39 research outputs found
Temporal analysis of the autophagic and apoptotic phenotypes in Leishmania parasites
The leishmaniases are worldwide neglected tropical diseases caused by parasitic protozoa of the Leishmania genus. Different stimuli induce Leishmania cell death, but the proteins involved remain poorly understood. Furthermore, confusion often appears between cell death and the cell survival process autophagy, whose phenotype is not clearly defined. In this article, we present a comprehensive and temporal analysis of the cellular events occurring during miltefosine-induced cell death and autophagy in L. major. We also provide a list of features in order to clearly identify apoptotic cells, autophagic cells and to distinguish both processes. Furthermore, we demonstrate that autophagy is followed by apoptosis in the absence of nutrients. Finally, we show that cells treated with the generic kinase inhibitor staurosporine express apoptotic as well as autophagic markers and therefore cannot be used as an apoptosis inducer in Leishmania. These descriptions lead to a better recognition and understanding of apoptosis and autophagy, enabling their targeting in the development of new anti-leishmanial drugs. These researches also make it possible to better understand these processes in general, through the study of an ancestral eukaryote
Nongenotoxic 3-Nitroimidazo[1,2-a]pyridines Are NTR1 Substrates That Display Potent in Vitro Antileishmanial Activity
Twenty nine original 3-nitroimidazo[1,2-a]pyridine derivatives, bearing a phenylthio (or benzylthio) moiety at position 8 of the scaffold, were synthesized. In vitro evaluation highlighted compound 5 as an antiparasitic hit molecule displaying low cytotoxicity for the human HepG2 cell line (CC50 > 100 mu M) alongside good antileishmanial activities (IC50 = 1-2.1 mu M) against L. donovani, L. infantum, and L. major; and good antitrypanosomal activities (IC50 = 1.3-2.2 mu M) against T. brucei brucei and T. cruzi, in comparison to several reference drugs such as miltefosine, fexinidazole, eflornithine, and benznidazole (IC50 = 0.6 to 13.3 mu M). Molecule 5, presenting a low reduction potential (E degrees = -0.63 V), was shown to be selectively bioactivated by the L. donovani type 1 nitroreductase (NTR1). Importantly, molecule 5 was neither mutagenic (negative Ames test), nor genotoxic (negative comet assay), in contrast to many other nitroaromatics. Molecule 5 showed poor microsomal stability; however, its main metabolite (sulfoxide) remained both active and nonmutagenic, making 5 a good candidate for further in vivo studies
Apoptosis and autophagy in Leishmania major
Leishmania est un protozoaire parasite de lâordre de KinĂ©toplastida de la famille des TrypanosomatidĂ©s, agent pathogĂšne des leishmanioses. Au cours de cette thĂšse nous nous sommes intĂ©ressĂ©s Ă lâapoptose chez L. major. Bien que ce processus soit phĂ©notypiquement identique Ă lâapoptose des mammifĂšres, les protĂ©ines clĂ©s et les voies mĂ©taboliques impliquĂ©es restent largement inconnues. Lâautophagie Ă©tant paradoxalement Ă©troitement liĂ©e Ă lâapoptose, nous nous sommes Ă©galement intĂ©ressĂ©s Ă ce processus de survie cellulaire. La premiĂšre partie de ce travail a permis de dĂ©crire le phĂ©notype autophagique et de montrer quâapoptose et autophagie sont deux processus distincts mais Ă©galement en lien Ă©troit. Dans la deuxiĂšme partie, nous nous sommes focalisĂ©s sur la mĂ©tacaspase de L. major LmjMCA. Nous avons observĂ© un rĂŽle de LmjMCA similaire Ă celui des caspases humaines au cours de lâapoptose, en lien avec son domaine catalytique. Nous avons montrĂ© que la surexpression de LmjMCA induit lâentrĂ©e en autophagie des cellules. Enfin, la troisiĂšme partie de ce travail sâest concentrĂ©e sur lâĂ©tude du cytosquelette. Nous avons ainsi pu Ă©tablir un lien entre (dĂ©)glutamylation, apoptose et autophagie. Nous avons mis en Ă©vidence que la polyglutamylation du cytosquelette, entraĂźne lâentrĂ©e en apoptose des cellules tandis que la dĂ©glutamylation du cytosquelette est associĂ©e au processus de survie cellulaire. Ce travail a permis de mieux caractĂ©riser les processus dâautophagie et dâapoptose chez Leishmania. Ces rĂ©sultats permettent dâouvrir des perspectives dans le dĂ©veloppement de nouveaux outils thĂ©rapeutiques.Leishmania is a protozoan parasite of the Kinetoplastida order and the Trypansomatid family and is the causative agent of leishmaniasis. In this thesis we focused on apoptosis in L. major. Although this process is phenotypically similar to mammal apoptosis, key proteins and metabolic pathways involved remain largely unknown. Autophagy being paradoxically closely linked to apoptosis, we were also interested in this cell survival process and its relationship with programmed cell death. The first part of this thesis has described the