Identification of new immune-stimulators and anti-microbial compounds

La Leishmaniose est une maladie tropicale négligée que l’on retrouve dans plus de 88 pays à travers le monde. On compte par an jusqu’à 1 000 000 de nouveaux cas, menant à près de 30 000 morts. Chez l’homme, il existe plusieurs formes de la maladie, allant d’une forme cutanée pouvant guérir spontanément, à la forme viscérale la plus grave.La leishmaniose viscérale est conférée par plusieurs espèces de leishmanies, dont L. infantum dans le pourtour du bassin Méditerranéen. Ce parasite dimorphique envahit les macrophages de ses hôtes notamment l’humain et le chien où il est responsable de la maladie. En l’absence de traitement, la leishmaniose viscérale est mortelle et les traitements existants aujourd’hui sont toxiques, coûteux, et font face à l’apparition croissante de résistances.Trouver de nouveaux traitements est donc aujourd’hui une priorité, et ce projet de thèse vise à identifier des alternatives naturelles aux molécules anti-Leishmania. En prenant une approche écologique, nous récupérons des déchets de la parfumerie et des plantes issues de la biodiversité locale et nous en testons les extraits sur les parasites afin d’identifier des composés immunostimulateurs, qui permettraient de favoriser une élimination du parasite par l’hôte. Afin de pouvoir suivre l’évolution au cours du temps d’une infection in vitro et/ou in vivo, nous avons également cherché à créer de nouveaux outils pour visualiser la présence du parasite Leishmania en développant de nouvelles souches rapportrices fluorescentes et bioluminescentes.Au cours de ma thèse, nous avons identifié une plante dont l’extrait favorise l’élimination de la forme amastigote intracellulaire retrouvée dans les macrophages hôtes. A l’aide de nos collaborateurs à l’Institut de Chimie de Nice (ICN), nous avons réalisé un fractionnement bioguidé et obtenu des sous-fractions de cette plante ainsi que des molécules pures capables d’éliminer le parasite Leishmania infantum.Une seule molécule, dont le nom est soumis à confidentialité pour des raisons de dépôt de brevet, a montré un effet sur le parasite intracellulaire, sans montrer de toxicité. Cette dernière module la sécrétion de cytokines de la cellule hôte. Actuellement, l’étude de 20 dérivés structuraux de cette molécule est en cours. Des études préliminaires chez la souris ont permis de montrer que la prise de cette molécule par voie orale, de façon préventive, pouvait diminuer la charge parasitaire dans l’animal.Nous avons en parallèle développé la construction de nouvelles souches rapportrices de Leishmania exprimant la luciférase teLuc, le fluorophore rouge mRuby ou rouge lointain mMaroon1, et des souches exprimant fluorescence et bioluminescence, eFFly-mCherry. Cette dernière construction a été intégrée dans 3 espèces différentes de leishmanies : L. infantum, responsable de leishmaniose viscérale, L. major responsable de leishmaniose cutanée, et L. tarentolae qui est une souche non pathogène pour l’homme.Leishmaniasis is a neglected tropical disease found in more than 88 countries around the world. There are up to 1,000,000 new cases per year, leading to nearly 30,000 deaths. In humans, there are several forms of the disease, ranging from a cutaneous form that can heal spontaneously, to the most severe visceral form.Visceral leishmaniasis is conferred by several species of Leishmania, including L. infantum around the Mediterranean basin. This dimorphic parasite invades the macrophages of its hosts, particularly humans and dogs, in which it is responsible for the disease. Left untreated, visceral leishmaniasis is fatal, and treatments available today are toxic, expensive, and face growing resistance.Finding new treatments is therefore a priority, and this thesis project aims to identify natural alternatives to anti-leishmanial molecules. By taking an ecological approach, we recover waste from the perfumery industry and plants from the local biodiversity. We then test the extracts on the parasites in order to identify immunostimulatory compounds, which would favor promotion of elimination of the parasite by the host’s own immune system. In order to follow the evolution over time of the infection in vitro and/or in vivo, we have also sought to create new tools to visualize the presence of Leishmania parasites by developing new fluorescent and bioluminescent reporter strains.We have identified a plant whose extract promotes the elimination of the intracellular amastigote form found in host macrophages. With the help of our collaborators at the Institut de Chimie de Nice (ICN), we performed a bioguided fractioning. We obtained sub-fractions of this plant as well as pure molecules capable of eliminating the parasite Leishmania infantum.One single molecule, whose name is confidential because of a pending patent registration, showed an effect on the intracellular parasite, without showing toxicity. It is capable of modulating the secretion of cytokines from the host cell. We are currently studying 20 structural derivatives of this molecule. Preliminary studies in mice have shown that taking this molecule orally, as a preventive measure, can reduce the parasite load in animals.In the meantime, we have developed the construction of new reporter strains of Leishmania expressing the luciferase teLuc, the red mRuby or far red mMaroon1 fluorophore, and strains expressing fluorescence and bioluminescence, eFFly-mCherry. This last construction has been integrated into 3 different species of Leishmania: L. infantum, responsible for visceral leishmaniasis, L. major responsible for cutaneous leishmaniasis, and L. tarentolae, which is a non-pathogenic strain for humans

New method for screening anti-Leishmania compounds in plants extracts by HPTLC-bioautography

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Identification of adipocytes as target cells for Leishmania infantum parasites

International audienceLeishmania infantum is the causative agent of visceral leishmaniasis transmitted by the bite of female sand flies. According to the WHO, the estimated annual incidence of leishmaniasis is one million new cases, resulting in 30,000 deaths per year. The recommended drugs for treating leishmaniasis include Amphotericin B. But over the course of the years, several cases of relapses have been documented. These relapses cast doubt on the efficiency of actual treatments and raise the question of potential persistence sites. Indeed, Leishmania has the ability to persist in humans for long periods of time and even after successful treatment. Several potential persistence sites have already been identified and named as safe targets. As adipose tissue has been proposed as a sanctuary of persistence for several pathogens, we investigated whether Leishmania infantum could be found in this tissue. We demonstrated both in cell cultures and in vivo that Leishmania infantum was able to infect adipocytes. Altogether our results suggest adipocytes as a ‘safe target’ for Leishmania infantum parasites

Escherichia coli Rho GTPase-activating toxin CNF1 mediates NLRP3 inflammasome activation via p21-activated kinases-1/2 during bacteraemia in mice

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