Inhibition du développement intraérythrocytaire in vitro de plasmodium falciparum par les phospholipases A2 sécrétées d'origine venineuse ou humaine (rôle des lipoprotéines plasmatiques)

La recherche de mécanismes originaux de cytotoxicité contre l'agent du paludisme, Plasmodium falciparum, par utilisation de toxines animales, a permis d'isoler de puissants inhibiteurs du parasite: les phospholipases A2 sécrétées (sPLA2), qui catalysent l'hydrolyse d'un glycérophospholipide (GL), libérant un acide gras et un lysophospholipide. Leurs CI50 peuvent être de l'ordre du pM. La toxicite n'est pas médiée par une interaction directe entre l'enzyme et le globule rouge infecté, mais par l'hydrolyse des GL du milieu de culture portés par les lipoprotéines (Lp). Les lipides générés sont indispensables à la toxicité. De plus l'oxydation des Lp hydrolysées aggraverait leur toxicité. Des 9 sPLA2 humaines, seules les IIF, V et X tuent le parasite. La X aurait une toxicité indépendante de l'apport de GL exogènes et la V serait plus active sur les Lp oxydées que les fraîches. Nos analyses posent donc la question du rôle potentiel des sPLA2 humaines dans la réponse innée au paludisme.As part of a general screening of animal toxins for antimalarial drugs, we discovered that secreted phospholipases A2 (sPLA2) had great inhibitory properties against Plasmodium falciparum, the malarial agent. These enzymes catalyse hydrolysis of a glycerophospholipid (GL) releasing a fatty acid and a lysophospholipid. Their IC50 can reach the 1 pM rate. The toxicity is not mediated by a direct interaction between the enzyme and the infected erythrocyte but from enzymatic hydrolysis of the culture medium GL carried by the lipoproteins (Lp). Generated lipids are obligatory actors of the anti-Plasmodium toxicity. Moreover oxidation of the hydrolysed Lp enhances its toxicity. Of the 9 human sPLA2 only the IIF, V and X kill the parasite. The X is the sole sPLA2 tested that is toxic in the absence of exogenously added GL and the V toxicity was higher upon hydrolysis of oxidized Lp. Our analyses raise the question of the potential role of the human sPLA2 in the innate response to malaria.PARIS-Museum Hist.Naturelle (751052304) / SudocSudocFranceF

Expression de l'aminopeptidase PfA-M1 au cours du cycle érythrocytaire de plasmodium falciparum et inhibition sélective

LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

Métabolisme des sphingolipides chez Plasmodium falciparum, agent du paludisme (Incidence chimiothérapeutique)

Plusieurs voies du métabolisme des sphingolipides ont été décrites chez Plasmodium falciparum, agent du paludisme, lors de son développement intraérythrocytaire : voie de synthèse de novo du céramide (Cer), voies de synthèse des glycosphingolipides et de la sphingomyéline (SM), et la voie d'hydrolyse de la SM. Ce métabolisme paraît être impliqué dans des processus fondamentaux pour le développement parasitaire. Nous avons réalisé une étude structure-activité d'analogues de Cer et de SM potentiellement inhibiteurs du développement de P. falciparum. Les analogues de Cer possédant une liaison méthylène liant l'azote de la structure sphingosyle à une chaine de 12-14 carbones sont les plus actifs avec des CI50 de 17-30 nM. Les activités SM synthase et sphingomyélinase (SMase) du parasite ne semblent pas être la cible de ces analogues. Les analogues de SM de type R-sphingosyl-phosphorylcholine inhibent également le développement parasitaire avec une CI50 de 0,3 æM pour le meilleur (analogue en liaison thiourée). Ces inhibiteurs semblent agir en inhibant l'activité SMase du parasite. Parallèlement, une approche bioinformatique nous a permis de caractériser le gène putatif de la SMase de P. falciparum. Au-delà de leur potentiel chimio-thérapeutique, ces analogues constituent des outils pour explorer le rôle du métabolisme des sphingolipides, et son importance chez P. falciparumSeveral sphingolipid pathways were described in Plasmodium falciparum, the malaria agent, during its intraerythrocytic stages : ceramide (Cer) de novo synthesis, glycosphingolipid and sphingomyelin (SM) synthesis, and SM hydrolysis. Metabolism of sphingolipids appears to be involved in fundamental processes for the parasite development. We realized a study of structure-activity relationships of Cer and SM analogues on the inhibition of the P. falciparum intraerythrocytic development. Among the Cer analogues tested, those presenting a methylene linkage between the amino group of sphingosyl skeleton and a 12-14 carbon chain, exhibit the best inhibitory activity with IC50s of 17-30 nM. Parasite SM synthase and sphingomyelinase (SMase) do not seem to be the target of these analogues. SM analogues of R-sphingosylphosphorylcholine type also inhibit the parasite development with IC50 of 0.3 æm for the best (analogue with a thiourea linkage). These inhibitors seem to act by inhibiting the parasite SMase. In parallel, we characterized the putative gene of the P. falciparum SMase. Beyond their chemotherapeutic potential, these analogues constitute useful tools to explore the role(s) of sphingolipid metabolism and its importance for P. falciparumPARIS-Museum Hist.Naturelle (751052304) / SudocSudocFranceF

Redescription and phylogenetic analyses of Durchoniella spp. (Ciliophora, Astomatida) associated with the polychaete Cirriformia tentaculata (Montagu, 1808)

Microscopic and phylogenetic analyses were performed on endocommensal astome ciliates retrieved from the middle intestine of a marine cirratulid polychaete, Cirriformia tentaculata, collected in the bay of Roscoff (English Channel, Northwest French coast) and on the Southwest English coast. Three morphotypes of the astome genus Durchoniella were identified, two corresponding to described species (the type species Durchoniella brasili (Léger and Duboscq, 1904) de Puytorac, 1954 and Durchoniella legeriduboscqui de Puytorac, 1954) while a third morphotype remains undescribed. Their small subunit (SSU) rRNA gene sequences showed at least 97.2% identity and phylogenetic analyses grouped them at the base of the subclass Scuticociliatia (Oligohymenophorea), as a sister lineage to all astomes from terrestrial oligochaete annelids. Ultrastructural examination by transmission electron microscopy and fluorescence in situ hybridization analyses revealed the presence of endocytoplasmic cocci and rod-shaped bacteria surrounded by a very thin membrane. These endocytoplasmic bacteria may play a role in the association between endocommensal astome ciliates and cirratulid polychaetes inhabiting in anoxic coastal sediments

A large multigene family expressed during the erythrocytic schizogony of Plasmodium falciparum

International audienceWe report the identification of a large multigene family of Plasmodium falciparum using a clone isolated with a polyclonal antiserum raised to a Babesia divergens merozoite protein. The recombinant antigen reacted with human sera collected from individuals exposed to malaria. The deduced protein sequence contains a motif homologous to the consensus sequence of merozoite rhoptry proteins encoded by multigene families in several Babesia species. Antibodies raised to the recombinant protein reacted with a 60-kDa merozoite protein both on B. divergens and on P. falciparum immunoblots. The insert hybridized to a large number of fragments on P. falciparum Southern blots and to most chromosomes of the parasite. Specifically, approx. 3-kb RNAs were detected in 4-16-nucleus schizonts. Ten distinct cDNAs were isolated that differed in the size, position and number of restriction sites in the region homologous to the original genomic clone. With about 140 copies per haploid genome, this is the first large multigene family described in malaria parasites. The existence of a multigene family encoding proteins present in the invasive stage of malaria parasites suggests an important role in invasion and denotes a significant potential for generating diversity