Towards an In Vitro Model of Plasmodium Hypnozoites Suitable for Drug Discovery

Contains fulltext : 96475.pdf (publisher's version ) (Open Access)BACKGROUND: Amongst the Plasmodium species in humans, only P. vivax and P. ovale produce latent hepatic stages called hypnozoites, which are responsible for malaria episodes long after a mosquito bite. Relapses contribute to increased morbidity, and complicate malaria elimination programs. A single drug effective against hypnozoites, primaquine, is available, but its deployment is curtailed by its haemolytic potential in glucose-6-phosphate dehydrogenase deficient persons. Novel compounds are thus urgently needed to replace primaquine. Discovery of compounds active against hypnozoites is restricted to the in vivo P. cynomolgi-rhesus monkey model. Slow growing hepatic parasites reminiscent of hypnozoites had been noted in cultured P. vivax-infected hepatoma cells, but similar forms are also observed in vitro by other species including P. falciparum that do not produce hypnozoites. METHODOLOGY: P. falciparum or P. cynomolgi sporozoites were used to infect human or Macaca fascicularis primary hepatocytes, respectively. The susceptibility of the slow and normally growing hepatic forms obtained in vitro to three antimalarial drugs, one active against hepatic forms including hypnozoites and two only against the growing forms, was measured. RESULTS: The non-dividing slow growing P. cynomolgi hepatic forms, observed in vitro in primary hepatocytes from the natural host Macaca fascicularis, can be distinguished from similar forms seen in P. falciparum-infected human primary hepatocytes by the differential action of selected anti-malarial drugs. Whereas atovaquone and pyrimethamine are active on all the dividing hepatic forms observed, the P. cynomolgi slow growing forms are highly resistant to treatment by these drugs, but remain susceptible to primaquine. CONCLUSION: Resistance of the non-dividing P. cynomolgi forms to atovaquone and pyrimethamine, which do not prevent relapses, strongly suggests that these slow growing forms are hypnozoites. This represents a first step towards the development of a practical medium-throughput in vitro screening assay for novel hypnozoiticidal drugs

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

Identification de protéines hépatocytaires impliquées dans l'infection hépatique de plasmodium par une approche d'interférence à ARN à grande échelle

Afin de mettre en évidence des protéines de l hôte nécessaires au développement des formes intra-hépatiques de Plasmodium chez l Homme, nous avons utilisé comme outil l ARN interférence, pour atténuer l expression de 6080 gènes de l hôte dans le modèle d infection HepG2-A16/hCD81EGFP / P. yoelii. Après deux étapes de sélection successives, vinqt-sept gènes ont été retenus. L invalidation de ces gènes par ARNi induit une diminution de l infection d au moins 30% par rapport aux contrôles sans altérer la viabilité des cellules hôtes. Nous en avons étudié cinq d entre eux : ALDH18A1, LIPG, TMEM30A, SLC25A45 et GPR39. Les quatre premiers jouent principalement un rôle dans le développement des formes intra-hépatiques tandis que le cinquième intervient dans le phénomène d invasion des cellules par les sporozoïtes. Ce travail a donc permis de mettre en évidence des protéines nécessaires à l infection intra-hépatique de Plasmodium, leur rôle exact reste à présent à déterminer.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

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

New Approach for High-Throughput Screening of Drug Activity on Plasmodium Liver Stages

Plasmodium liver stages represent potential targets for antimalarial prophylactic drugs. Nevertheless, there is a lack of molecules active on these stages. We have now developed a new approach for the high-throughput screening of drug activity on Plasmodium liver stages in vitro, based on an infrared fluorescence scanning system. This method allowed us to count automatically and rapidly Plasmodium-infected hepatocytes, using different hepatic cells and different Plasmodium species, including Plasmodium falciparum. This new technique is well adapted for high-throughput drug screening and should facilitate the identification of new antimalarial compounds active on Plasmodium liver stages

Skin-draining lymph node priming is sufficient to induce sterile immunity against pre-erythrocytic malaria

International audienceThe Plasmodium-infected hepatocyte has been considered necessary to prime the immune responses leading to sterile protection after vaccination with attenuated sporozoites. However, it has recently been demonstrated that priming also occurs in the skin. We wished to establish if sterile protection could be obtained in the absence of priming by infected hepatocytes. To this end, we developed a subcutaneous (s.c.) immunization protocol where few, possibly none, of the immunizing irradiated Plasmodium yoelii sporozoites infect hepatocytes, and also used CD81-deficient mice non-permissive to productive hepatocyte infections. We then compared and contrasted the patterns of priming with those obtained by intradermal immunization, where priming occurs in the liver. Using sterile immunity as a primary read-out, we exploited an inhibitor of T-cell migration, transgenic mice with conditional depletion of dendritic cells and adoptive transfers of draining lymph node-derived T cells, to provide evidence that responses leading to sterile immunity can be primed in the skin-draining lymph nodes with little, if any, contribution from the infected hepatocyte

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

International audienceHepatocyte invasion by Plasmodium sporozoites represents a promising target for innovative antimalarial therapy, but the molecular events mediating this process are still largely uncharacterized. We previously showed that Plasmodium falciparum sporozoite entry into hepatocytes strictly requires CD81. However, CD81-overexpressing human hepatoma cells remain refractory to P. falciparum infection, suggesting the existence of additional host factors necessary for sporozoite entry. Here, through differential transcriptomic analysis of human hepatocytes and hepatoma HepG2-CD81 cells, the transmembrane protein Aquaporin-9 (AQP9) was found to be among the most downregulated genes in hepatoma cells. RNA silencing showed that sporozoite invasion of hepatocytes requires AQP9 expression. AQP9 overexpression in hepatocytes increased their permissiveness to P. falciparum. Moreover, chemical disruption with the AQP9 inhibitor phloretin markedly inhibited hepatocyte infection. Our findings identify AQP9 as a novel host factor required for P. falciparum sporozoite hepatocyte-entry and indicate that AQP9 could be a potential therapeutic target

Scavenger receptor BI boosts hepatocyte permissiveness to Plasmodium infection.

Contains fulltext : 71106.pdf (publisher's version ) (Closed access)Infection of hepatocytes by Plasmodium falciparum sporozoites requires the host tetraspanin CD81. CD81 is also predicted to be a coreceptor, along with scavenger receptor BI (SR-BI), for hepatitis C virus. Using SR-BI-knockout, SR-BI-hypomorphic and SR-BI-transgenic primary hepatocytes, as well as specific SR-BI-blocking antibodies, we demonstrate that SR-BI significantly boosts hepatocyte permissiveness to P. falciparum, P. yoelii, and P. berghei entry and promotes parasite development. We show that SR-BI, but not the low-density lipoprotein receptor, acts as a major cholesterol provider that enhances Plasmodium infection. SR-BI regulates the organization of CD81 at the plasma membrane, mediating an arrangement that is highly permissive to penetration by sporozoites. Concomitantly, SR-BI upregulates the expression of the liver fatty-acid carrier L-FABP, a protein implicated in Plasmodium liver-stage maturation. These findings establish the mechanistic basis of the CD81-dependent Plasmodium sporozoite invasion pathway