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This work was supported by the International Division of the Institute Pasteur (ACIP A13-2013 project), the Action “KRIPIS I” (MIS 450598) co-financed by European Union and the National Ministry of Education and Religion Affairs under the Operational Strategic Reference Framework (NSRF 2007-2013) and the General Secretariat of Research and Technology (GSRT) and FAPESB/CAPES grant # PET0042/2013 from the Brazilian State and Federal governments respectively. Although the multiplicative and growth‐arrested states play key roles in Leishmania development, the regulators of these transitions are largely unknown. In an attempt to gain a better understanding of these processes, we characterised one member of a family of protein kinases with dual specificity, LinDYRK1, which acts as a stasis regulator in other organisms. LinDYRK1 over‐expressing parasites displayed a decrease in proliferation and in cell cycle re‐entry of arrested cells. Parasites lacking LinDYRK1 displayed distinct fitness phenotypes in logarithmic and stationary growth phases. In logarithmic growth‐phase, LinDYRK1‐/‐ parasites proliferated better than control lines, supporting a role of this kinase in stasis, while in stationary growth‐phase, LinDYRK1‐/‐ parasites had important defects as they rounded up, accumulated vacuoles and lipid bodies and displayed subtle but consistent differences in lipid composition. Moreover, they expressed less metacyclic‐enriched transcripts, displayed increased sensitivity to complement lysis and a significant reduction in survival within peritoneal macrophages. The distinct LinDYRK1‐/‐ growth phase phenotypes were mirrored by the distinct LinDYRK1 localisations in logarithmic (mainly in flagellar pocket area and endosomes) and late stationary phase (mitochondrion). Overall, this work provides first evidence for the role of a DYRK family member in sustaining promastigote stationary phase phenotype and infectivity. Postprint

Summary: Aberrant macrophage activation during intracellular infection generates immunopathologies that can cause severe human morbidity. A better understanding of immune subversion strategies and macrophage phenotypic and functional responses is necessary to design host-directed intervention strategies. Here, we uncover a fine-tuned transcriptional response that is induced in primary and lesional macrophages infected by the parasite Leishmania amazonensis and dampens NF-κB and NLRP3 inflammasome activation. Subversion is amastigote-specific and characterized by a decreased expression of activating and increased expression of de-activating components of these pro-inflammatory pathways, thus revealing a regulatory dichotomy that abrogates the anti-microbial response. Changes in transcript abundance correlate with histone H3K9/14 hypoacetylation and H3K4 hypo-trimethylation in infected primary and lesional macrophages at promoters of NF-κB-related, pro-inflammatory genes. Our results reveal a Leishmania immune subversion strategy targeting host cell epigenetic regulation to establish conditions beneficial for parasite survival and open avenues for host-directed, anti-microbial drug discovery. : Lecoeur et al. demonstrate that the parasite Leishmania amazonensis modifies expression of NF-kB-related genes and prevents NLRP3 inflammasome activation of host macrophages in vitro and in vivo by changing histone H3 modifications at the promotor of pro-inflammatory genes. This mechanism of immune subversion opens avenues for host-directed, anti-leishmanial therapies. Keywords: Leishmania amazonensis, amastigotes, lesional macrophage, NF-κB, NLRP3 inflammasome, H3 acetylation, H3 methylation, epigenetics, histone

International audience; The parasite Leishmania~ donovani causes a fatal disease termed visceral leishmaniasis. The process through which the parasite adapts to environmental change remains largely unknown. Here we show that aneuploidy is integral for parasite adaptation and that karyotypic fluctuations allow for selection of beneficial haplotypes, which impact transcriptomic output and correlate with phenotypic variations in proliferation and infectivity. To avoid loss of diversity following karyotype and haplotype selection, L. donovani utilizes two mechanisms: polyclonal selection of beneficial haplotypes to create coexisting subpopulations that preserve the original diversity, and generation of new diversity as aneuploidy-prone chromosomes tolerate higher mutation rates. Our results reveal high aneuploidy turnover and haplotype selection as a unique evolutionary adaptation mechanism that L. donovani uses to preserve genetic diversity under strong selection. This unexplored process may function in other human diseases, including fungal infection and cancer, and stimulate innovative treatment options.

International audience; All eukaryotic genomes encode multiple members of the heat shock protein 70 (HSP70) family, which evolved distinctive structural and functional features in response to specific environmental constraints. Phylogenetic analysis of this protein family thus can inform on genetic and molecular mechanisms that drive species-specific environmental adaptation. Here we use the eukaryotic pathogen Leishmania spp. as a model system to investigate the evolution of the HSP70 protein family in an early-branching eukaryote that is prone to gene amplification and adapts to cytotoxic host environments by stress-induced and chaperone-dependent stage differentiation. Combining phylogenetic and comparative analyses of trypanosomatid genomes, draft genome of Paratrypanosoma and recently published genome sequences of 204 L. donovani field isolates, we gained unique insight into the evolutionary dynamics of the Leishmania HSP70 protein family. We provide evidence for (i) significant evolutionary expansion of this protein family in Leishmania through gene amplification and functional specialization of highly conserved canonical HSP70 members, (ii) evolution of trypanosomatid-specific, non-canonical family members that likely gained ATPase-independent functions, and (iii) loss of one atypical HSP70 member in the Trypanosoma genus. Finally, we reveal considerable copy number variation of canonical cytoplasmic HSP70 in highly related L. donovani field isolates, thus identifying this locus as a potential hot spot of environment-genotype interaction. Our data draw a complex picture of the genetic history of HSP70 in trypanosomatids that is driven by the remarkable plasticity of the Leishmania genome to undergo massive intra-chromosomal gene amplification to compensate for the absence of regulated transcriptional control in these parasites.

International audience; Staphylococcus aureus is one of the versatile Gram positive bacteria causing a range of diseases. Upon challenge, host immune cells recognize S. aureus and mount diverse immune responses including production of pro-inflammatory cytokines such as IL-1β and TNF-α. These cytokines are important mediators of inflammation which can be detected via various immunological methods such as enzyme linked immunosorbent assay (ELISA) and immunoblotting. In the current study, we found that a number of clinical isolates as well as laboratory strains of S. aureus exhibited cross reactivity with ELISA antibodies for murine IL-1β and TNF-α assays. This cross reactivity generates exaggerated false positive signals which can be a source of discrepancy for the understanding of real immune responses against S. aureus infection by host immune cells.

Existing therapies for leishmaniases present significant limitations, such as toxic side effects, and are rendered inefficient by parasite resistance. It is of utmost importance to develop novel drugs targeting Leishmania that take these two limitations into consideration. We thus chose a target-based approach using an exoprotein kinase, Leishmania casein kinase 1.2 (LmCK1.2) that was recently shown to be essential for intracellular parasite survival and infectivity. We developed a four-step pipeline to identify novel selective antileishmanial compounds. In step 1, we screened 5,018 compounds from kinase-biased libraries with Leishmania and mammalian CK1 in order to identify hit compounds and assess their specificity. For step 2, we selected 88 compounds among those with the lowest 50% inhibitory concentration to test their biological activity on host-free parasites using a resazurin reduction assay and on intramacrophagic amastigotes using a high content phenotypic assay. Only 75 compounds showed antileishmanial activity and were retained for step 3 to evaluate their toxicity against mouse macrophages and human cell lines. The four compounds that displayed a selectivity index above 10 were then assessed for their affinity to LmCK1.2 using a target deconvolution strategy in step 4. Finally, we retained two compounds, PP2 and compound 42, for which LmCK1.2 seems to be the primary target. Using this four-step pipeline, we identify from several thousand molecules, two lead compounds with a selective antileishmanial activity.

International audience; Upon infection, parasites secrete effector molecules particularly kinases, that modify their host cells to create a permissive environment. For instance, Leishmania secretes casein kinase 1.2 (L-CK1.2) into the host cell via exosomes. Previous studies in the lab demonstrated that L-CK1.2 interacts with and phosphorylates more than 200 host proteins in vitro. Moreover, L-CK1.2 when ectopically expressed alters the phosphorylation of 771 proteins in J774 macrophages. Pathway analysis of these host targets indicated that L-CK1.2 might have an impact on a multitude of host pathways, including a few known to be modified during Leishmania infection. During Eimeriatenella infection, rhoptry kinase 1 (EtROP1) inhibits host apoptosis by interacting with host p53. On comparison, it appears that these two exo-kinases from divergent parasites, impact similar host pathways such as apoptosis and cell cycle. Our hypothesis states that there might exist convergence in the host pathways modified by Leishmania and Eimeria. To investigate this hypothesis, we will perform phosphoproteomic and transcriptomic studies. For Leishmania, we have just established the first ever phosphoproteome for L donovani infected mouse BMDMs. We quantified about 12,000 phosphosites comparing infected versus uninfected, including 663 sites with a p-value below 0.05. In order to uncover L-CK1.2 specific effects, we also had infected cells treated or untreated with the L-CK1.2 inhibitor, D4476. Further analysis is ongoing. For E. tenella, chicken lung epithelial cells (CLEC213) were infected with either wild type or EtROP1 overexpressing parasites; the samples were collected and will soon be sent for transcriptomic analysis. Once we have the complete set, including the phosphoproteome for Eimeria-infected CLEC213 cells and the transcriptome for L. donovani-infected macrophages, we will then assess which pathways are similarly or differently modified during both infections and decipher their role by interfering with important genes of these pathways using CRISPR-Cas knockout, RNAi or overexpression.

