Differential activity of methylene blue against erythrocytic and hepatic stages of Plasmodium

Background: In the context of malaria elimination/eradication, drugs that are effective against the different developmental stages of the parasite are highly desirable. The oldest synthetic anti-malarial drug, the thiazine dye methylene blue (MB), is known for its activity against Plasmodium blood stages, including gametocytes. The aim of the present study was to investigate a possible effect of MB against malaria parasite liver stages. Methods: MB activity was investigated using both in vitro and in vivo models. In vitro assays consisted of testing MB activity on Plasmodium falciparum, Plasmodium cynomolgi and Plasmodium yoelii parasites in human, simian or murine primary hepatocytes, respectively. MB in vivo activity was evaluated using intravital imaging in BALB/c mice infected with a transgenic bioluminescent P. yoelii parasite line. The transmission-blocking activity of MB was also addressed using mosquitoes fed on MB-treated mice. Results: MB shows no activity on Plasmodium liver stages, including hypnozoites, in vitro in primary hepatocytes. In BALB/c mice, MB has moderate effect on P. yoelii hepatic development but is highly effective against blood stage growth. MB is active against gametocytes and abrogates parasite transmission from mice to mosquitoes. Conclusion: While confirming activity of MB against both sexual and asexual blood stages, the results indicate that MB has only little activity on the development of the hepatic stages of malaria parasites

Rapid and Specific Action of Methylene Blue against Plasmodium Transmission Stages

Methylene blue (MB) is the oldest synthetic anti-infective. Its high potency against asexual and sexual stages of malaria parasites is well documented. This study aimed to investigate possible additional activities of MB in interfering with parasite transmission and determine target stages in Anopheles vectors and humans. MB’s transmission-blocking activity was first evaluated by an ex vivo direct membrane feeding assay (DMFA) using Plasmodium falciparum field isolates. To investigate anti-mosquito stage activity, Plasmodium berghei-infected Anopheles stephensi mosquitoes were fed a second blood meal on mice that had been treated with methylene blue, 3, 6- and 15-days after the initial infectious blood meal. Anti-sporozoite and liver stage activities were evaluated in vitro and in vivo via sporozoite invasion and liver stage development assays, respectively. MB exhibited a robust inhibition of P. falciparum transmission in An. gambiae, even when added shortly before the DMFA but only a moderate effect against P. berghei oocyst development. Exposure of mature P. berghei and P. falciparum sporozoites to MB blocked hepatocyte invasion, yet P. berghei liver stage development was unaffected by MB. Our results indicate previously underappreciated rapid specific activities of methylene blue against Plasmodium transmission stages, preventing the establishment of both mosquito midgut and liver infections as the first essential steps in both hosts

A Plant-Derived Morphinan as a Novel Lead Compound Active against Malaria Liver Stages

BACKGROUND: The global spread of multidrug–resistant malaria parasites has led to an urgent need for new chemotherapeutic agents. Drug discovery is primarily directed to the asexual blood stages, and few drugs that are effective against the obligatory liver stages, from which the pathogenic blood infection is initiated, have become available since primaquine was deployed in the 1950s. METHODS AND FINDINGS: Using bioassay-guided fractionation based on the parasite's hepatic stage, we have isolated a novel morphinan alkaloid, tazopsine, from a plant traditionally used against malaria in Madagascar. This compound and readily obtained semisynthetic derivatives were tested for inhibitory activity against liver stage development in vitro (P. falciparum and P. yoelii) and in vivo (P. yoelii). Tazopsine fully inhibited the development of P. yoelii (50% inhibitory concentration [IC(50)] 3.1 μM, therapeutic index [TI] 14) and P. falciparum (IC(50) 4.2 μM, TI 7) hepatic parasites in cultured primary hepatocytes, with inhibition being most pronounced during the early developmental stages. One derivative, N-cyclopentyl-tazopsine (NCP-tazopsine), with similar inhibitory activity was selected for its lower toxicity (IC(50) 3.3 μM, TI 46, and IC(50) 42.4 μM, TI 60, on P. yoelii and P. falciparum hepatic stages in vitro, respectively). Oral administration of NCP-tazopsine completely protected mice from a sporozoite challenge. Unlike the parent molecule, the derivative was uniquely active against Plasmodium hepatic stages. CONCLUSIONS: A readily obtained semisynthetic derivative of a plant-derived compound, tazopsine, has been shown to be specifically active against the liver stage, but inactive against the blood forms of the malaria parasite. This unique specificity in an antimalarial drug severely restricts the pressure for the selection of drug resistance to a parasite stage limited both in numbers and duration, thus allowing researchers to envisage the incorporation of a true causal prophylactic in malaria control programs