phenotypical changes during autophagy and shown that apoptosis and autophagy are two separate processes, but there is also a close relationship between these two mechanisms. In the second part of this thesis, we have demonstrated that LmjMCA has a role similar to the one of human caspases during apoptosis, through its catalytic domain. In addition, we have shown that LmjMCA overexpression induces autophagy entry after nutrient deprivation via its C-terminal domain.The third part of this work has focused on the study of the cytoskeleton. We could establish a link between (de)glutamylation, apoptosis and autophagy. Indeed, we have shown that cytoskeleton polyglutamylation induces cell death while cytoskeleton deglutamylation is associated with the cell survival process autophagy. This thesis allowed us to better characterize the autophagy and apoptosis processes in Leishmania and to identify the metacaspase as involved in the corresponding pathways. These results open perspectives in the development of new therapeutic tools
La mort cellulaire chez Leishmania
International audienceLeishmaniases still represent a global scourge and new therapeutic tools are necessary to replace the current expensive, difficult to administer treatments that induce numerous adverse effects and for which resistance is increasingly worrying. In this context, the particularly original organization of the Leishmania parasite in comparison to higher eukaryotes is a great advantage. It allows for the development of new, very specific, and thus non-cytotoxic treatments. Among these originalities, Leishmania cell death can be cited. Despite a classic pattern of apoptosis, key mammalian apoptotic proteins are not present in Leishmania, such as caspases, cell death receptors, and anti-apoptotic molecules. Recent studies have helped to develop a better understanding of parasite cell death, identifying new proteins or even new apoptotic pathways. This review provides an overview of the current knowledge on Leishmania cell death, describing its physiological roles and its phenotype, and discusses the involvement of various proteins: endonuclease G, metacaspase, aquaporin Li-BH3AQP, calpains, cysteine proteinase C, LmjHYD36 and Lmj.22.0600. From these data, potential apoptotic pathways are suggested. This review also offers tools to identify new Leishmania cell death effectors. Lastly, different approaches to use this knowledge for the development of new therapeutic tools are suggested: either inhibition of Leishmania cell death or activation of cell death for instance by treating cells with proteins or peptides involved in parasite death fused to a cell permeant peptide or encapsulated into a lipidic vector to target intra-macrophagic Leishmania cells.Alors que les leishmanioses reprĂ©sentent toujours un flĂ©au mondial, de nouveaux outils thĂ©rapeutiques sont nĂ©cessaires pour remplacer les traitements actuels qui sont chers, difficiles Ă administrer, qui induisent de nombreux effets secondaires et pour lesquels la rĂ©sistance est de plus en plus inquiĂ©tante. Pour cela, lâorganisation trĂšs originale du parasite Leishmania par rapport aux eucaryotes supĂ©rieurs est un grand avantage. En effet, cela permet le dĂ©veloppement de nouveaux traitements trĂšs spĂ©cifiques et donc non cytotoxiques. Parmi ces originalitĂ©s, la mort cellulaire de Leishmania peut ĂȘtre citĂ©e. MalgrĂ© un aspect classique dâapoptose, les protĂ©ines apoptotiques clefs des mammifĂšres ne sont pas prĂ©sentes chez Leishmania, telles que les caspases, les rĂ©cepteurs de mort et les molĂ©cules anti-apoptotiques. Des Ă©tudes rĂ©centes ont participĂ© Ă une meilleure comprĂ©hension de la mort cellulaire du parasite, identifiant de nouvelles protĂ©ines ou mĂȘme de nouvelles voies apoptotiques. Cette revue fait le point sur lâĂ©tat actuel des connaissances sur la mort cellulaire de Leishmania, dĂ©crivant ses rĂŽles physiologiques et son phĂ©notype et discutant lâimplication de diffĂ©rentes protĂ©ines : endonuclĂ©ase G, mĂ©tacaspase, aquaporine Li-BH3AQP, calpaĂŻnes, cystĂ©ine protĂ©inase C, LmjHYD36 et Lmj.22.0600. Ă partir de ces donnĂ©es, diffĂ©rentes voies apoptotiques potentielles sont suggĂ©rĂ©es. Cette revue fournit Ă©galement des outils pour identifier de nouveaux effecteurs de la mort cellulaire de Leishmania. Pour finir, diffĂ©rentes approches pour utiliser ces connaissances pour le dĂ©veloppement de nouveaux outils thĂ©rapeutiques sont suggĂ©rĂ©es : soit inhibition de la mort cellulaire de Leishmania, soit activation de celle-ci par exemple en traitant les cellules avec des protĂ©ines/peptides impliquĂ©s dans la mort du parasite fusionnĂ©s Ă un peptide pĂ©nĂ©trant dans les cellules ou encapsulĂ©s dans un vecteur lipidique pour cibler les cellules de Leishmania intra-macrophagiques
Calcein+/PI- as an early apoptotic feature in Leishmania.