International audience; Leishmaniasis is a severe public health problem, caused by the protozoan Leishmania . This parasite has two developmental forms, extracellular promastigote in the insect vector and intracellular amastigote in the mammalian host where it resides inside the phagolysosome of macrophages. Little is known about the virulence factors that regulate host-pathogen interactions and particularly host signalling subversion. All the proteomes of Leishmania extracellular vesicles identified the presence of Leishmania casein kinase 1 (L-CK1.2), a signalling kinase. L-CK1.2 is essential for parasite survival and thus might be essential for host subversion. To get insights into the functions of L-CK1.2 in the macrophage, the systematic identification of its host substrates is crucial, we thus developed an easy method to identify substrates, combining phosphatase treatment, in vitro kinase assay and Stable Isotope Labelling with Amino acids in Cell (SILAC) culture-based mass spectrometry. Implementing this approach, we identified 225 host substrates as well as a potential novel phosphorylation motif for CK1. We confirmed experimentally the enrichment of our substratome in bona fide L-CK1.2 substrates and showed they were also phosphorylated by human CK1δ. L-CK1.2 substratome is enriched in biological processes such as “viral and symbiotic interaction,” “actin cytoskeleton organisation” and “apoptosis,” which are consistent with the host pathways modified by Leishmania upon infection, suggesting that L-CK1.2 might be the missing link. Overall, our results generate important mechanistic insights into the signalling of host subversion by these parasites and other microbial pathogens adapted for intracellular survival.

Genome instability has been recognized as a key driver for microbial and cancer adaptation and thus plays a central role in many human pathologies. Even though genome instability encompasses different types of genomic alterations, most available genome analysis software are limited to just one kind mutation or analytical step. To overcome this limitation and better understand the role of genetic changes in enhancing pathogenicity we established GIP, a novel, powerful bioinformatic pipeline for comparative genome analysis. Here we show its application to whole genome sequencing datasets of Leishmania, Plasmodium, Candida, and cancer. Applying GIP on available data sets validated our pipeline and demonstrated the power of our analysis tool to drive biological discovery. Applied to Plasmodium vivax genomes, our pipeline allowed us to uncover the convergent amplification of erythrocyte binding proteins and to identify a nullisomic strain. Re-analyzing genomes of drug adapted Candida albicans strains revealed correlated copy number variations of functionally related genes, strongly supporting a mechanism of epistatic adaptation through interacting gene-dosage changes. Our results illustrate how GIP can be used for the identification of aneuploidy, gene copy number variations, changes in nucleic acid sequences, and chromosomal rearrangements. Altogether, GIP can shed light on the genetic bases of cell adaptation and drive disease biomarker discovery.One Sentence SummaryGIP - a novel pipeline for detecting, comparing and visualizing genome instability.

How genome instability is harnessed for fitness gain despite its potential deleterious effects is largely elusive. An ideal system to address this important open question is provided by the protozoan pathogen Leishmania, which exploits frequent variations in chromosome and gene copy number to regulate expression levels. Using ecological genomics and experimental evolution approaches we provide first evidence that Leishmania adaptation relies on epistatic interactions between functionally associated gene copy number variations in pathways driving fitness gain in a given environment. We further uncover post-transcriptional regulation as a key mechanism that compensates for deleterious gene dosage effects and provides phenotypic robustness to genetically heterogenous parasite populations. Finally, we correlate dynamic variations in snoRNA gene dosage with changes in rRNA 2’-O-methylation and pseudouridylation, suggesting translational control is an additional layer of parasite adaptation. Leishmania genome instability is thus harnessed for fitness gain by genome-dependent variations in gene expression, and genome-independent, compensatory mechanisms. This allows for polyclonal adaptation and maintenance of genetic heterogeneity despite strong selective pressure. The epistatic adaptation described here needs to be considered in Leishmania epidemiology and biomarker discovery, and may be relevant to other fast evolving, eukaryotic cells that exploit genome instability for adaptation, such as fungal pathogens or cancer.One Sentence SummaryEpistatic interactions harness genome instability for Leishmania fitness gain.

International audience; The efficient manipulation of their host cell is an essential feature of intracellular parasites. Most molecular mechanisms governing the subversion of host cell by protozoan parasites involve the release of parasite-derived molecules into the host cell cytoplasm and direct interaction with host proteins. Among these released proteins, kinases are particularly important as they govern the subversion of important host pathways, such as signalling or metabolic pathways. These enzymes, which catalyse the transfer of a phosphate group from ATP onto serine, threonine, tyrosine or histidine residues to covalently modify proteins, are involved in numerous essential biological processes such as cell cycle or transport. Although little is known about the role of most of the released parasite-derived kinases in the host cell, they are examples of kinases hijacking host cellular pathways such as signal transduction or apoptosis, which are essential for immune response evasion as well as parasite survival and development. Here we present the current knowledge on released protozoan kinases and their involvement in host-pathogen interactions. We also highlight the knowledge gaps remaining before considering those kinases - involved in host signalling subversion - as antiparasitic drug targets.

International audience; Intracellular parasites have evolved intricate strategies to subvert host cell functions for their own survival. These strategies are particularly damaging to the host if the infection involves immune cells, as illustrated by protozoan parasites of the genus Leishmania that thrive inside mononuclear phagocytic cells, causing devastating immunopathologies. While the impact of Leishmania infection on host cell phenotype and functions has been well documented, the regulatory mechanisms underlying host cell subversion were only recently investigated. Here we summarize the current knowledge on how Leishmania infection affects host nuclear activities and propose thought-provoking new concepts on the reciprocal relationship between epigenetic and transcriptional regulation in host cell phenotypic plasticity, its potential subversion by the intracellular parasite, and its relevance for host-directed therapy.

Algeria ranks second after Afghanistan for the incidence of cutaneous leishmaniasis (CL) worldwide. Here, we report a 34-years retrospective analysis of CL in Algeria and focused on the most affected region, the M’Sila province. All 66 cutaneous isolates corresponded to Leishmania (L.) major. Our study of the sandfly and rodent fauna further highlighted the high density of Phlebotomus papatasi and additional phlebotomine species of medical importance, not previously identified in M’Sila. Wild rodents belonging to nine species were trapped in M’Sila, and Psammomys obesus and Meriones shawi were found infected by L. major. In addition, Leishmania infantum was isolated from two visceral leishmaniasis cases, one dog and its proven vectors (P. perniciosus, P. longicuspis, and P. perfiliewi) inventoried during the survey. The high incidence of CL in the M’Sila province is likely a consequence of the increase in minimum temperatures recorded that constitutes suitable conditions for establishing a high endemicity and leads to an explosive rise in leishmaniases cases in this region. A thorough investigation of the underlying risk factors is urgently needed to detect new cases earlier. All these would improve the preparedness to fight the disease.

The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network driving parasite fitness, with genome instability causing highly reproducible, gene dosage-dependent changes in protein linked to post-transcriptional regulation. These in turn were associated with a gene dosage-independent reduction in flagellar transcripts and a coordinated increase in abundance of coding and non-coding RNAs known to regulate ribosomal biogenesis and protein translation. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain, where differential regulation of mRNA stability and the generation of fitness-adapted ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania non-coding small RNome as a potential novel source for biomarker discovery.

International audience; Casein Kinase 1 (CK1) family members are serine/threonine protein kinases that are involved in many biological processes and highly conserved in eukaryotes from protozoan to humans. Even though pathogens exploit host CK1 signaling pathways to survive, the role of CK1 in infectious diseases and host/pathogen interaction is less well characterized compared to other diseases, such as cancer or neurodegenerative diseases. Here we present the current knowledge on CK1 in protozoan parasites highlighting their essential role for parasite survival and their importance for host-pathogen interactions. We also discuss how the dual requirement of CK1 family members for parasite biological processes and host subversion could be exploited to identify novel antimicrobial interventions.

International audience; Chalcones (1,3-diphenyl-2-propen-1-ones) either natural or synthetic have a plethora of biological properties including antileishmanial activities, but their development as drugs is hampered by their largely unknown mechanisms of action. We demonstrate herein that our previously described benzochalcone fluorogenic probe (HAB) could be imaged by fluorescence microscopy in live Leishmania amazonensis promastigotes where it targeted the parasite acidocalcisomes, lysosomes and the mitochondrion. As in the live zebrafish model, HAB formed yellow-emitting fluorescent complexes when associated with biological targets in Leishmania. Further, we used HAB as a reversible probe to study the binding of a portfolio of diverse chalcones and analogues in live promastigotes, using a combination of competitive flow cytometry analysis and cell microscopy. This pharmacological evaluation suggested that the binding of HAB in promastigotes was representative of chalcone pharmacology in Leishmania, with certain exogenous chalcones exhibiting competitive inhibition (ca. 20-30%) towards HAB whereas non-chalconic inhibitors showed weak capacity (ca. 3-5%) to block the probe intracellular binding. However, this methodology was restricted by the strong toxicity of several competing chalcones at high concentration, in conjunction with the limited sensitivity of the HAB fluorophore. This advocates for further optimization of this undirect target detection strategy using pharmacophore-derived reversible fluorescent probes.