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

A Role for Immune Responses against Non-CS Components in the Cross-Species Protection Induced by Immunization with Irradiated Malaria Sporozoites

Immunization with irradiated Plasmodium sporozoites induces sterile immunity in rodents, monkeys and humans. The major surface component of the sporozoite the circumsporozoite protein (CS) long considered as the antigen predominantly responsible for this immunity, thus remains the leading candidate antigen for vaccines targeting the parasite's pre-erythrocytic (PE) stages. However, this role for CS was questioned when we recently showed that immunization with irradiated sporozoites (IrrSpz) of a P. berghei line whose endogenous CS was replaced by that of P. falciparum still conferred sterile protection against challenge with wild type P. berghei sporozoites. In order to investigate the involvement of CS in the cross-species protection recently observed between the two rodent parasites P. berghei and P. yoelii, we adopted our gene replacement approach for the P. yoelii CS and exploited the ability to conduct reciprocal challenges. Overall, we found that immunization led to sterile immunity irrespective of the origin of the CS in the immunizing or challenge sporozoites. However, for some combinations, immune responses to CS contributed to the acquisition of protective immunity and were dependent on the immunizing IrrSpz dose. Nonetheless, when data from all the cross-species immunization/challenges were considered, the immune responses directed against non-CS parasite antigens shared by the two parasite species played a major role in the sterile protection induced by immunization with IrrSpz. This opens the perspective to develop a single vaccine formulation that could protect against multiple parasite species

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

Identification de facteurs de la cellule hôte impliqués dans l'infection hépatique de Plasmodium (Rôle de l'aquaporine 9)

Plasmodium, transmis au mammifère sous sa forme sporozoïte lors de la piqûre d un moustique anophèle infecté, est l agent pathogène du paludisme, la plus mortelle des infections parasitaires. Afin d identifier des facteurs de l hôte nécessaires lors de sa première étape d infection, dans le foie, nous avons comparé le transcriptome de cellules hépatiques permissives et réfractaires à l infection par les sporozoïtes du parasite humain, P. falciparum. Des gènes dont l expression est associée à la permissivité des hépatocytes humain ont ainsi pu être identifiés. Parmi ces gènes, nous avons montré que l aquaporine 9 (AQP9) est nécessaire à l étape d invasion des sporozoïtes, et non à leur développement intra-hépatocytaire. Dans des modèles murins, nous avons observé que le niveau de dépendance des sporozoïtes de Plasmodium de rongeur varie suivant l espèce plasmodiale et la cellule hôte utilisée. La fonction d AQP9 semble impliquée, puisqu un inhibiteur de la fonction de transport d AQP9, la phlorétine, bloque l invasion des cellules hépatiques par les sporozoïtes de Plasmodium. Enfin, nous avons observé la localisation d AQP9 sur la membrane de la vacuole parasitophore (MVP) des schizontes hépatiques, où elle se retrouve probablement internalisée pendant ou peu après l étape d invasion. Si plusieurs protéines du globule rouge ont déjà pu être mises en évidence sur la MVP des parasites érythrocytaires, AQP9 est à ce jour la première protéine de l hôte identifiée sur la MVP des schizontes hépatiques. L ensemble de ces résultats apporte un nouvel élément impliquant une seconde protéine de l hôte, après CD81, pour l infection des hépatocytes par Plasmodium.Plasmodium, transmitted to the mammal in its sporozoite form through the bite of an infected Anopheles mosquito, is the causative agent of malaria, the most deadly parasitic disease. To identify host cell factors required during the first stage of infection in the liver, we compared the transcriptome of permissive and refractory liver cells for the infection by the sporozoite of the human parasite, P. falciparum. Genes whose expression is associated with human hepatocyte permissiveness were identified. Among them, we have shown that aquaporin 9 (AQP9) is required for the process of sporozoite invasion, but not for its intra-hepatocytic development. In murine models, we observed that the requirement of rodent Plasmodium sporozoites for AQP9 depends on the parasite species and the host cell used. AQP9 function appears to be involved, since phloretin, an inhibitor of the transport function of AQP9, blocks the invasion of liver cells by Plasmodium sporozoites. Finally, we observed the localization of AQP9 on the parasitophorous vacuole membrane (PVM) of the hepatic schizonts, where it is probably internalized during or soon after the invasion step. If several red blood cell proteins have been identified on the PVM of erythrocytic parasites, AQP9 is so far the first host protein identified on the PVM of hepatic schizonts. Taken together, these results provide a new element involving a second protein of the host, after CD81, for the infection of hepatocytes by Plasmodium.PARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Phase hépatique du paludisme : voies d’entrée des sporozoïtes de Plasmodium

