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First published online May 10, 2006
doi: 10.1242/10.1242/jcs.02911

Journal of Cell Science 119, 1992-2002 (2006)
Published by The Company of Biologists 2006
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Cholesterol contributes to the organization of tetraspanin-enriched microdomains and to CD81-dependent infection by malaria sporozoites

Olivier Silvie1,2,*, Stéphanie Charrin3,4,5,§, Martine Billard3,4,5, Jean-François Franetich1,2, Krista L. Clark6,{ddagger}, Geert-Jan van Gemert7, Robert W. Sauerwein7, François Dautry4,5,8, Claude Boucheix3,4,5, Dominique Mazier1,2 and Eric Rubinstein3,4,5,*

1 Inserm, U511, 91 Bd de l'Hôpital, F-75013 Paris, France
2 Université Pierre et Marie Curie-Paris 6, Faculté de Médecine Pitié-Salpêtrière, 91 Bd de l'Hôpital, F-75013 Paris, France
3 Inserm, U602, 14 Av Paul Vaillant Couturier, F-94807 Villejuif, France
4 Université Paris-Sud 11, 14 Av Paul Vaillant Couturier, F-94807 Villejuif, France
5 Institut André Lwoff, IFR 89, 7 rue Guy Môquet, F-94801 Villejuif, France
6 Kansas State University, Division of Biology, Manhattan, KS 66506, USA
7 University Medical Centre St Radboud, Department of Medical Microbiology, 6500HB Nijmegen, The Netherlands
8 CNRS, UPR1983, 7 rue Guy Môquet, F-94801 Villejuif, France


Figure 1
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  		Fig. 1. Generation of new mAbs against mouse tetraspanin complexes. Biotin-labeled Hepa1-6 cells were lysed in 1% Brij 97 before immunoprecipitation with the anti-mouse CD9 mAb 4.1F12 (CD9), with the mAbs MT81 and MT81w, or with an anti-{alpha}5 integrin mAb ({alpha}5-int.).

 

Figure 2
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  		Fig. 2. MT81 and MT81w are anti-mouse CD81 mAbs. Flow cytometry analysis of different cell types stained with the mAbs MT81 (left panels) and MT81w (right panels). Upper panels: Embryonic fibroblasts from wild-type (wt), CD81 knockout (Cd81-/-, thick line) or CD9 knockout (Cd9-/-) mice. The labeling of Cd81-/- cells with mAbs MT81 and MT81w was the same as the control staining. Middle panels: Hepa1-6 cells transfected with a siRNA oligonucleotide targeting CD81 (siCD81, thick line) or with a control siRNA (cont, thin line). Lower panels: HeLa cells transfected with a mouse CD81 cDNA (mCD81, thick line) or with the vector alone (mock, thin line). The thin gray line represents the control staining.

 

Figure 3
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  		Fig. 3. Binding of MT81 and MT81w mAbs to Hepa1-6 cells. Hepa1-6 cells labeled with increasing concentrations of mAbs MT81 (solid squares) and MT81w (open squares) were analyzed by flow cytometry. Results are expressed as the mean fluorescence intensity (MFI) as a function of mAb concentration.

 

Figure 4
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  		Fig. 4. MT81w only binds to CD81 associated with tetraspanins. (A) Hepa1-6 cells were lysed in the presence of CHAPS, Brij 97, digitonin or Triton X-100 before immunoprecipitation with the anti-CD81 mAbs MT81 and MT81w or with the anti-CD9 mAb 4.1F12. Immunoprecipitates were analyzed by western blotting using biotin-labeled anti-CD81 (MT81) and anti-CD9 (4.1F12) mAbs. Note that MT81w precipitates both CD81 and CD9 in CHAPS and Brij 97, but not in digitonin or Triton X-100. (B) Hepa1-6 cells were lysed in 1% Brij 97 before five rounds of immunoprecipitation with anti-CD9 mAb 4.1F12 to deplete CD9 (depl. +) or with beads alone as a control (depl. -). The supernatants were then used for immunoprecipitations as in A. The membranes in each panel were given the same exposure.

 

Figure 5
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  		Fig. 5. Precipitation of tetraspanins by digitonin. Biotin-labeled Hepa1-6 cells were lysed in 1% Brij 97 and the lysates were treated with digitonin (digit. +) or with vehicle alone as a control (-). After elimination of the precipitated material, the supernatants were used for immunoprecipitation using anti-CD81 mAbs MT81 and MT81w or anti-{alpha}5 integrin ({alpha}5-int.). The panels are from the same membranes and were given the same exposure.