Leishmania infantum causes visceral leishmaniasis, a deadly vector-borne disease introduced to the Americas during the colonial era. This non-native trypanosomatid parasite has since established widespread transmission cycles using alternative vectors, and human infection has become a significant concern to public health, especially in Brazil. A multi-kilobase deletion was recently detected in Brazilian L. infantum genomes and is suggested to reduce susceptibility to the anti-leishmanial drug miltefosine. We show that deletion-carrying strains occur in at least 15 Brazilian states and describe diversity patterns suggesting that these derive from common ancestral mutants rather than from recurrent independent mutation events. We also show that the deleted locus and associated enzymatic activity is restored by hybridization with non-deletion type strains. Genetic exchange appears common in areas of secondary contact but also among closely related parasites. We examine demographic and ecological scenarios underlying this complex L. infantum population structure and discuss implications for disease control., Philipp Schwabl, Mariana Boité, and colleagues analyze 126 Leishmania infantum genomes to determine how demographic and selective consequences of the parasite’s invasive history have contributed to intricate population genetic heterogeneity across Brazil. Their data suggest a complex interplay of population expansion, secondary contact and genetic exchange events underlying diversity patterns at short and long-distance scales. These processes also appear pivotal to the proliferation of a drug resistance-associated multi-gene deletion on chromosome 31.

Summary Nitric oxide (NO) is an important antimicrobial effector but also prevents unnecessary tissue damage by shutting down the recruitment of monocyte-derived phagocytes. Intracellular pathogens such as Leishmania major can hijack these cells as a niche for replication. Thus, NO might exert containment by restricting the availability of the cellular niche required for efficient pathogen proliferation. However, such indirect modes of action remain to be established. By combining mathematical modeling with intravital 2-photon biosensors of pathogen viability and proliferation, we show that low L. major proliferation results not from direct NO impact on the pathogen but from reduced availability of proliferation-permissive host cells. Although inhibiting NO production increases recruitment of these cells, and thus pathogen proliferation, blocking cell recruitment uncouples the NO effect from pathogen proliferation. Therefore, NO fulfills two distinct functions for L. major containment: permitting direct killing and restricting the supply of proliferation-permissive host cells., Graphical abstract, Highlights • Direct killing of L. major by NO occurs only during the peak of the immune response • Efficient L. major proliferation requires newly recruited monocyte-derived cells • Loss of NO production increases both pathogen proliferation and monocyte recruitment • NO dampens L. major proliferation indirectly, limiting the pathogen’s cellular niche, Besides direct antimicrobial activity, nitric oxide (NO) can inhibit the entry of inflammatory cells into infected tissues. Intracellular pathogens can hijack such cells as niches for proliferation. Formaglio et al. show that restriction of proliferation-permissive host cell recruitment by NO represents a mechanism that controls Leishmania major infection.

Background One of the major challenges to leishmaniasis treatment is the emergence of parasites resistant to antimony. To study differentially expressed genes associated with drug resistance, we performed a comparative transcriptomic analysis between wild-type and potassium antimonyl tartrate (SbIII)-resistant Leishmania infantum lines using high-throughput RNA sequencing. Methods All the cDNA libraries were constructed from promastigote forms of each line, sequenced and analyzed using STAR for mapping the reads against the reference genome (L. infantum JPCM5) and DESeq2 for differential expression statistical analyses. All the genes were functionally annotated using sequence similarity search. Results The analytical pipeline considering an adjusted p-value < 0.05 and fold change > 2.0 identified 933 transcripts differentially expressed (DE) between wild-type and SbIII-resistant L. infantum lines. Out of 933 DE transcripts, 504 presented functional annotation and 429 were assigned as hypothetical proteins. A total of 837 transcripts were upregulated and 96 were downregulated in the SbIII-resistant L. infantum line. Using this DE dataset, the proteins were further grouped in functional classes according to the gene ontology database. The functional enrichment analysis for biological processes showed that the upregulated transcripts in the SbIII-resistant line are associated with protein phosphorylation, microtubule-based movement, ubiquitination, host–parasite interaction, cellular process and other categories. The downregulated transcripts in the SbIII-resistant line are assigned in the GO categories: ribonucleoprotein complex, ribosome biogenesis, rRNA processing, nucleosome assembly and translation. Conclusions The transcriptomic profile of L. infantum showed a robust set of genes from different metabolic pathways associated with the antimony resistance phenotype in this parasite. Our results address the complex and multifactorial antimony resistance mechanisms in Leishmania, identifying several candidate genes that may be further evaluated as molecular targets for chemotherapy of leishmaniasis. Graphical Abstract

Although several studies have investigated genetic diversity ofLeishmania infantumin North Africa, genome-wide analyses are lacking. Here, we conducted comparative analyses of nuclear and mitochondrial genomes of sevenL.infantumisolates from Tunisia with the aim to gain insight into factors that drive genomic and phenotypic adaptation. Isolates were from cured (n=4) and recurrent (n=3) visceral leishmaniasis (VL) cases, originating from northern (n=2) and central (n=5) Tunisia, where respectively stable and emerging VL foci are observed. All isolates from relapsed patients were from Kairouan governorate (Centre); one showing resistance to the anti-leishmanial drug Meglumine antimoniate. Nuclear genome diversity of the isolates was analysed by comparison to theL. infantumJPCM5 reference genome. Kinetoplast maxi and minicircle sequences (1 and 59, respectively) were extracted from unmapped reads and identified byblastanalysis against public data sets. The genome variation analysis grouped together isolates from the same geographical origins. Strains from the North were very different from the reference showing more than 34 587 specific single nucleotide variants, with one isolate representing a full genetic hybrid as judged by variant frequency. Composition of minicircle classes within isolates corroborated this geographical population structure. Read depth analysis revealed several significant gene copy number variations correlating with either geographical origin (amastin and Hsp33 genes) or relapse (CLN3 gene). However, no specific gene copy number variation was found in the drug-resistant isolate. In contrast, resistance was associated with a specific minicircle pattern suggestingLeishmaniamitochondrial DNA as a potential novel source for biomarker discovery.

International audience; Leishmania parasites are the causative agents of human leishmaniases. They infect professional phagocytes of their mammalian hosts, including dendritic cells (DCs) that are essential for the initiation of adaptive immune responses. These immune functions strictly depend on the DC's capacity to differentiate from immature, antigen-capturing cells to mature, antigen-presenting cells-a process accompanied by profound changes in cellular phenotype and expression profile. Only little is known on how intracellular Leishmania affects this important process and DC transcriptional regulation. Here, we investigate these important open questions analyzing phenotypic, cytokine profile and transcriptomic changes in murine, immature bone marrow-derived DCs (iBMDCs) infected with antibody-opsonized and non-opsonized Leishmania amazonensis (L.am) amastigotes. DCs infected by non-opsonized amastigotes remained phenotypically immature whereas those infected by opsonized parasites displayed a semi-mature phenotype. The low frequency of infected DCs in culture led us to use DsRed2-transgenic parasites allowing for the enrichment of infected BMDCs by FACS. Sorted infected DCs were then subjected to transcriptomic analyses using Affymetrix GeneChip technology. Independent of parasite opsonization, Leishmania infection induced expression of genes related to key DC processes involved in MHC Class I-restricted antigen presentation and alternative NF-κB activation. DCs infected by non-opsonized parasites maintained an immature phenotype and showed a small but significant down-regulation of gene expression related to pro-inflammatory TLR signaling, the canonical NF-kB pathway and the NLRP3 inflammasome. This transcriptomic profile was further enhanced in Lecoeur et al. DC Subversion by Leishmania Amazonensis DCs infected with opsonized parasites that displayed a semi-mature phenotype despite absence of inflammasome activation. This paradoxical DC phenotype represents a Leishmania-specific signature, which to our knowledge has not been observed with other opsonized infectious agents. In conclusion, systems-analyses of our transcriptomics data uncovered important and previously unappreciated changes in the DC transcription factor landscape, thus revealing a novel Leishmania immune subversion strategy directly acting on transcriptional control of gene expression. Our data raise important questions on the dynamic and reciprocal interplay between transacting and epigenetic regulators in establishing permissive conditions for intracellular Leishmania infection and polarization of the immune response.

International audience; Leishmania spp are obligate intracellular parasites that infect phagocytes, notably macrophages. No information is available on how Leishmania parasites respond to pyroptosis of their host cell, known to limit microbial infection. Here, we analyzed the pyroptotic process and the fate of intracellular amastigotes at the single cell level using high-content, real-time imaging. Bone marrow-derived macrophages were infected with virulent L. amazonensis amastigotes and sequentially treated with lipopolysaccharide and adenosine triphosphate for pyroptosis induction. Real-time monitoring identified distinct pyroptotic phases, including rapid decay of the parasitophorous vacuole (PV), progressive cell death, and translocation of the luminal PV membrane to the cell surface in 40% of macrophages, resulting in the extracellular exposure of amastigotes that remained anchored to PV membranes. Electron microscopy analyses revealed an exclusive polarized orientation of parasites, with the anterior pole exposed toward the extracellular milieu, and the parasite posterior pole attached to the PV membrane. Exposed parasites retain their full infectivity towards naïve macrophages suggesting that host cell pyroptosis may contribute to parasite dissemination.

