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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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The A and B helices of CD81 LEL confer CD9/CD81 chimeric molecules the ability to support infection by P. yoelii sporozoites.A: Amino acid sequence alignment of CD81, CD9, and chimeras. Only the sequence of the LEL is shown. The origin of the flanking domains (TM3 and TM4) is shown on both sides of the sequence. The position of CD81 helices is indicated on the top of the alignment. CD81 residues are shown in red capital letters and CD9 residues in blue small letters. The CCG consensus site and other conserved cysteines, as well as a functionally important site (VVDDD) are underlined. CD81 LEL residues presumably in contact with the SEL are indicated with an asterisk. Open circles shows residues known to be involved in the interaction with HCV E2 glycoprotein. B and C: HepG2-A16 cells were transiently transfected with plasmids expressing CD9, CD81, or CD81/CD9 chimeras 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 and *, p<0.05 as compared to CD9-transfected cells.
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ppat-1000010-g001: The A and B helices of CD81 LEL confer CD9/CD81 chimeric molecules the ability to support infection by P. yoelii sporozoites.A: Amino acid sequence alignment of CD81, CD9, and chimeras. Only the sequence of the LEL is shown. The origin of the flanking domains (TM3 and TM4) is shown on both sides of the sequence. The position of CD81 helices is indicated on the top of the alignment. CD81 residues are shown in red capital letters and CD9 residues in blue small letters. The CCG consensus site and other conserved cysteines, as well as a functionally important site (VVDDD) are underlined. CD81 LEL residues presumably in contact with the SEL are indicated with an asterisk. Open circles shows residues known to be involved in the interaction with HCV E2 glycoprotein. B and C: HepG2-A16 cells were transiently transfected with plasmids expressing CD9, CD81, or CD81/CD9 chimeras 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 and *, p<0.05 as compared to CD9-transfected cells.

Mentions: HepG2-A16 cells express very little CD81 at their surface and are not permissive to P. yoelii invasion. The few EEF observed were small and intranuclear, resulting from CD81-independent sporozoite entry through disruption of the cell plasma membrane [12]. We have recently shown that stable expression of human CD81 is sufficient to allow P. yoelii sporozoite entry and further differentiation into replicative exo-erythrocytic forms (EEF) [13],[14]. As shown in Fig. 1, transient expression of CD81, but not of CD9, rendered HepG2-A16 cells susceptible to P. yoelii infection. This differential ability of CD81 and CD9 to support infection opened the way to investigating CD81 domains important for infection using CD9/CD81 chimeras. Two previously characterized chimeras were first tested [35]. CD9×81 consists of the first three transmembrane domains of CD9 joined to the second half of CD81, comprising most of its large extracellular domain (excluding the very first residues) and the fourth transmembrane region (Fig 1A). This chimera rendered HepG2-A16 cells susceptible to P. yoelii infection while the reciprocal construction CD81×9 did not (Fig. 1B). This second chimera is known to be functional since it potentiated the toxicity of the diphtheria toxin to the same extent as CD9 [35]. Furthermore, a construct where CD9 LEL was exchanged with CD81 LEL (CD9LEL81) (Fig. 1A) was also able to support infection to the same extent as CD81 (Fig. 1B), pointing out the importance of CD81 LEL.


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)

The A and B helices of CD81 LEL confer CD9/CD81 chimeric molecules the ability to support infection by P. yoelii sporozoites.A: Amino acid sequence alignment of CD81, CD9, and chimeras. Only the sequence of the LEL is shown. The origin of the flanking domains (TM3 and TM4) is shown on both sides of the sequence. The position of CD81 helices is indicated on the top of the alignment. CD81 residues are shown in red capital letters and CD9 residues in blue small letters. The CCG consensus site and other conserved cysteines, as well as a functionally important site (VVDDD) are underlined. CD81 LEL residues presumably in contact with the SEL are indicated with an asterisk. Open circles shows residues known to be involved in the interaction with HCV E2 glycoprotein. B and C: HepG2-A16 cells were transiently transfected with plasmids expressing CD9, CD81, or CD81/CD9 chimeras 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 and *, p<0.05 as compared to CD9-transfected cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2279262&req=5

ppat-1000010-g001: The A and B helices of CD81 LEL confer CD9/CD81 chimeric molecules the ability to support infection by P. yoelii sporozoites.A: Amino acid sequence alignment of CD81, CD9, and chimeras. Only the sequence of the LEL is shown. The origin of the flanking domains (TM3 and TM4) is shown on both sides of the sequence. The position of CD81 helices is indicated on the top of the alignment. CD81 residues are shown in red capital letters and CD9 residues in blue small letters. The CCG consensus site and other conserved cysteines, as well as a functionally important site (VVDDD) are underlined. CD81 LEL residues presumably in contact with the SEL are indicated with an asterisk. Open circles shows residues known to be involved in the interaction with HCV E2 glycoprotein. B and C: HepG2-A16 cells were transiently transfected with plasmids expressing CD9, CD81, or CD81/CD9 chimeras 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 and *, p<0.05 as compared to CD9-transfected cells.
Mentions: HepG2-A16 cells express very little CD81 at their surface and are not permissive to P. yoelii invasion. The few EEF observed were small and intranuclear, resulting from CD81-independent sporozoite entry through disruption of the cell plasma membrane [12]. We have recently shown that stable expression of human CD81 is sufficient to allow P. yoelii sporozoite entry and further differentiation into replicative exo-erythrocytic forms (EEF) [13],[14]. As shown in Fig. 1, transient expression of CD81, but not of CD9, rendered HepG2-A16 cells susceptible to P. yoelii infection. This differential ability of CD81 and CD9 to support infection opened the way to investigating CD81 domains important for infection using CD9/CD81 chimeras. Two previously characterized chimeras were first tested [35]. CD9×81 consists of the first three transmembrane domains of CD9 joined to the second half of CD81, comprising most of its large extracellular domain (excluding the very first residues) and the fourth transmembrane region (Fig 1A). This chimera rendered HepG2-A16 cells susceptible to P. yoelii infection while the reciprocal construction CD81×9 did not (Fig. 1B). This second chimera is known to be functional since it potentiated the toxicity of the diphtheria toxin to the same extent as CD9 [35]. Furthermore, a construct where CD9 LEL was exchanged with CD81 LEL (CD9LEL81) (Fig. 1A) was also able to support infection to the same extent as CD81 (Fig. 1B), pointing out the importance of CD81 LEL.

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