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

Temperature Shift and Host Cell Contact Up-Regulate Sporozoite Expression of Plasmodium falciparum Genes Involved in Hepatocyte Infection

Plasmodium sporozoites are deposited in the skin by Anopheles mosquitoes. They then find their way to the liver, where they specifically invade hepatocytes in which they develop to yield merozoites infective to red blood cells. Relatively little is known of the molecular interactions during these initial obligatory phases of the infection. Recent data suggested that many of the inoculated sporozoites invade hepatocytes an hour or more after the infective bite. We hypothesised that this pre-invasive period in the mammalian host prepares sporozoites for successful hepatocyte infection. Therefore, the genes whose expression becomes modified prior to hepatocyte invasion would be those likely to code for proteins implicated in the subsequent events of invasion and development. We have used P. falciparum sporozoites and their natural host cells, primary human hepatocytes, in in vitro co-culture system as a model for the pre-invasive period. We first established that under co-culture conditions, sporozoites maintain infectivity for an hour or more, in contrast to a drastic loss in infectivity when hepatocytes were not included. Thus, a differential transcriptome of salivary gland sporozoites versus sporozoites co-cultured with hepatocytes was established using a pan-genomic P. falciparum microarray. The expression of 532 genes was found to have been up-regulated following co-culture. A fifth of these genes had no orthologues in the genomes of Plasmodium species used in rodent models of malaria. Quantitative RT-PCR analysis of a selection of 21 genes confirmed the reliability of the microarray data. Time-course analysis further indicated two patterns of up-regulation following sporozoite co-culture, one transient and the other sustained, suggesting roles in hepatocyte invasion and liver stage development, respectively. This was supported by functional studies of four hitherto uncharacterized proteins of which two were shown to be sporozoite surface proteins involved in hepatocyte invasion, while the other two were predominantly expressed during hepatic parasite development. The genome-wide up-regulation of expression observed supports the hypothesis that the shift from the mosquito to the mammalian host contributes to activate quiescent salivary gland sporozoites into a state of readiness for the hepatic stages. Functional studies on four of the up-regulated genes validated our approach as one means to determine the repertoire of proteins implicated during the early events of the Plasmodium infection, and in this case that of P. falciparum, the species responsible for the severest forms of malaria

Gene Disruption of Plasmodium falciparum p52 Results in Attenuation of Malaria Liver Stage Development in Cultured Primary Human Hepatocytes

Difficulties with inducing sterile and long lasting protective immunity against malaria with subunit vaccines has renewed interest in vaccinations with attenuated Plasmodium parasites. Immunizations with sporozoites that are attenuated by radiation (RAS) can induce strong protective immunity both in humans and rodent models of malaria. Recently, in rodent parasites it has been shown that through the deletion of a single gene, sporozoites can also become attenuated in liver stage development and, importantly, immunization with these sporozoites results in immune responses identical to RAS. The promise of vaccination using these genetically attenuated sporozoites (GAS) depends on translating the results in rodent malaria models to human malaria. In this study, we perform the first essential step in this transition by disrupting, p52, in P. falciparum an ortholog of the rodent parasite gene, p36p, which we had previously shown can confer long lasting protective immunity in mice. These P. falciparum P52 deficient sporozoites demonstrate gliding motility, cell traversal and an invasion rate into primary human hepatocytes in vitro that is comparable to wild type sporozoites. However, inside the host hepatocyte development is arrested very soon after invasion. This study reveals, for the first time, that disrupting the equivalent gene in both P. falciparum and rodent malaria Plasmodium species generates parasites that become similarly arrested during liver stage development and these results pave the way for further development of GAS for human use

Rôle de protéines de l'hépatocyte dans sa permissivité à l'infection par les sporozoïtes de Plasmodium