Protozoan parasites of the genus Leishmania are the causative agents of leishmaniasis, a spectrum of a disease that threatens public health worldwide. Although next-generation therapeutics are urgently needed, the early stage of the drug discovery process is hampered by very low hit rates from intracellular Leishmania phenotypic high-throughput screenings. Designing and applying a physiologically relevant in vitro assay is therefore in high demand. In this study, we characterized the infectivity, morphology, and drug susceptibility of different Leishmania and host cell infection combinations. Primary bone marrow-derived macrophage (BMDM) and differentiated human acute monocytic leukemia (THP-1) cells were infected with amastigote or promastigote forms of Leishmania amazonensis and Leishmania donovani. Regardless of host cell types, amastigotes were generally well phagocytosed and showed high infectivity, whereas promastigotes, especially those of L. donovani, had predominantly remained in the extracellular space. In the drug susceptibility test, miltefosine and sodium stibogluconate (SSG) showed varying ranges of activity with 14 and &gt, 10-fold differences in susceptibility, depending on the host-parasite pairs, indicating the importance of assay conditions for evaluating antileishmanial activity. Overall, our results suggest that combinations of Leishmania species, infection forms, and host cells must be carefully optimized to evaluate the activity of potential therapeutic compounds against Leishmania.

Casein Kinase 1 (CK1) family members are serine/threonine protein kinases ubiquitously expressed in eukaryotic organisms. They are involved in a wide range of important cellular processes, such as membrane trafficking, or vesicular transport in organisms from yeast to humans. Due to its broad spectrum of action, CK1 activity and expression is tightly regulated by a number of mechanisms, including subcellular sequestration. Defects in CK1 regulation, localisation or the introduction of mutations in the CK1 coding sequence are often associated with important diseases such as cancer. Increasing evidence suggest that the manipulation of host cell CK1 signalling pathways by intracellular pathogens, either by exploiting the host CK1 or by exporting the CK1 of the pathogen into the host cell may play an important role in infectious diseases. Leishmania CK1.2 is essential for parasite survival and released into the host cell, playing an important role in host pathogen interactions. Although Leishmania CK1.2 has dual role in the parasite and in the host cell, nothing is known about its parasitic localisation and organelle-specific functions. In this study, we show that CK1.2 is a ubiquitous kinase, which is present in the cytoplasm, associated to the cytoskeleton and localised to various organelles, indicating potential roles in kinetoplast and nuclear segregation, as well as ribosomal processing and motility. Furthermore, using truncated mutants, we show for the first time that the two low complexity regions (LCR) present in the C-terminus of CK1.2 are essential for the subcellular localisation of CK1.2 but not for its kinase activity, whereas the deletion of the N-terminus leads to a dramatic decrease in CK1.2 abundance. In conclusion, our data on the localisation and regulation of Leishmania CK1.2 contribute to increase the knowledge on this essential kinase and get insights into its role in the parasite.

International audience; Leishmaniases are major vector-borne tropical diseases responsible for great human morbidity and mortality, caused by protozoan, trypanosomatid parasites of the genus Leishmania. In the mammalian host, parasites survive and multiply within mononuclear phagocytes, especially macrophages. However, the underlying mechanisms by which Leishmania spp. affect their host are not fully understood. Herein, proteomic alterations of primary, bone marrow-derived BALB/c macrophages are documented after 72 h of infection with Leishmania donovani insect-stage promastigotes, applying a SILAC-based, quantitative proteomics approach. The protocol was optimised by combining strong anion exchange and gel electrophoresis fractionation that displayed similar depth of analysis (combined total of 6189 mouse proteins). Our analyses revealed 86 differentially modulated proteins (35 showing increased and 51 decreased abundance) in response to Leishmania donovani infection. The proteomics results were validated by analysing the abundance of selected proteins. Intracellular Leishmania donovani infection led to changes in various host cell biological processes, including primary metabolism and catabolic process, with a significant enrichment in lysosomal organisation. Overall, our analysis establishes the first proteome of bona fide primary macrophages infected ex vivo with Leishmania donovani, revealing new mechanisms acting at the host/pathogen interface. SIGNIFICANCE: Little is known on proteome changes that occur in primary macrophages after Leishmania donovani infection. This study describes a SILAC-based quantitative proteomics approach to characterise changes of bone marrow-derived macrophages infected with L. donovani promastigotes for 72 h. With the application of SILAC and the use of SAX and GEL fractionation methods, we have tested new routes for proteome quantification of primary macrophages. The protocols developed here can be applicable to other diseases and pathologies. Moreover, this study sheds important new light on the "proteomic reprogramming" of infected macrophages in response to L. donovani promastigotes that affects primary metabolism, cellular catabolic processes, and lysosomal/vacuole organisation. Thus, our study reveals key molecules and processes that act at the host/pathogen interface that may inform on new immuno- or chemotherapeutic interventions to combat leishmaniasis.

The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth in promastigote culture (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network associated with parasite fitness gain, with genome instability causing highly reproducible, gene dosage-independent and -dependent changes. Reduction of flagellar transcripts and increase in coding and non-coding RNAs implicated in ribosomal biogenesis and protein translation were not correlated to dosage changes of the corresponding genes, revealing a gene dosage-independent, post-transcriptional mechanism of regulation. In contrast, abundance of gene products implicated in post-transcriptional regulation itself correlated to corresponding gene dosage changes. Thus, RNA abundance during parasite adaptation is controled by direct and indirect gene dosage changes. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain in culture may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain in culture, where differential regulation of mRNA stability and the generation of modified ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania transcriptome and non-coding small RNome as potential novel sources for the discovery of biomarkers that may be associated with parasite phenotypic adaptation in clinical settings.

International audience; Pathogen fitness landscapes change when transmission cycles establish in non-native environments or spill over into new vectors and hosts. The introduction of Leishmania infantum in the Americas into the Neotropics during European colonization represents a unique case study to investigate the mechanisms of ecological adaptation of this important parasite. Defining the evolutionary trajectories that drive L. infantum fitness in this new environment are of great public health importance as they will allow unique insight into pathways of host/pathogen co-evolution and their consequences for region-specific changes in disease manifestation. This review summarizes current knowledge on L. infantum genetic and phenotypic diversity in the Americas and its possible role in the unique epidemiology of visceral leishmaniasis (VL) in the New World. We highlight the importance of appreciating adaptive molecular mechanisms in L. infantum to understand the parasites' successful establishment on the continent.

Ticks are the most important vectors of pathogens affecting both domestic and wild animals worldwide. Hard tick feeding is a slow process&mdash, taking up to several days&mdash, and necessitates extended control over the host response. The success of the feeding process depends upon injection of tick saliva, which not only controls host hemostasis and wound healing, but also subverts the host immune response to avoid tick rejection that creates a favorable niche for the survival and propagation of diverse tick-borne pathogens. Here, we report on the molecular and biochemical features and functions of an Ixodes ricinus serine protease inhibitor (IrSPI). We characterize IrSPI as a Kunitz elastase inhibitor that is overexpressed in several tick organs&mdash, especially salivary glands&mdash, during blood-feeding. We also demonstrated that when IrSPI is injected into the host through saliva, it had no impact on tissue factor pathway-induced coagulation, fibrinolysis, endothelial cell angiogenesis or apoptosis, but the protein exhibits immunomodulatory activity. In particular, IrSPI represses proliferation of CD4+ T lymphocytes and proinflammatory cytokine secretion from both splenocytes and macrophages. Our study contributes valuable knowledge to tick-host interactions and provides insights that could be further exploited to design anti-tick vaccines targeting this immunomodulator implicated in I. ricinus feeding.

International audience; Members of the highly conserved pleiotropic CK1 family of serine/threonine-specific kinases are tightly regulated in the cell and play crucial regulatory roles in multiple cellular processes from protozoa to human. Since their dysregulation as well as mutations within their coding regions contribute to the development of various different pathologies, including cancer and neurodegenerative diseases, they have become interesting new drug targets within the last decade. However, to develop optimized CK1 isoform-specific therapeutics in personalized therapy concepts, a detailed knowledge of the regulation and functions of the different CK1 isoforms, their various splice variants and orthologs is mandatory. In this review we will focus on the stress-induced CK1 isoform delta (CK1δ), thereby addressing its regulation, physiological functions, the consequences of its deregulation for the development and progression of diseases, and its potential as therapeutic drug target.

