There has been a very limited number of high-throughput screening campaigns carried out with Leishmania drug targets. In part, this is due to the small number of suitable target genes that have been shown by genetic or chemical methods to be essential for the parasite. In this perspective, we discuss the state of genetic target validation in the field of Leishmania research and review the 200 Leishmania genes and 36 Trypanosoma cruzi genes for which gene deletion attempts have been made since the first published case in 1990. We define a quality score for the different genetic deletion techniques that can be used to identify potential drug targets. We also discuss how the advances in genome-scale gene disruption techniques have been used to assist target-based and phenotypic-based drug development in other parasitic protozoa and why Leishmania has lacked a similar approach so far. The prospects for this scale of work are considered in the context of the application of CRISPR/Cas9 gene editing as a useful ...

Genetically Validated Drug Targets in Leishmania: Current Knowledge and Future Prospects

Leishmaniases 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.

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Cell death in Leishmania.

Rice false smut fungus (teleomorph: Villosiclava virens; anamorph: Ustilaginoidea virens) can generate chlamydospores and survive winter under field conditions. The chlamydospore is considered as an important infection source of the disease. However, little is known about the regulatory mechanism of the chlamydospore production. In this study, we identified a defective homeobox transcription factor (designated as UvHOX2) gene in a U. virens random insertional mutant B-766 that could not form chlamydospores. To confirm the regulatory function of UvHOX2, an agrobacterium tumefaciens mediated transformation (ATMT)- and CRISPR/Cas9- based targeted gene replacement method was developed. The UvHox2 deletion mutants completely failed to produce chlamydospores , showed reduced conidia production and decreased virulence, and was hyper-sensitive to oxidative, osmotic, and cell wall stresses. We confirmed that UvHOX2 is located in the nulei of U. virens, and the expression of UvHox2 was the strongest during the early stage of chlamydospore and conidium formation. Global transcription pattern of UvHOX2 was also determined by RNA-seq in this study, and several genes that might be down-stream of UvHOX2 regulation were identified. The results will better our understanding of the molecular mechanism of chlamydospore formation in U. virens as a model fungus.

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A Homeobox Transcription Factor UvHOX2 Regulates Chlamydospore Formation, Conidiogenesis, and Pathogenicity in Ustilaginoidea virens.

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.

https://hal.archives-ouvertes.fr/hal-01888421/document

Temporal analysis of the autophagic and apoptotic phenotypes in Leishmania parasites

Extracellular vesicles (EVs) are minute particles secreted by the cells of living organisms. Although the functional role of EVs is not yet clear, recent work has highlighted their role in intercellular communication. Here, we expand on this view by suggesting that EVs can also mediate communication among interacting organisms such as hosts, pathogens and vectors. This inter-kingdom communication via EVs is likely to have important evolutionary consequences ranging from adaptation of parasites to specialized niches in the host, to host resistance and evolution and maintenance of parasite virulence and transmissibility. A potential system to explore these consequences is the interaction among the human host, the mosquito vector and Plasmodium parasite involved in the malaria disease. Indeed, recent studies have found that EVs derived from Plasmodium infected red blood cells in humans are likely mediating the parasite's transition from the asexual to sexual stage, which might facilitate transmission to the mosquito vector. However, more work is needed to establish the adaptive consequences of this EV signaling among different taxa. We suggest that an integrative molecular approach, including a comparative phylogenetic analysis of the molecules (e.g., proteins and nucleic acids) derived from the EVs of interacting organisms (and their closely-related species) in the malaria system will prove useful for understanding interkingdom communication. Such analyses will also shed light on the evolution and persistence of host, parasite and vector interactions, with implications for the control of vector borne infectious diseases.

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Extracellular Vesicles Could Carry an Evolutionary Footprint in Interkingdom Communication.

Metacaspases (MCAs) are cysteine peptidases expressed in plants, fungi and protozoa, with a caspase-like histidine-cysteine catalytic dyad, but differing from caspases, for example, in their substrate specificity. The role of MCAs is subject to debate: roles in cell cycle control, in cell death or even in cell survival have been suggested. In this study, using a Leishmania major MCA-deficient strain, we showed that L. major MCA (LmjMCA) not only had a role similar to caspases in cell death but also in autophagy and this through different domains. Upon cell death induction by miltefosine or H2O2, LmjMCA is processed, releasing the catalytic domain, which activated substrates via its catalytic dyad His/Cys and a proline-rich C-terminal domain. The C-terminal domain interacted with proteins, notably proteins involved in stress regulation, such as the MAP kinase LmaMPK7 or programmed cell death like the calpain-like cysteine peptidase. We also showed a new role of LmjMCA in autophagy, acting on or upstream of ATG8, involving Lmjmca gene overexpression and interaction of the C-terminal domain of LmjMCA with itself and other proteins. These results allowed us to propose two models, showing the role of LmjMCA in the cell death and also in the autophagy pathway, implicating different protein domains.

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Mariko Dacher

Papers

Implication of different domains of the Leishmania major metacaspase in cell death and autophagy