Although leishmaniases are responsible for high morbidity and mortality all over the world, no really satisfying treatment exists. Furthermore, the corresponding parasite Leishmania undergoes a very characteristic form of programmed cell death. Indeed, different stimuli can induce morphological and biochemical apoptotic-like features. However, the key proteins involved in mammal apoptosis, such as caspases and death receptors, are not encoded in the genome of this parasite. Currently, little is known about Leishmania apoptosis, notably owing to the lack of specific tools for programmed cell death analysis in these parasites. Furthermore, there is a need for a better understanding of Leishmania programmed cell death in order (i) to better understand the role of apoptosis in unicellular organisms, (ii) to better understand apoptosis in general through the study of an ancestral eukaryote, and (iii) to identify new therapeutic targets against leishmaniases. To advance understanding of apoptosis in Leishmania, in this study we developed a new tool based on the quantification of calcein and propidium iodide by flow cytometry. This double labeling can be employed to distinguish early apoptosis, late apoptosis and necrosis in Leishmania live cells with a very simple and rapid assay. This paper should, therefore, be of interest for people working on Leishmania and related parasites
A potential acetyltransferase involved in Leishmania major metacaspase-dependent cell death
International audienceCurrently, there is no satisfactory treatment for leishmaniases, owing to the cost, mode of administration, side effects and to the increasing emergence of drug resistance. As a consequence, the proteins involved in Leishmania apoptosis seem a target of choice for the development of new therapeutic tools against these neglected tropical diseases. Indeed, Leishmania cell death, while phenotypically similar to mammalian apoptosis, is very peculiar, involving no homologue of the key mammalian apoptotic proteins such as caspases and death receptors. Furthermore, very few proteins involved in Leishmania apoptosis have been identified
(De)glutamylation and cell death in Leishmania parasites
International audienceTrypanosomatids are flagellated protozoan parasites that are very unusual in terms of cyto-skeleton organization but also in terms of cell death. Most of the Trypanosomatid cytoskele-ton consists of microtubules, forming different substructures including a subpellicular corset. Oddly, the actin network appears structurally and functionally different from other eukaryotic actins. And Trypanosomatids have an apoptotic phenotype under cell death conditions, but the pathways involved are devoid of key mammal proteins such as caspases or death receptors, and the triggers involved in apoptotic induction remain unknown. In this article, we have studied the role of the post-translational modifications, deglutamylation and poly-glutamylation, in Leishmania. We have shown that Leishmania apoptosis was linked to poly-glutamylation and hypothesized that the cell survival process autophagy was linked to deglutamylation. A balance seems to be established between polyglutamylation and deglu-tamylation, with imbalance inducing microtubule or other protein modifications characterizing either cell death if polyglutamylation was prioritized, or the cell survival process of autophagy if deglutamylation was prioritized. This emphasizes the role of post-translational modifications in cell biology, inducing cell death or cell survival of infectious agents
A novel hydrolase with a pro-death activity from the protozoan parasite Leishmania major
International audienceApoptosis is a cell death process generally described as involving a cascade of caspase activation, death receptors and/or pro- and antiapoptotic molecules from the BcL-2 family. But about 20 years ago, a caspase-independent apoptotic pathway has been described. Regarding this pathway, we can learn a lot from Leishmania parasites. Indeed, these parasitic protozoa enter, in response to different stimuli, in a form of cell death phenotypically similar to mammalian apoptosis but without involving caspases or death receptors. So far, only two proteins have been clearly identified as being involved in Leishmania-regulated cell