Leishmania parasites are responsible for leishmaniases. During their life cycle, they alternate between extracellular promastigotes in the insect vector and intracellular amastigotes in mammalian macrophages. Survival in the mammalian host implies to subvert the macrophages. Leishmania casein kinase 1 isoform 2 (LmCK1.2), as a signalling kinase, could be involved in the manipulation of the host cell, because: (i) it is released into the host cell via exosomes, (ii) it is essential for intracellular parasite survival, (iii) it phosphorylates host proteins. However, little is known about its functions in the parasites and the pathways it may regulate in the macrophage. I first showed that LmCK1.2 was found in the cytoplasm, associated to the cytoskeleton and to various organelles such as the basal body or the mitotic spindle. The subcellular localisations of LmCK1.2 required the presence of the C-terminal low complexity regions, suggesting the importance of protein-protein interactions for this process. To gain insights into LmCK1.2 functions, I then used proteomics to identify 171 LmCK1.2 associated proteins (LmCKAPs) in the parasite, which were involved in various pathways including endocytosis through the regulation of the AP2 complex and cytokinesis through the regulation of a novel cell-cycle regulated protein, LmCKAP1. Finally, I identified 146 mammalian host proteins associated with LmCK1.2 (LmCKAPhost), which are over-represented in proteins involved in cellular trafficking and protein metabolism, highlighting pathways that might be important for intracellular parasite survival. This work considerably increased our knowledge on Leishmania CK1.2. These data demonstrate singular similarities in localization and interactome composition between LmCK1.2 and human CK1s, suggesting that Leishmania CK1.2 has evolved to mimic mammalian CK1s and particularly CK1α. Similarly to other pathogens such as viruses, controlling CK1α pathways might be one of the keys to Leishmania intracellular survival., Les parasites Leishmania sont les agents responsables des leishmanioses. Au cours de leur cycle de vie, ils alternent entre une forme promastigote, extracellulaire, chez l'insecte vecteur et une forme intracellulaire, amastigote, dans les macrophages de mammifères. Pour survivre dans l'hôte mammifère, les amastigotes doivent manipuler le macrophage à leur avantage. La caséine kinase 1.2 de Leishmanie (LmCK1.2) pourrait être impliquée dans la manipulation de la cellule-hôte, car : (i) elle est exportée dans la cellule-hôte via des exosomes, (ii) elle est essentielle à la survie du parasite intracellulaire, et (iii) elle phosphoryle des protéines de la cellule hôte. Cependant, il existe peu d’informations sur ses fonctions dans le parasite et sur les voies de signalisations qu’elle pourrait réguler dans le macrophage. Lors de ce travail de thèse, j’ai montré que LmCK1.2 était présente dans le cytoplasme, qu’elle était associée au cytosquelette et à divers organelles tels que le corps basal ou le fuseau mitotique. J’ai montré que la présence de régions de faible complexité, présentes dans la partie C-terminale de LmCK1.2, était nécessaire à sa localisation subcellulaire. Afin de mieux comprendre les fonctions de LmCK1.2, j’ai ensuite réalisé des analyses protéomiques qui m’ont permis d’identifier 171 protéines associées à LmCK1.2 (LmCKAPs) dans le parasite. Ces LmCKAPs sont impliquées dans diverses voies. J’ai pu montrer l’implication de LmCK1.2 dans deux voies particulières : l’endocytose via la régulation du complexe AP2, et la cytokinèse via la régulation d’une nouvelle protéine mitotique, LmCKAP1. J’ai également identifié 146 protéines du macrophage associées à LmCK1.2 (LmCKAPhost). Elles sont surreprésentées dans deux voies particulières : le trafic cellulaire et le métabolisme des protéines, qui pourraient correspondre à des voies importantes pour la survie du parasite intracellulaire. Ce travail a considérablement enrichi nos connaissances sur LmCK1.2. En particulier, ces résultats montrent la singulière ressemblance entre LmCK1.2 et les CK1 humaines en ce qui concerne la localisation et la composition des protéines partenaires, suggérant que CK1.2 de leishmanie ait évolué pour imiter les CK1 de mammifères, particulièrement CK1α. Le contrôle des voies de signalisation de CK1α pourrait être l'une des clés de la survie intracellulaire de la leishmanie, comme cela a été montré pour d'autres agents pathogènes tels que les virus.

Members of the casein kinase 1 (CK1) family are key regulators in numerous cellular signal transduction pathways and in order to prevent the development of certain diseases, CK1 kinase activity needs to be tightly regulated. Modulation of kinase activity by site-specific phosphorylation within the C-terminal regulatory domain of CK1δ has already been shown for several cellular kinases. By using biochemical methods, we now identified residues T161, T174, T176, and S181 within the kinase domain of CK1δ as target sites for checkpoint kinase 1 (Chk1). At least residues T176 and S181 show full conservation among CK1δ orthologues from different eukaryotic species. Enzyme kinetic analysis furthermore led to the hypothesis that site-specific phosphorylation within the kinase domain finally contributes to fine-tuning of CK1δ kinase activity. These data provide a basis for the extension of our knowledge about the role of site-specific phosphorylation for regulation of CK1δ and associated signal transduction pathways.

International audience; CRISPR/Cas9 technology has been developing rapidly in the field of parasitology, allowing for the dissection of molecular processes with unprecedented efficiency. Optimization and implementation of a new technology like CRISPR, especially in nonmodel organisms, requires communication and collaboration throughout the field. Recently, a 'CRISPR in Parasitology' symposium was held at the Institut Pasteur Paris, bringing together scientists studying Leishmania, Plasmodium, Trypanosoma, and Anopheles. Here we share technological advances and challenges in using CRISPR/Cas9 in the parasite and vector systems that were discussed. As CRISPR/Cas9 continues to be applied to diverse parasite systems, the community should now focus on improvement and standardization of the technique as well as expanding the CRISPR toolkit to include Cas9 alternatives/derivatives for more advanced applications like genome-wide functional screens.

Leishmania parasites are thought to control protein activity at the post-translational level, e.g. by protein phosphorylation. In the pathogenic amastigote, the mammalian stage of Leishmania parasites, heat shock proteins show increased phosphorylation, indicating a role in stage-specific signal transduction. Here we investigate the impact of phosphosites in the L. donovani heat shock protein 90. Using a chemical knock-down/genetic complementation approach, we mutated 11 confirmed or presumed phosphorylation sites and assessed the impact on overall fitness, morphology and in vitro infectivity. Most phosphosite mutations affected the growth and morphology of promastigotes in vitro, but with one exception, none of the phosphorylation site mutants had a selective impact on the in vitro infection of macrophages. Surprisingly, aspartate replacements mimicking the negative charge of phosphorylated serines or threonines had mostly negative impacts on viability and infectivity. HSP90 is a substrate for casein kinase 1.2-catalysed phosphorylation in vitro. While several putative phosphosite mutations abrogated casein kinase 1.2 activity on HSP90, only Ser289 could be identified as casein kinase target by mass spectrometry. In summary, our data show HSP90 as a downstream client of phosphorylation-mediated signalling in an organism that depends on post-transcriptional gene regulation.

International audience; The limited success of recent phenotypic anti-leishmanial drug screening campaigns calls for new screening strategies for the discovery of clinically relevant hits. Here we present such a novel strategy based on physiologically relevant, ex vivo biology. We established high content phenotypic assays that combine primary murine macrophages and lesion-derived, virulent L. donovani and L. amazonensis amastigotes, which we applied to validate previously identified, anti-leishmanial hit compounds referred to as 'GSK Leish-Box'. Together with secondary screens using cultured promastigotes, our pipeline distinguished stage-and/or species-specific compounds, including 20 hits with broad activity at 10 µM against intracellular amastigotes of both viscerotropic and dermotropic Leishmania. Even though the GSK Leish-Box hits were identified by phenotypic screening using THP-1 macrophage-like cells hosting culture-derived L. donovani LdBob parasites, our ex vivo assays only validated anti-leishmanial activity at 10 µM on intra-macrophagic L. donovani for 23 out of the 188 GSK Leish-Box hits. In conclusion, our comparative approach allowed the identification of hits with broad anti-leishmanial activity that represent interesting novel candidates to be tested in animal models. Physiologically more relevant screening approaches such as described here may reduce the very high attrition rate observed during pre-clinical and clinical phases of the drug development process. Leishmaniases are neglected diseases caused by protozoan parasites of the genus Leishmania that are transmitted by the bite of female Phlebotomine sandflies. Almost 1 billion people are at risk of infection in close to 100 endemic countries throughout the tropical and subtropical regions of Africa, Asia, the Mediterranean countries and South and Central America 1. Leishmaniasis clinical manifestations range from self-healing cutaneous lesions with possible mucosal dissemination to severe visceral forms, causing death if untreated. In the absence of efficient reservoir and vector control strategies, and of preventive or therapeutic human vaccines , the mainstay of current intervention strategy to limit the disease is chemotherapy. Leishmaniases remain the only trypanosomatid diseases for which therapeutics are largely based on repurposed drugs, including anti-fungal (amphotericin B), anticancer (miltefosine), antibiotic (paromomycin) and antimalarial (sitamaquine) molecules 2,3. However, all current treatments are limited and show serious drawbacks, including high cost that are prohibitory for resource-limited countries, poor compliance due to constraining mode of administration 4 , poor safety with important adverse effects due to toxicity 5 , treatment failure and drug resistance 6. Similarly, current efforts to overcome the drug resistance by developing drug combination therapies 7,8 may fail in light of the identification of an L. infantum strain that gained resistance against antimony and amphotericin B 9. Multi-drug resistance was also documented in Indian field isolates 10 , and was confirmed experimentally by the in vitro selection of L. donovani parasites showing resistance to different drug combinations and even cross-resistance to unrelated drugs 11. Combination therapy therefore may be of only limited use, raising important concerns on the current

Protozoan parasites of the genus Leishmania cause severe human and veterinary diseases worldwide, termed leishmaniases. A hallmark of Leishmania biology is its capacity to adapt to a variety of unpredictable fluctuations inside its human host, notably pharmacological interventions, thus, causing drug resistance. Here we investigated mechanisms of environmental adaptation using a comparative genomics approach by sequencing 10 new clinical isolates of the L. donovani, L. major, and L. tropica complexes that were sampled across eight distinct geographical regions. Our data provide new evidence that parasites adapt to environmental change in the field and in culture through a combination of chromosome and gene amplification that likely causes phenotypic variation and drives parasite fitness gains in response to environmental constraints. This novel form of gene expression regulation through genomic change compensates for the absence of classical transcriptional control in these early-branching eukaryotes and opens new venues for biomarker discovery., Protozoan parasites of the genus Leishmania adapt to environmental change through chromosome and gene copy number variations. Only little is known about external or intrinsic factors that govern Leishmania genomic adaptation. Here, by conducting longitudinal genome analyses of 10 new Leishmania clinical isolates, we uncovered important differences in gene copy number among genetically highly related strains and revealed gain and loss of gene copies as potential drivers of long-term environmental adaptation in the field. In contrast, chromosome rather than gene amplification was associated with short-term environmental adaptation to in vitro culture. Karyotypic solutions were highly reproducible but unique for a given strain, suggesting that chromosome amplification is under positive selection and dependent on species- and strain-specific intrinsic factors. We revealed a progressive increase in read depth towards the chromosome ends for various Leishmania isolates, which may represent a nonclassical mechanism of telomere maintenance that can preserve integrity of chromosome ends during selection for fast in vitro growth. Together our data draw a complex picture of Leishmania genomic adaptation in the field and in culture, which is driven by a combination of intrinsic genetic factors that generate strain-specific phenotypic variations, which are under environmental selection and allow for fitness gain.