 

Figure 6
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  		Fig. 6. CD81 localization in tetraspanin-enriched microdomains depends on cholesterol. (A) Hepa1-6 cell monolayers were treated with 10 mM MßCD for 15 minutes at 37°C (or left untreated as a control) before fixation and double labeling with anti-CD9 mAb 4.1F12 (right panel) and anti-CD81 mAbs MT81 or MT81w (left panel). Bars, 40 µm. (B) Flow cytometry analysis of Hepa1-6 cells stained with the anti-CD81 mAbs MT81 and MT81w or the anti-CD9 mAb 4.1F12. Upper panels: cells were treated with MßCD (thick black line) or left untreated (thin black line). Middle panels: cells were treated with MßCD (thin black line) followed by MßCD/cholesterol (thick black line). Lower panels: cells were treated with MßCD/cholesterol (thick black line) or left untreated (thin black line).

 

Figure 7
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  		Fig. 7. Microdomain-associated CD81 supports P. yoelii sporozoite infection. (A) Effects of increasing concentrations of mAbs MT81 (solid squares) and MT81w (open squares) on P. yoelii infection of Hepa1-6 cells, expressed as the mean percentage of inhibition in triplicate wells (± s.d.). (B) Untreated (control) and MßCD/cholesterol treated Hepa1-6 cells were infected with P. yoelii sporozoites in the presence of MT81 and MT81w mAbs. Results are expressed as the mean percentage inhibition in triplicate wells (± s.d.), as compared to wells without mAbs.

 

Figure 8
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  		Fig. 8. CD81 palmitoylation is dispensable for P. yoelii sporozoite infection. Hepa1-6 cells were transfected with plasmids coding for wild-type human CD81 (hCD81) or palmitoylation-defective human CD81 (hCD81plm), or with the vector alone (mock), 24 hours before infection with P. yoelii sporozoites in the presence of MT81 as indicated. The number of exo-erythrocytic form (EEF)-infected cells (mean ± s.d.) was determined as described in the Materials and Methods section.

 

Figure 9
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  		Fig. 9. Host cholesterol is required for P. yoelii sporozoite invasion. (A-C) Hepa1-6 cells were treated with MßCD followed by MßCD/cholesterol when indicated, before infection by P. yoelii sporozoites and quantification of exo-erythrocytic forms (EEFs) at 24 hours (A) or intracellular sporozoites at 3 hours (B). (C) Cells were treated either before (black bars) or 3 hours after (open bars) infection by sporozoites. (D) Primary mouse hepatocytes were treated with MßCD followed by MßCD/cholesterol when indicated before infection with P. yoelii sporozoites. The number of infected cells was determined as described in the Materials and Methods section. **P<0.01, as compared to untreated control.

 

Figure 10
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  		Fig. 10. CD81 and cholesterol are required for P. falciparum sporozoite invasion of human hepatocytes. (A) Primary human hepatocytes transfected with a siRNA oligonucleotide targeting CD81 (siCD81) or with a control siRNA (cont) were stained with anti-CD81 (mAb TS81, left panel) or anti-CD9 (mAb TS9, right panel) and analyzed by flow cytometry. (B) Quantification of P. falciparum exo-erythrocytic forms (EEFs; mean of triplicate wells ± s.d.) in human hepatocytes transfected with siRNA oligonucleotides targeting CD81 (siCD81) or CD9 (siCD9), or with a control siRNA (control). *P<0.05 as compared to control. (C) Primary human hepatocytes were treated with MßCD followed by MßCD/cholesterol, when indicated, before infection by P. falciparum sporozoites. The number of EEF-infected cells was determined as described in the Materials and Methods section. **P<0.01, as compared to untreated control.

 

Figure 11
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  		Fig. 11. Cholesterol and CD81 are functionally linked during Plasmodium sporozoite invasion. HepG2 (A) or HepG2/CD81 cells (B,C) were treated with MßCD followed by MßCD/cholesterol when indicated before infection by P. berghei (A,C) or P. yoelii (B) sporozoites. The number of exo-erythrocytic form (EEF)-infected cells was determined as described in the Materials and Methods section. **P<0.01, as compared to untreated control.

 

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© The Company of Biologists Ltd 2006