Les sporozoïtes de Plasmodium sont transmis par des moustiques du genre Anopheles, migrent jusqu’au foie et infectent les hépatocytes de l’hôte, où les parasites se multiplient et se différencient avant d’infecter les globules rouges : il s’agit de la phase pré-érythrocytaire de l’infection, asymptomatique et obligatoire. L’invasion des hépatocytes constitue une cible idéale pour des approches anti-paludiques prophylactiques, mais les mécanismes impliqués dans cette étape restent mal connus. Les parasites du phylum Apicomplexa infectent leurs cellules cibles à travers une structure spécifique, appelée jonction, pour former une vacuole parasitophore, essentielle au développement du parasite. La tétraspanine CD81, l’un des récepteurs du virus de l’hépatite C, joue un rôle crucial lors de l’infection du foie par les sporozoïtes de Plasmodium. CD81 intervient à la phase précoce de l’infection, probablement au niveau de l’entrée du sporozoïte. Le rôle précis de CD81 au cours de l’infection reste à définir, mais l’hypothèse actuelle est que CD81 intervient indirectement, en régulant l’activité d’un ou plusieurs autres facteurs essentiels, d’origine hépatocytaire ou parasitaire, au sein de microdomaines membranaires enrichis en tétraspanines

Antiplasmodial activity of traditional polyherbal remedy from Odisha, India: Their potential for prophylactic use

Objective: To evaluate the potential prophylactic activity of traditional polyherbal remedy against malaria. Methods: A traditional polyherbal remedy against malaria from Odisha, India was evaluated for its potential prophylactic activity using in vitro hepatic cell lines assay and the murine malaria system Plasmodium yoelii yoelii/Anopheles stephensi. Results: The polyherbal extract inhibited the Plasmodium yoelii hepatic stages in vitro (IC50 0.74 mg/mL), a therapeutic index of 9.54. In mice treated with the aqueous extract (2000 mg/kg/day), peak parasitaemia values were 81% lower in the experimental 2.35% ± 0.14% as compared to controls 12.62% ± 0.52% (P < 0.001), suggesting significant prophylactic activity. Conclusions: The observations provide a proof of concept for a traditional malaria prophylactic remedy used by tribal populations in India

Phase hépatique du paludisme : voies d’entrée des sporozoïtes de Plasmodium

Les sporozoïtes de Plasmodium sont transmis par des moustiques du genre Anopheles, migrent jusqu’au foie et infectent les hépatocytes de l’hôte, où les parasites se multiplient et se différencient avant d’infecter les globules rouges : il s’agit de la phase pré-érythrocytaire de l’infection, asymptomatique et obligatoire. L’invasion des hépatocytes constitue une cible idéale pour des approches anti-paludiques prophylactiques, mais les mécanismes impliqués dans cette étape restent mal connus. Les parasites du phylum Apicomplexa infectent leurs cellules cibles à travers une structure spécifique, appelée jonction, pour former une vacuole parasitophore, essentielle au développement du parasite. La tétraspanine CD81, l’un des récepteurs du virus de l’hépatite C, joue un rôle crucial lors de l’infection du foie par les sporozoïtes de Plasmodium. CD81 intervient à la phase précoce de l’infection, probablement au niveau de l’entrée du sporozoïte. Le rôle précis de CD81 au cours de l’infection reste à définir, mais l’hypothèse actuelle est que CD81 intervient indirectement, en régulant l’activité d’un ou plusieurs autres facteurs essentiels, d’origine hépatocytaire ou parasitaire, au sein de microdomaines membranaires enrichis en tétraspanines