International audience; Leishmania are obligatory intracellular parasites that cycle between the sand fly midgut (extracellular promastigotes) and mammalian macrophage phagolysosomes (intracellular amastigotes). They have developed mechanisms of adaptation to the distinct environments of host and vector that favor utilization of both proline and alanine. LdAAP24 is the L. donovani proline-alanine transporter. It is a member of Leishmania system A that translocates neutral amino acids. Since system A is promastigote-specific, we aimed to assess whether LdAAP24 is also expressed exclusively in promastigotes. Herein, we established that upon exposing L. donovani promastigotes to amastigote differentiation signal (pH 5.5 and 37 °C), parasites rapidly and completely degrade LdAAP24 protein in both axenic and in spleen-derived amastigotes. In contrast, LdAAP24 mRNA remained unchanged throughout differentiation. Addition of either MG132 or Bafilomycin A1 partially inhibited LdAAP24 protein degradation, indicating a role for both lysosome- and proteasome-mediated degradation. This work provides the first evidence for post-translational regulation of stage-specific expression of LdAAP24.

Visceral leishmaniasis is an insidious neglected disease with worldwide distribution. It is caused by parasites from the Leishmania donovani complex, which are able to be transmitted by different species of phlebotomine sand flies and to infect numerous mammal hosts. Despite the high number of people infected or at risk, and the remarkable quantity of studies focusing on this disease, a proper experimental model to efficiently decipher the infectious process of visceral leishmaniasis taking into account the nuances of parasite’s virulence and the duration of the infection is still lacking. Therefore, using golden Syrian hamsters and BALB/c mice, state-of-the-art genetic manipulation applied on a fully virulent L. donovani strain and in vivo imaging approaches, we describe herein three benefits for experimental visceral leishmaniasis: (i) the development of a double transfected bioluminescent (firefly luciferase) and fluorescent (E2-crimson) virulent strain of L. donovani (Ld1S_luci_E2-crimson), favoring a wide range of both in vivo and in vitro investigations, (ii) the establishment of a non-invasive mouse model to evaluate the infectious process during visceral leishmaniasis and the parasite’s virulence in real time, allowing longitudinal studies with the same animals, and (iii) the elaboration of a suitable method to reinstate (and verify anew) the virulence in a population of attenuated parasites, by recovering persistent parasites from chronic infected mice. Consequently, these results open up new perspectives on the study of visceral leishmaniasis, especially in the fields of therapeutics and vaccinology, since the model described herein renders now possible long-lasting follow up studies, with easy and accurate day-by-day verifications of the infection status along with a reduced number of laboratory animals. Trial registration ClinicalTrials.gov 2013-0047., Author summary Visceral leishmaniasis is a chronic disease caused by the life-threatening parasite Leishmania donovani. This chronicity character is a major concern in experimental models of visceral leishmaniasis, since infected animals do not develop any visible sign or symptom before one/two months after infection. Consequently, there is an urge to develop a model that allow the early identification of the parasite in the host’s tissues and an easy follow up of the infection. Therefore, we generated genetically-modified virulent parasites that are concomitantly fluorescent and bioluminescent, and using highly performant imaging approaches, we could detect these parasites in tissues of infected hamsters and mice within a week post-infection. With these parasites, we established, then, a mouse model of visceral leishmaniasis that allows prolonged longitudinal studies and enables the investigation of parasite’s survival, using non-invasive in vivo imaging techniques. These features are of extreme importance, particularly for the development of new therapies and vaccines and open up new perspectives on the study of this disease.

Aneuploidy is usually deleterious in multicellular organisms but appears to be tolerated and potentially beneficial in unicellular organisms, including pathogens. Leishmania , a major protozoan parasite, is emerging as a new model for aneuploidy, since in vitro -cultivated strains are highly aneuploid, with interstrain diversity and intrastrain mosaicism. The alternation of two life stages in different environments (extracellular promastigotes and intracellular amastigotes) offers a unique opportunity to study the impact of environment on aneuploidy and gene expression. We sequenced the whole genomes and transcriptomes of Leishmania donovani strains throughout their adaptation to in vivo conditions mimicking natural vertebrate and invertebrate host environments. The nucleotide sequences were almost unchanged within a strain, in contrast to highly variable aneuploidy. Although high in promastigotes in vitro , aneuploidy dropped significantly in hamster amastigotes, in a progressive and strain-specific manner, accompanied by the emergence of new polysomies. After a passage through a sand fly, smaller yet consistent karyotype changes were detected. Changes in chromosome copy numbers were correlated with the corresponding transcript levels, but additional aneuploidy-independent regulation of gene expression was observed. This affected stage-specific gene expression, downregulation of the entire chromosome 31, and upregulation of gene arrays on chromosomes 5 and 8. Aneuploidy changes in Leishmania are probably adaptive and exploited to modulate the dosage and expression of specific genes; they are well tolerated, but additional mechanisms may exist to regulate the transcript levels of other genes located on aneuploid chromosomes. Our model should allow studies of the impact of aneuploidy on molecular adaptations and cellular fitness. IMPORTANCE Aneuploidy is usually detrimental in multicellular organisms, but in several microorganisms, it can be tolerated and even beneficial. Leishmania —a protozoan parasite that kills more than 30,000 people each year—is emerging as a new model for aneuploidy studies, as unexpectedly high levels of aneuploidy are found in clinical isolates. Leishmania lacks classical regulation of transcription at initiation through promoters, so aneuploidy could represent a major adaptive strategy of this parasite to modulate gene dosage in response to stressful environments. For the first time, we document the dynamics of aneuploidy throughout the life cycle of the parasite, in vitro and in vivo . We show its adaptive impact on transcription and its interaction with regulation. Besides offering a new model for aneuploidy studies, we show that further genomic studies should be done directly in clinical samples without parasite isolation and that adequate methods should be developed for this.

International audience; The state of antileishmanial chemotherapy is strongly compromised by the emergence of drug-resistant Leishmania. The evolution of drug-resistant phenotypes has been linked to the parasites’ intrinsic genome instability, with frequent gene and chromosome amplifications causing fitness gains that are directly selected by environmental factors, including the presence of antileishmanial drugs. Thus, even though the unique eukaryotic biology of Leishmania and its dependence on parasite-specific virulence factors provide valid opportunities for chemotherapeutical intervention, all strategies that target the parasite in a direct fashion are likely prone to select for resistance. Here, we review the current state of antileishmanial chemotherapy and discuss the limitations of ongoing drug discovery efforts. We finally propose new strategies that target Leishmania viability indirectly via mechanisms of host–parasite interaction, including parasite-released ectokinases and host epigenetic regulation, which modulate host cell signaling and transcriptional regulation, respectively, to establish permissive conditions for intracellular Leishmania survival.

International audience; Cutaneous leishmaniasis (CL) is a main public health problem in Morocco, Algeria, Tunisia and Iran, known as the MATI region. During the biennial meeting of Pasteur Institutes of the MATI region in November 2016 in Tehran, a Leishmania panel formed of scientists with different expertise in CL research from the MATI and Institut Pasteur in Paris, proposed a new consortium to develop an integrative research approach to better understand this disease. The proposed consortium synergizes three complementary thematics on Leishmania genetics, host immune response, and vector transmission. Moreover, assessing the social impact of CL with the aim to raise awareness at both the national and international levels will be a major focus of this consortium.

The recent adaptation of CRISPR Cas9 genome editing toLeishmaniaspp. has opened a new era in decipheringLeishmaniabiology. The method was recently improved using a PCR-based CRISPR Cas9 approach, which eliminated the need for cloning. This new approach, which allows high-throughput gene deletion, was successfully validated inL. mexicanaandL. major. In this study, we validated the toolkit inLeishmania donovanitargeting the flagellar protein PF16, confirming that the tagged protein localizes to the flagellum and that null mutants lose their motility. We then used the technique to characterise CK1.1, a member of the casein kinase 1 family, which is involved in the regulation of many cellular processes. We showed that CK1.1 is a low-abundance protein present in promastigotes and in amastigotes. We demonstrated that CK1.1 is not essential for promastigote and axenic amastigote survival or for axenic amastigote differentiation, although it may have a role during stationary phase. Altogether, our data validate the use of PCR-based CRISPR Cas9 toolkit inL. donovani, which will be crucial for genetic modification of hamster-derived, disease-relevant parasites.

