Hepatocyte Permissiveness to Plasmodium Infection Is Conveyed by a Short and Structurally Conserved Region of the CD81 Large Extracellular Domain

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

In Vitro Activities of 25 Quinolones and Fluoroquinolones against Liver and Blood Stage Plasmodium spp.

The in vitro activities of 25 quinolones and fluoroquinolones against erythrocytic stages of Plasmodium falciparum and against liver stages of Plasmodium yoelii yoelii and P. falciparum were studied. All compounds were inhibitory for chloroquine-sensitive and chloroquine-resistant P. falciparum grown in red blood cells. This inhibitory effect increased with prolonged incubation and according to the logarithm of the drug concentration. Grepafloxacin, trovafloxacin, and ciprofloxacin were the most effective drugs, with 50% inhibitory concentrations of <10 μg/ml against both strains. Only grepafloxacin, piromidic acid, and trovafloxacin had an inhibitory effect against hepatic stages of P. falciparum and P. yoelii yoelii; this effect combined reductions of the numbers and the sizes of schizonts in treated cultures. Thus, quinolones have a potential for treatment or prevention of malaria through their unique antiparasitic effect against erythrocytic and hepatic stages of Plasmodium

New Active Drugs against Liver Stages of Plasmodium Predicted by Molecular Topology▿ †

We conducted a quantitative structure-activity relationship (QSAR) study based on a database of 127 compounds previously tested against the liver stage of Plasmodium yoelii in order to develop a model capable of predicting the in vitro antimalarial activities of new compounds. Topological indices were used as structural descriptors, and their relation to antimalarial activity was determined by using linear discriminant analysis. A topological model consisting of two discriminant functions was created. The first function discriminated between active and inactive compounds, and the second identified the most active among the active compounds. The model was then applied sequentially to a large database of compounds with unknown activity against liver stages of Plasmodium. Seventeen drugs that were predicted to be active or inactive were selected for testing against the hepatic stage of P. yoelii in vitro. Antiretroviral, antifungal, and cardiotonic drugs were found to be highly active (nanomolar 50% inhibitory concentration values), and two ionophores completely inhibited parasite development. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed on hepatocyte cultures for all compounds, and none of these compounds were toxic in vitro. For both ionophores, the same in vitro assay as those for P. yoelii has confirmed their in vitro activities on Plasmodium falciparum. A similar topological model was used to estimate the octanol/water partition of each compound. These results demonstrate the utility of the QSAR and molecular topology approaches for identifying new drugs that are active against the hepatic stage of malaria parasites. We also show the remarkable efficacy of some drugs that were not previously reported to have antiparasitic activity

The A and B helices of CD81 LEL confer CD9/CD81 chimeric molecules the ability to support infection by <i>P. yoelii</i> sporozoites.

<p>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 <i>P. yoelii</i> 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.</p

3D structure of CD81 LEL.

<p>The drawing of CD81 LEL (PDB #1g8q) was generated in MolMol. Four helices (A, C, D, E) are drawn in red while the B helix, crucial for <i>P. yoelii</i> infection is displayed in blue. The black balls indicate the CCG ubiquitous motif. The crucial D137 as well as D138 and D139 are in purple while V135 and V136 are in royal blue. Residues V146, T149, F150, T153 and L154 putatively involved in contact with the SEL are indicated in dark blue. T163, F186 and D196 residues, in yellow, have been reported to play a role in the HCV E2 glycoprotein binding to CD81-LEL. Residues V135, V136, T163, F186 and D196 projected backward, behind the drawing plane. The two disulfides bridges are colored light coral. Hydrophilic residues K144, K148 and E152 located on the top of the B helix are in green. The SEL, in cyan, is in front of the drawing plane.</p

The VVD (135–137)→AAA and DDD (137–139)→AAA mutants unable to support infection by <i>P. yoelii</i> sporozoites interact with CD9P-1 and EWI-2.

<p>CHO cells were transiently transfected with WT or mutant CD81 plasmids (in pEGFP-N3), together with a CD9P-1 (top) or a EWI-2 (bottom) cDNA. After 48 h, the cells were lysed with digitonin and immunoprecipitations with antibodies against CD81, CD9P-1 and EWI-2 were performed. After electrophoresis and transfer, the membranes were incubated with biotin-labelled mAbs to CD81 (TS81), CD9P-1 (1F11) and EWI-2 (8A12). The mutants are designed as follows: VVD: VVD (135–137)→AAA; DDD: DDD (137–139)→AAA</p

A CD81 mAb binds poorly to the non-functional mutant VVD (135–137)→AAA but does not block infection.

<p>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 <i>P. yoelii</i> 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 <i>P. yoelii</i> infection.</p

21 residues of CD81 in a CD9 backbone are sufficient to render hepatocytic cells susceptible to <i>P. yoelii</i> sporozoites infection.

<p>A: Amino acid sequence alignment of CD9, CD81 and chimeras. Only the sequence of the large extracellular loop of the different chimeras is shown. The origin of the flanking domains (TM3 and TM4) is shown on both sides of the sequence. The position of CD81 helices are 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 B: HepG2-A16 cells were transiently transfected with plasmids expressing CD9, CD81, or CD81/CD9 chimeras and infected two days later with <i>P. yoelii</i> 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. **, p<0.01 as compared to mock-transfected cells. C: HepG2-A16 cells stably expressing CD81, CD9, CD81ccg9 or CD9[81B] were infected with <i>P. yoelii</i> sporozoites. After two days incubation, the number of EEF-infected cells was determined in triplicate wells by immunofluorescence. Results are expressed as mean±s.d. **, p<0.01 as compared to mock-transfected cells.</p