EEF-infected cells in triplicate wells was determined by immunofluorescence. Results are expressed as mean±s.d. B: single mutations. **, p<0.01 as compared to cells transfected with WT CD81. C: triple or double mutations. **, p<0.01 as compared to mock-transfected cells
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ppat-1000010-g003: Mutations of residues at the A-B helix junction impair the ability of CD81 to support infection by P. yoelii sporozoites.A: Alignment of human CD81, mouse CD81 and human CD9. Residues that contribute to the stability of the subunit fold as reported by Kitadokoro et al. [30] are boxed in red. Additional residues that are buried according to the modelling of Seigneuret et al. [31] are boxed in pink. Some of the buried residues are also involved in the stabilization of the molecule and are therefore boxed in red. Residues potentially in contact with the small extracellular domain, according to the modelling of Seigneuret [36] and not fitting in the previous categories, are in blue. Finally, additional residues identical in both CD9 and CD81 sequences are in parma. The different mutants are also indicated on this alignment. The multiple mutants are designed as follow: VVD: VVD (135–137)→AAA; DDD: DDD (137–139)→AAA; NN: NN (141–142)→AA; EEE: DDD (137–139)→EEE; KDE: DDD (137–139)→KDE B and C: HepG2-A16 cells were transfected with the indicated mutants 48 hours before infection with P. yoelii sporozoites. After two days incubation, the number of EEF-infected cells in triplicate wells was determined by immunofluorescence. Results are expressed as mean±s.d. B: single mutations. **, p<0.01 as compared to cells transfected with WT CD81. C: triple or double mutations. **, p<0.01 as compared to mock-transfected cells

Mentions: The above data indicated that in a CD9 backbone, the A and B helices of CD81 were necessary and sufficient to confer susceptibility to P. yoelii infection. We used the crystal structure of CD81 as well as published molecular modelling of tetraspanins to guide site-directed mutagenesis [30],[31],[36]. We reasoned that if CD81 plays a role as a receptor for a parasite protein, or as a partner molecule for such receptor, residues critically involved in these interactions would be solvent-exposed. We therefore excluded from mutagenesis analysis residues that contribute to the stability of the domain fold [30] or are buried in the molecule [31]. Recently, Seigneuret proposed that the hydrophobic face of CD81 LEL is in contact with the small extracellular loop [36], we therefore also excluded residues predicted to be in contact with the small domain from further analysis. Finally, since CD9 does not support Plasmodium sporozoite infection, residues common to both CD9 and CD81 were also not considered. From this analysis, we uncovered a stretch of 14 amino acids located around CD81 A–B helix junction, that are solvent-exposed and strongly different in CD9 (Fig. 3A). Notably, the loop corresponding to the A–B junction is highly acidic in CD81 with a row of 3 aspartic acids.


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)

Mutations of residues at the A-B helix junction impair the ability of CD81 to support infection by P. yoelii sporozoites.A: Alignment of human CD81, mouse CD81 and human CD9. Residues that contribute to the stability of the subunit fold as reported by Kitadokoro et al. [30] are boxed in red. Additional residues that are buried according to the modelling of Seigneuret et al. [31] are boxed in pink. Some of the buried residues are also involved in the stabilization of the molecule and are therefore boxed in red. Residues potentially in contact with the small extracellular domain, according to the modelling of Seigneuret [36] and not fitting in the previous categories, are in blue. Finally, additional residues identical in both CD9 and CD81 sequences are in parma. The different mutants are also indicated on this alignment. The multiple mutants are designed as follow: VVD: VVD (135–137)→AAA; DDD: DDD (137–139)→AAA; NN: NN (141–142)→AA; EEE: DDD (137–139)→EEE; KDE: DDD (137–139)→KDE B and C: HepG2-A16 cells were transfected with the indicated mutants 48 hours before infection with P. yoelii sporozoites. After two days incubation, the number of EEF-infected cells in triplicate wells was determined by immunofluorescence. Results are expressed as mean±s.d. B: single mutations. **, p<0.01 as compared to cells transfected with WT CD81. C: triple or double mutations. **, p<0.01 as compared to mock-transfected cells
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2279262&req=5

ppat-1000010-g003: Mutations of residues at the A-B helix junction impair the ability of CD81 to support infection by P. yoelii sporozoites.A: Alignment of human CD81, mouse CD81 and human CD9. Residues that contribute to the stability of the subunit fold as reported by Kitadokoro et al. [30] are boxed in red. Additional residues that are buried according to the modelling of Seigneuret et al. [31] are boxed in pink. Some of the buried residues are also involved in the stabilization of the molecule and are therefore boxed in red. Residues potentially in contact with the small extracellular domain, according to the modelling of Seigneuret [36] and not fitting in the previous categories, are in blue. Finally, additional residues identical in both CD9 and CD81 sequences are in parma. The different mutants are also indicated on this alignment. The multiple mutants are designed as follow: VVD: VVD (135–137)→AAA; DDD: DDD (137–139)→AAA; NN: NN (141–142)→AA; EEE: DDD (137–139)→EEE; KDE: DDD (137–139)→KDE B and C: HepG2-A16 cells were transfected with the indicated mutants 48 hours before infection with P. yoelii sporozoites. After two days incubation, the number of EEF-infected cells in triplicate wells was determined by immunofluorescence. Results are expressed as mean±s.d. B: single mutations. **, p<0.01 as compared to cells transfected with WT CD81. C: triple or double mutations. **, p<0.01 as compared to mock-transfected cells
Mentions: The above data indicated that in a CD9 backbone, the A and B helices of CD81 were necessary and sufficient to confer susceptibility to P. yoelii infection. We used the crystal structure of CD81 as well as published molecular modelling of tetraspanins to guide site-directed mutagenesis [30],[31],[36]. We reasoned that if CD81 plays a role as a receptor for a parasite protein, or as a partner molecule for such receptor, residues critically involved in these interactions would be solvent-exposed. We therefore excluded from mutagenesis analysis residues that contribute to the stability of the domain fold [30] or are buried in the molecule [31]. Recently, Seigneuret proposed that the hydrophobic face of CD81 LEL is in contact with the small extracellular loop [36], we therefore also excluded residues predicted to be in contact with the small domain from further analysis. Finally, since CD9 does not support Plasmodium sporozoite infection, residues common to both CD9 and CD81 were also not considered. From this analysis, we uncovered a stretch of 14 amino acids located around CD81 A–B helix junction, that are solvent-exposed and strongly different in CD9 (Fig. 3A). Notably, the loop corresponding to the A–B junction is highly acidic in CD81 with a row of 3 aspartic acids.

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