International audience; Trypanosomatid parasites of the genus Leishmania cause severe human diseases collectively termed leishmaniasis. Parasite ATP-binding proteins have emerged as potent targets for chemotherapeutic intervention. However, many parasite-specific ATP-binding proteins may escape current efforts in drug target identification, validation and deconvolution due to the lack of sequence conservation and functional annotation of these proteins in early branching eukaryotic trypanosomatids. Here, we selectively enriched for ATP-binding proteins from Leishmania donovani axenic promastigote and amastigote total protein extracts utilizing a Capture Compound™ (CC) linked to the ATP-competitive inhibitor staurosporine. As judged by in-gel kinase activity assay and competitive inhibition with free staurosporine, the CC specifically enriched for parasite phosphotransferases. Comparative nanoLC-MS(n) analysis identified 70 captured proteins, including 24 conserved protein kinases, and 32 hypothetical proteins with potential ATP-binding function. We identified conserved signature sequence motifs characteristic for staurosporine-binding protein kinases, and identified the hypothetical proteins LinJ.20.0280 and LinJ.09.1630 as novel ATP-binding proteins. Thus, functional enrichment procedures such as described here, combined with bio-informatics analyses and activity assays, provide powerful tools for the discovery of parasite-specific ATP-binding proteins that escape homology-based identification, which can be subsequently targeted for pharmacological intervention.BIOLOGICAL SIGNIFICANCE: Functional enrichment using a Capture Compound™ linked to the ATP-competitive inhibitor staurosporine provides a powerful new tool for the discovery of parasite-specific ATP-binding proteins that escape homology-based identification, which can be subsequently targeted for pharmacological intervention.

Malaria, schistosomiasis and leishmaniases are among the most prevalent tropical parasitic diseases and each requires new innovative treatments. Targeting essential parasite pathways, such as those that regulate gene expression and cell cycle progression, is a key strategy for discovering new drug leads. In this study, four clinically approved anti-cancer drugs (Vorinostat, Belinostat, Panobinostat and Romidepsin) that target histone/lysine deacetylase enzymes were examined for in vitro activity against Plasmodium knowlesi, Schistosoma mansoni, Leishmania amazonensis and L. donovani parasites and two for in vivo activity in a mouse malaria model. All four compounds were potent inhibitors of P. knowlesi malaria parasites (IC50 9–370 nM), with belinostat, panobinostat and vorinostat having 8–45 fold selectivity for the parasite over human neonatal foreskin fibroblast (NFF) or human embryonic kidney (HEK 293) cells, while romidepsin was not selective. Each of the HDAC inhibitor drugs caused hyperacetylation of P. knowlesi histone H4. None of the drugs was active against Leishmania amastigote or promastigote parasites (IC50 > 20 μM) or S. mansoni schistosomula (IC50 > 10 μM), however romidepsin inhibited S. mansoni adult worm parings and egg production (IC50 ∼10 μM). Modest in vivo activity was observed in P. berghei infected mice dosed orally with vorinostat or panobinostat (25 mg/kg twice daily for four days), with a significant reduction in parasitemia observed on days 4–7 and 4–10 after infection (P, Graphical abstract Image 1, Highlights • HDAC inhibitor cancer drugs have in vitro activity against P. knowlesi. • HDAC inhibitor drugs cause hyperacetylation of P. knowlesi histone H4. • HDAC inhibitor drugs are not active against Leishmania amastigotes or promastigotes. • Romidepsin inhibited S. mansoni adult worm parings and egg production. • Oral Panobinostat has better activity in P. berghei infected mice than Vorinostat.

Currently, medicines used to treat leishmaniasis have many serious side effects and their use is greatly compromised by the appearance of resistant parasites. It is therefore urgent and necessary to obtain new drugs to fight the disease effectively. In recent years, pharmaceutical companies have carried out screening campaigns that have identified thousands of compounds with a leishmanicidal effect. However, since their targets are unknown, these compounds are difficult to optimize, which in the long term could lead to side effects for the drugs derived from these compounds. Thus, deconvolution of targets has become a major issue, since it allows the identification of the primary and / or secondary targets of the compounds in order to understand their mechanism of action and to promote their optimization. We carried out a screening campaign which allowed us to identify a dozen potential inhibitors of kinases effective against the parasite. In order to determine their targets, I have developed a deconvolution strategy adapted to these inhibitors which consists initially in a selective enrichment of the Leishmania ATP binding proteins (ATPome) by ATP affinity chromatography , Then in a second stage in a competition by the compound of interest. The method of enrichment was very effective since it made it possible to obtain the first ATPome never realized in the trypanosomatids with the identification of 1177 proteins with potential ATP binding, including 205 annotated proteins for the binding function to ATP (Gene Ontology: 0005524), including 80 kinases and about 80 hypothetical proteins with an ATP binding domain or known ATPase function but not annotated. I then optimized the stage of competition and established the proof of concept using a known inhibitor-kinase pair. Finally, by combining this method with 2D-DiGE, I established a fast, visual and semi-quantitative method that allows me to determine whether inhibitors are broad-spectrum or specific; Only the latter will be optimized. This method thus makes it possible to visually determine the efficiency and the specificity of the competitive compounds of ATP. Using this approach, of the ten compounds initially obtained by screening, I was able to select two that were specific. For these two compounds, the targets will be identified by mass spectrometry.; Aujourd’hui, les médicaments utilisés pour traiter la leishmaniose ont de nombreux effets secondaires graves et leur utilisation est fortement compromise par l’apparition de parasites résistants. Il devient donc urgent et indispensable d’obtenir de nouveaux médicaments pour combattre efficacement la maladie. Ces dernières années, les compagnies pharmaceutiques ont réalisés des campagnes de criblages qui ont permis d’identifier des milliers de composés présentant un effet leishmanicide. Toutefois, comme leurs cibles sont inconnues, ces composés sont difficiles à optimiser, ce qui à terme pourrait entrainer des effets secondaires pour les médicaments dérivés de ces composés. Ainsi, la déconvolution de cibles, est devenue un enjeu majeur, puisqu’elle permet l’identification des cibles primaires et/ou secondaires des composés afin de comprendre leur mécanisme d’action et de favoriser leur optimisation. Nous avons réalisés une campagne de criblage qui nous a permis d’identifier une dizaine d’inhibiteurs potentiels de kinases efficaces contre le parasite. Afin de déterminer leurs cibles, j’ai développé une stratégie de déconvolution adaptée à ces inhibiteurs qui consiste dans un premier temps en un enrichissement sélectif des protéines de liaison à l’ATP de Leishmania (ATPome) par chromatographie d’affinité à l’ATP, puis dans un deuxième temps en une compétition par le composé d’intérêt. La méthode d’enrichissement a été très efficace puisqu’elle a permis d’obtenir le premier ATPome jamais réalisé chez les trypanosomatidés avec l’identification de 1177 protéines de liaisons à l’ATP potentielles, comprenant 205 protéines annotées pour la fonction de liaison à l’ATP (Gene Ontology : 0005524), dont 80 kinases et environ 80 protéines hypothétiques avec un domaine de liaison à l’ATP ou une fonction ATPase connue, mais non annotés. J’ai ensuite optimisé l’étape de compétition et établi la preuve de concept en utilisant un couple inhibiteur-kinase connu. Finalement, en combinant cette méthode avec la 2D-DiGE, j’ai établi une méthode rapide, visuelle et semi-quantitative qui me permet de déterminer si les inhibiteurs sont à large spectre ou spécifiques ; seuls ces derniers seront optimisés. Cette méthode permet donc de déterminer visuellement l’efficacité et la spécificité des composés compétitifs de l’ATP. En utilisant cette approche, sur les dix composés initialement obtenus par criblage, j’ai pu en sélectionner deux qui soient spécifiques. Pour ces deux composés, les cibles seront identifiées par spectrométrie de masse.Sciences de la Vie et de la Terre Etablissement d'une approche de déconvolution de cibles pour des composés leishmanicides Olivier LECLERCQ 8 décembre 2016 Soutenu devant le jury suivant : Flore RENAUD, présidente Gerald SPÄTH, tuteur scientifique Najma RACHIDI, co-tutrice scientifique Sylvie DEMIGNOT, tutrice pédagogique Sébastien POMEL, rapporteur Christian MITRI, examinateur

International audience; Protozoan parasites of the genus Leishmania adapt to their arthropod and vertebrate hosts through the development of defined life cycle stages. Stage differentiation is triggered by environmental stress factors and has been linked to parasite chaperone activities. Using a null mutant approach we previously revealed important, nonredundant functions of the cochaperone cyclophilin 40 in L. donovani-infected macrophages. Here, we characterized in more detail the virulence defect of cyp40-/- null mutants. In vitro viability assays, infection tests using macrophages, and mixed infection experiments ruled out a defect of cyp40-/- parasites in resistance to oxidative and hydrolytic stresses encountered inside the host cell phagolysosome. Investigation of the CyP40-dependent proteome by quantitative 2D-DiGE analysis revealed up regulation of various stress proteins in the null mutant, presumably a response to compensate for the lack of CyP40. Applying transmission electron microscopy we showed accumulation of vesicular structures in the flagellar pocket of cyp40-/- parasites that we related to a significant increase in exosome production, a phenomenon previously linked to the parasite stress response. Together these data suggest that cyp40-/- parasites experience important intrinsic homeostatic stress that likely abrogates parasite viability during intracellular infection.