death: the metacaspase and the endonuclease G. We report here the identification of a new protein modeled as a potential hydrolase, highly conserved among Leishmania species and absent in the very close parasite Trypanosoma brucei. This protein is involved in L. major-regulated cell death induced by curcumin, miltefosine and pentamidine, after gene overexpression and/or protein translocation to the nucleus. The identification of proteins involved in Leishmania-regulated cell death will provide a better understanding of nonconventional apoptotic pathways in higher eukaryotes. It will also allow the development of new therapeutic tools via the identification of new specific targets
Ăvaluation du kit de PCR en temps rĂ©el MycoGENIE Âź Pneumocystis jirovecii pour le diagnostic molĂ©culaire de la pneumocystose
International audiencePneumocystis jirovecii (Pj) est un champignon pathogĂšne opportuniste strictement humain. Il colonise transitoirement et sans symptĂŽme lâarbre respiratoire des patients immunocompĂ©tents, et peut ĂȘtre responsable dâinfections pulmonaires sĂ©vĂšres chez les patients immunodĂ©primĂ©s. Le diagnostic biologique de pneumocystose repose sur la mise en Ă©vidence du pathogĂšne Ă lâexamen direct dâun prĂ©lĂšvement pulmonaire profond. Si lâexamen direct manque souvent de sensibilitĂ©, de nombreuses techniques de PCR se sont dĂ©veloppĂ©es pour amĂ©liorer la sensibilitĂ© de dĂ©tection du champignon. Les techniques de PCR quantitatives (qPCR) permettent de quantifier la charge fongique dans le prĂ©lĂšvement, et potentiellement de diffĂ©rencier la colonisation de lâarbre respiratoire par Pj dâune vĂ©ritable infection gĂ©nĂ©ralement associĂ©e Ă une charge fongique plus Ă©levĂ©e.Lâobjectif de notre travail Ă©tait dâĂ©valuer les performances du kit MycoGENIEÂź P. jirovecii (Ademtech) dans le diagnostic de la pneumocystose en comparaison avec la technique de qPCR utilisĂ©e en routine au laboratoire de parasitologie-mycologie du CHU de Dijon.Soixante-dix-huit Ă©chantillons respiratoires (72 LBA, 3 ATP, 3 crachats) ont Ă©tĂ© testĂ©s en parallĂšle : 46 Ă©chantillons rĂ©trospectifs (prĂ©levĂ©s entre janvier 2016 et juin 2016 et conservĂ©s Ă â80 °C) et 32 Ă©chantillons prospectifs prĂ©levĂ©s entre juillet 2016 et septembre 2016. Les 78 Ă©chantillons ont Ă©tĂ© extraits par lâautomate Magtration System 12GCÂź (Bionobis). Les deux techniques de PCR utilisĂ©es dans notre Ă©tude ciblaient la mĂȘme sĂ©quence dâADN de 79 pb du gĂšne mitochondrial codant la grosse sous-unitĂ© de lâARN ribosomal (mtLSU rRNA). Les PCR ont Ă©tĂ© rĂ©alisĂ©es sur lâautomate LC480 II (Roche Diagnostics). Le test MycoGENIEÂź Pj contenait une gamme Ă©talon permettant de quantifier la charge fongique du prĂ©lĂšvement en nombre de copies/mL ainsi quâun contrĂŽle interne dâinhibition. Le kit est marquĂ© CE-IVD.Sur les 46 Ă©chantillons testĂ©s rĂ©trospectivement, 36 Ă©taient positifs et 9 Ă©taient nĂ©gatifs par les deux techniques. Un seul rĂ©sultat Ă©tait discordant (nĂ©gatif avec la technique de notre laboratoire et positif avec le kit MycoGENIEÂź Pj). Les rĂ©sultats des 32 Ă©chantillons testĂ©s prospectivement Ă©taient concordants (12 positifs et 20 nĂ©gatifs). La concordance entre les 2 tests est donc de 98,7 %. Aucun inhibiteur nâa Ă©tĂ© dĂ©tectĂ© avec le kit MycoGENIEÂź Pj. La dĂ©tection de lâADN a Ă©tĂ© plus prĂ©coce pour lâensemble des Ă©chantillons positifs avec le kit MycoGENIEÂź Pj, avec un seuil de dĂ©tection plus prĂ©coce en moyenne de 1,2 cycle. Le rĂ©sultat discordant concernait un patient atteint dâune leucĂ©mie aiguĂ« myĂ©loblastique en aplasie post-inductive intensive pour lequel la radiologie et la clinique orientaient le diagnostic vers une pneumopathie interstitielle hypoxĂ©miante et bilatĂ©rale.La quantification de lâADN nâa pas permis de dĂ©finir de seuil pour diffĂ©rencier pneumocystose maladie et colonisation : des taux trĂšs faibles ont Ă©tĂ© retrouvĂ©s chez des patients dont le diagnostic retenu Ă©tait une pneumocystose ; Ă lâinverse, des taux Ă©levĂ©s dâADN de Pj Ă©taient observĂ©s chez des patients considĂ©rĂ©s comme colonisĂ©s. Dâautres Ă©lĂ©ments comme le terrain, lâaspect radiologique, la clinique et dâautres marqueurs biologiques (ex. ÎČ-glucanes) devraient ĂȘtre pris en compte pour prĂ©ciser le diagnostic de pneumocystose ou de colonisation Ă Pj