L infection des hépatocytes par les sporozoïtes de Plasmodium est la première étape obligatoire du cycle parasitaire chez l hôte vertébré. A ce jour, la nature des mécanismes moléculaires impliqués dans l invasion des hépatocytes par les sporozoïtes et le développement du schizonte hépatique restent peu connus. Dans un premier temps, nous avons montré que seuls 21 résidus du domaine B de CD81, particulièrement le résidu D137, portent la permissivité des cellules hépatocytaires à l infection plasmodiale. Ces résultats, couplés à l utilisation de différents anticorps monoclonaux dirigés contre CD81, appuient l idée d un rôle indirect de CD81, vraisemblablement par modulation de l activité d une protéine partenaire restant à identifier. Par la suite, nous avons démontré que le récepteur aux lipoprotéines SR-BI, exprimé à la surface de la cellule hôte, module l infection CD81-dépendante par les sporozoïtes de Plasmodium. SR-BI permet la formation de structures membranaires hautement permissives à l infection, appelées microdomaines enrichis en tétraspanines, selon deux mécanismes bien distincts : d un côté il régule le cholestérol cellulaire et membranaire, de l autre il favorise le positionnement de CD81 à la surface des cellules. De plus, SR-BI favorise le développement parasitaire en régulant la protéine L-FABP, nécessaire à la croissance du parasite. Enfin, 6 nouvelles protéines impliquées dans la permissivité de l hépatocyte à l infection par les sporozoïtes de Plasmodium ont été identifiées dont HP1, indispensable à l infection par les sporozoïtes de P. falciparum et P. berghei. Ces résultats ouvrent la voie à une meilleure compréhension du stade hépatique de Plasmodium et à la génération de nouveaux outils d études de l infection à P. falciparumPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Overview of the In-Space Propulsion (ISP-1) Project

The intent of this publication is to provide an overview of the progress of the ISP1 project over the 2011-2012 period. In the frame of the European FP7 Collaborative Project, Focused Research Project Theme 7, the In-Space Propulsion (ISP-1) project GA# 218849 was initiated in 2009 with the objective of improving the knowledge and the techniques which are necessary for future space missions relying on cryogenic propulsion. The ISP-1 program is structured into five main work packages which deal with various technological issues associated to the development of a Low Thrust Cryogenic Propulsion system. It concentrates on liquid oxygen, liquid hydrogen, and liquid methane propellants. The subject addressed by the work packages are the LOX-methane combustion, the hydrogen embrittlement, the material compatibility and tribology in liquid oxygen, the energy management of low thrust propulsion system, and the development of electrically driven cryogenic turbopumps. The scientific and technological goals assigned to each work package can be summarized as: - LOX/CH4 Combustion studies and test focused on low thrust LOX/CH4 space propulsion with an emphasis on low pressure liquid injection - Compatibility and tribology analysis and tests addressing both technological aspects (Foil bearings in cryogenic conditions , material for bearing retainer, graphite against a hard surface for dynamic seals) and more fundamental aspects (feasibility of CH4 tribological tests, theoretical analysis of local contact conditions) of tribology. - Hydrogen embrittlement studies and tests with the objective of testing new materials in a High pressure and medium range temperature environment - Heat Accumulators studies and tests, focused on energy management techniques, with testing of a low temperature accumulator - The Propellant Electric Pumps design and tests, to be concluded by the testing of a demonstrator pump in LN2 The work packages of project ISP1 rely on a combination of analysis and test, of theoretical activities which can serve as a basis for PhD theses or more application oriented activities such as the design and testing of a demonstrator pump or a demonstrator accumulator. These work packages are focused on technologies which are considered as the critical points of future cryogenic space propulsion systems. By increasing the technological readiness level of these technologies, these activities pave the way for the development of future propulsion systems and constitute an asset with respect to possible future international cooperation.JRC.F.4-Nuclear Reactor Integrity Assessment and Knowledge Managemen

The Host Protein Aquaporin-9 is Required for Efficient Plasmodium falciparum Sporozoite Entry into Human Hepatocytes

Contains fulltext : 235497.pdf (Publisher’s version ) (Open Access

The Ig Domain Protein CD9P-1 Down-regulates CD81 Ability to Support Plasmodium yoelii Infection*

Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a malaria natural infection. The molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that CD81 is required on hepatocytes for infection by Plasmodium falciparum and Plasmodium yoelii sporozoites. CD81 belongs to the tetraspanin superfamily of transmembrane proteins. By interacting with each other and with other transmembrane proteins, tetraspanins may play a role in the lateral organization of membrane proteins. In this study, we investigated the role of the two major molecular partners of CD81 in hepatocytic cells, CD9P-1/EWI-F and EWI-2, two transmembrane proteins belonging to a novel subfamily of immunoglobulin proteins. We show that CD9P-1 silencing increases the host cell susceptibility to P. yoelii sporozoite infection, whereas EWI-2 knock-down has no effect. Conversely, overexpression of CD9P-1 but not EWI-2 partially inhibits infection. Using CD81 and CD9P-1 chimeric molecules, we demonstrate the role of transmembrane regions in CD81-CD9P-1 interactions. Importantly, a CD9P-1 chimera that no longer associates with CD81 does not affect infection. Based on these data, we conclude that CD9P-1 acts as a negative regulator of P. yoelii infection by interacting with CD81 and regulating its function

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

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