Background Rosetting, namely the capacity of the Plasmodium falciparum-infected red blood cells to bind uninfected RBCs, is commonly observed in African children with severe malaria. Rosetting results from specific interactions between a subset of variant P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesins encoded by var genes, serum components and RBC receptors. Rosette formation is a redundant phenotype, as there exists more than one var gene encoding a rosette-mediating PfEMP1 in each genome and hence a diverse array of underlying interactions. Moreover, field diversity creates a large panel of rosetting-associated serotypes and studies with human immune sera indicate that surface-reacting antibodies are essentially variant-specific. To gain better insight into the interactions involved in rosetting and map surface epitopes, a panel of monoclonal antibodies (mAbs) was investigated. Methods Monoclonal antibodies were isolated from mice immunized with PfEMP1-VarO recombinant domains. They were characterized using ELISA and reactivity with the native PfEMP1-VarO adhesin on immunoblots of reduced and unreduced extracts, as well as SDS-extracts of Palo Alto 89F5 VarO schizonts. Functionality was assessed using inhibition of Palo Alto 89F5 VarO rosette formation and disruption of Palo Alto 89F5 VarO rosettes. Competition ELISAs were performed with biotinylated antibodies against DBL1 to identify reactivity groups. Specificity of mAbs reacting with the DBL1 adhesion domain was explored using recombinant proteins carrying mutations abolishing RBC binding or binding to heparin, a potent inhibitor of rosette formation. Results Domain-specific, surface-reacting mAbs were obtained for four individual domains (DBL1, CIDR1, DBL2, DBL4). Monoclonal antibodies reacting with DBL1 potently inhibited the formation of rosettes and disrupted Palo Alto 89F5 VarO rosettes. Most surface-reactive mAbs and all mAbs interfering with rosetting reacted on parasite immunoblots with disulfide bond-dependent PfEMP1 epitopes. Based on competition ELISA and binding to mutant DBL1 domains, two distinct binding sites for rosette-disrupting mAbs were identified in close proximity to the RBC-binding site. Conclusions Rosette-inhibitory antibodies bind to conformation-dependent epitopes located close to the RBC-binding site and distant from the heparin-binding site. These results provide novel clues for a rational intervention strategy that targets rosetting. Electronic supplementary material The online version of this article (doi:10.1186/s12936-015-1016-5) contains supplementary material, which is available to authorized users.

Currently, medicines used to treat leishmaniasis have many serious side effects and their use is greatly compromised by the appearance of resistant parasites. It is therefore urgent and necessary to obtain new drugs to fight the disease effectively. In recent years, pharmaceutical companies have carried out screening campaigns that have identified thousands of compounds with a leishmanicidal effect. However, since their targets are unknown, these compounds are difficult to optimize, which in the long term could lead to side effects for the drugs derived from these compounds. Thus, deconvolution of targets has become a major issue, since it allows the identification of the primary and / or secondary targets of the compounds in order to understand their mechanism of action and to promote their optimization. We carried out a screening campaign which allowed us to identify a dozen potential inhibitors of kinases effective against the parasite. In order to determine their targets, I have developed a deconvolution strategy adapted to these inhibitors which consists initially in a selective enrichment of the Leishmania ATP binding proteins (ATPome) by ATP affinity chromatography , Then in a second stage in a competition by the compound of interest. The method of enrichment was very effective since it made it possible to obtain the first ATPome never realized in the trypanosomatids with the identification of 1177 proteins with potential ATP binding, including 205 annotated proteins for the binding function to ATP (Gene Ontology: 0005524), including 80 kinases and about 80 hypothetical proteins with an ATP binding domain or known ATPase function but not annotated. I then optimized the stage of competition and established the proof of concept using a known inhibitor-kinase pair. Finally, by combining this method with 2D-DiGE, I established a fast, visual and semi-quantitative method that allows me to determine whether inhibitors are broad-spectrum or specific; Only the latter will be optimized. This method thus makes it possible to visually determine the efficiency and the specificity of the competitive compounds of ATP. Using this approach, of the ten compounds initially obtained by screening, I was able to select two that were specific. For these two compounds, the targets will be identified by mass spectrometry., Aujourd’hui, les médicaments utilisés pour traiter la leishmaniose ont de nombreux effets secondaires graves et leur utilisation est fortement compromise par l’apparition de parasites résistants. Il devient donc urgent et indispensable d’obtenir de nouveaux médicaments pour combattre efficacement la maladie. Ces dernières années, les compagnies pharmaceutiques ont réalisés des campagnes de criblages qui ont permis d’identifier des milliers de composés présentant un effet leishmanicide. Toutefois, comme leurs cibles sont inconnues, ces composés sont difficiles à optimiser, ce qui à terme pourrait entrainer des effets secondaires pour les médicaments dérivés de ces composés. Ainsi, la déconvolution de cibles, est devenue un enjeu majeur, puisqu’elle permet l’identification des cibles primaires et/ou secondaires des composés afin de comprendre leur mécanisme d’action et de favoriser leur optimisation. Nous avons réalisés une campagne de criblage qui nous a permis d’identifier une dizaine d’inhibiteurs potentiels de kinases efficaces contre le parasite. Afin de déterminer leurs cibles, j’ai développé une stratégie de déconvolution adaptée à ces inhibiteurs qui consiste dans un premier temps en un enrichissement sélectif des protéines de liaison à l’ATP de Leishmania (ATPome) par chromatographie d’affinité à l’ATP, puis dans un deuxième temps en une compétition par le composé d’intérêt. La méthode d’enrichissement a été très efficace puisqu’elle a permis d’obtenir le premier ATPome jamais réalisé chez les trypanosomatidés avec l’identification de 1177 protéines de liaisons à l’ATP potentielles, comprenant 205 protéines annotées pour la fonction de liaison à l’ATP (Gene Ontology : 0005524), dont 80 kinases et environ 80 protéines hypothétiques avec un domaine de liaison à l’ATP ou une fonction ATPase connue, mais non annotés. J’ai ensuite optimisé l’étape de compétition et établi la preuve de concept en utilisant un couple inhibiteur-kinase connu. Finalement, en combinant cette méthode avec la 2D-DiGE, j’ai établi une méthode rapide, visuelle et semi-quantitative qui me permet de déterminer si les inhibiteurs sont à large spectre ou spécifiques ; seuls ces derniers seront optimisés. Cette méthode permet donc de déterminer visuellement l’efficacité et la spécificité des composés compétitifs de l’ATP. En utilisant cette approche, sur les dix composés initialement obtenus par criblage, j’ai pu en sélectionner deux qui soient spécifiques. Pour ces deux composés, les cibles seront identifiées par spectrométrie de masse.Sciences de la Vie et de la Terre Etablissement d'une approche de déconvolution de cibles pour des composés leishmanicides Olivier LECLERCQ 8 décembre 2016 Soutenu devant le jury suivant : Flore RENAUD, présidente Gerald SPÄTH, tuteur scientifique Najma RACHIDI, co-tutrice scientifique Sylvie DEMIGNOT, tutrice pédagogique Sébastien POMEL, rapporteur Christian MITRI, examinateur

International audience; Reverse genetics in Leishmania spp has gained importance beyond basic research as efforts increase to discover and validate new drug targets. Often, the most promising targets are essential for viability of the parasites, defying a genetic analysis by current gene replacement strategies. Duncan et al. demonstrate the applicability of DiCre recombination in Leishmania for induced replacement of the kinase CRK3 gene in promastigotes. DiCre gene replacement leads to the rapid loss of the gene and allows monitoring the phenotypic effects of the loss of function, eliminating the need for prolonged cultivation and selection. Implementation of the DiCre approach will allow functional genetics of the most important of Leishmania genes and is likely to boost genetic research and drug target discovery.

International audience; 3,6-Disubstituted imidazo[1,2-b]pyridazine derivatives were synthesized to identify new inhibitors of various eukaryotic kinases, including mammalian and protozoan kinases. Among the imidazo[1,2-b]pyridazines tested as kinase inhibitors, several derivatives were selective for DYRKs and CLKs, with IC50

International audience; Across bacterial, archaeal and eukaryotic kingdoms, heat shock proteins (HSPs) are defined as a class of highly conserved chaperone proteins that are rapidly induced in response to temperature increase through dedicated heat shock transcription factors. While this transcriptional response governs cellular adaptation of fungal, plant and animal cells to thermic shock and other forms of stress, early-branching eukaryotes of the kinetoplastid order, including trypanosomatid parasites, lack classical mechanisms of transcriptional regulation and show largely constitutive expression of HSPs, thus raising important questions on the function of HSPs in the absence of stress and the regulation of their chaperone activity in response to environmental adversity. Understanding parasite-specific mechanisms of stress-response regulation is especially relevant for protozoan parasites of the genus Leishmania that are adapted for survival inside highly toxic phagolysosomes of host macrophages causing the various immuno-pathologies of leishmaniasis. Here we review recent advances on the function and regulation of chaperone activities in these kinetoplastid pathogens and propose a new model for stress-response regulation through a reciprocal regulatory relationship between stress kinases and chaperones that may be relevant for parasite-adaptive differentiation and infectivity.