Targeting essential pathways in trypanosomatids gives insights into protozoan mechanisms of cell death

Apoptosis is a normal component of the development and health of multicellular organisms. However, apoptosis is now considered a prerogative of unicellular organisms, including the trypanosomatids of the genera Trypanosoma spp. and Leishmania spp., causative agents of some of the most important neglected human diseases. Trypanosomatids show typical hallmarks of apoptosis, although they lack some of the key molecules contributing to this process in metazoans, like caspase genes, Bcl-2 family genes and the TNF-related family of receptors. Despite the lack of these molecules, trypanosomatids appear to have the basic machinery to commit suicide. The components of the apoptotic execution machinery of these parasites are slowly coming into light, by targeting essential processes and pathways with different apoptogenic agents and inhibitors. This review will be confined to the events known to drive trypanosomatid parasites to apoptosis

Leishmania Genome Dynamics during Environmental Adaptation Reveal Strain-Specific Differences in Gene Copy Number Variation, Karyotype Instability, and Telomeric Amplification.

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.IMPORTANCE 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

Transcriptional regulation of rat CYP2B genes

Cytochromes P450 (CYPs) are a large superfamily of haemoproteins involved in the metabolism of numerous endogenous and exogenous compounds. CYP2B1 and CYP2B2 expression is increased by a variety of xenobiotics including the anti-epileptic drug phenobarbital. The expression of CYP2B genes declines during hepatocyte culture. The profiles of liver-enriched and stress-induced transcription factors were examined during cell culture. No absolute correlation was found between the profiles of transcription factors (C/EBP, SP1, NFκB, AP1) and CYP2B mRNA amounts. The binding activity of SP1 varied in a manner similar to CYP2B mRNA expression up to 24 hours of cell culture. This led to the identification of two putative SP1 elements in the CYP2B1 promoter. Co-transfection studies of primary hepatocytes with an SP1 expression vector and a luciferase reporter gene linked to the CYP2B1 promoter (-2/-179) indicates that SP1 has a functional role in the regulation of CYP2B1 gene expression. Phenobarbital and other CYP2B inducers, such as the plant product picrotoxin, stimulate constitutive androstane receptor (CAR) activation of a reporter gene linked to the CYP2B1 gene PBRE (phenobarbital response element) in transfected, primary hepatocytes and liver, but not in the transfected, transformed cell lines, HEPG2, CV-1 and HeLa, which support only constitutive transactivation by CAR. Co-transfection experiments in primary hepatocytes demonstrated that the co-activator SRC-1, mediates the action of CAR. Gel shift assays show that phenobarbital is a direct ligand of CAR. Different classes of inducers were shown to mediate induction of CYP2B genes via different receptors. Pregnenolone 16a-carbonitrile and glucocorticoids mediate their actions on the CYP2B1 gene through the Pregnane X Receptor (PXR). Both CAR and PXR bind to the same PBRE direct repeat (DR)4 element to mediate xenobiotic-regulated CYP2B1 expression. Our results suggest that CAR also binds to the DR3 PXRE of the CYP3A1 promoter to mediate the phenobarbital response of this gene

Leishmania Protein Kinases: Important Regulators of the Parasite Life Cycle and Molecular Targets for Treating Leishmaniasis

Leishmania is a protozoan parasite of the trypanosomatid family, causing a wide range of diseases with different clinical manifestations including cutaneous, mucocutaneous and visceral leishmaniasis. According to WHO, one billion people are at risk of Leishmania infection as they live in endemic areas while there are 12 million infected people worldwide. Annually, 0.9–1.6 million new infections are reported and 20–50 thousand deaths occur due to Leishmania infection. As current chemotherapy for treating leishmaniasis exhibits numerous drawbacks and due to the lack of effective human vaccine, there is an urgent need to develop new antileishmanial therapy treatment. To this end, eukaryotic protein kinases can be ideal target candidates for rational drug design against leishmaniasis. Eukaryotic protein kinases mediate signal transduction through protein phosphorylation and their inhibition is anticipated to be disease modifying as they regulate all essential processes for Leishmania viability and completion of the parasitic life cycle including cell-cycle progression, differentiation and virulence. This review highlights existing knowledge concerning the exploitation of Leishmania protein kinases as molecular targets to treat leishmaniasis and the current knowledge of their role in the biology of Leishmania spp. and in the regulation of signalling events that promote parasite survival in the insect vector or the mammalian host

Leishmania donovani RAN-GTPase interacts at the nuclear rim with linker histone H1

International audienceRan-GTPase regulates multiple cellular processes such as nucleo-cytoplasmic transport, mitotic spindle assembly, nuclear envelope assembly, cell-cycle progression and the mitotic checkpoint. The leishmanial Ran protein contrary to its mammalian counterpart which is predominately nucleoplasmic is localised at the nuclear rim. The focus of this paper was to characterise the L.donovani Ran orthologue (LdRan) with emphasis on the Ran-histone association. LdRan was found to be developmentally regulated, expressed three times less in the amastigote stage. LdRan over-expression caused a growth defect linked to a delayed S-phase progression in promastigotes like its mammalian counterpart. We report for the first time that Ran interacts with a linker histone -histone H1- in vitro and that the two proteins co-localise at the parasite nuclear rim. Interaction of Ran with core histones H3 and H4, creating in metazoans a chromosomal Ran-GTP gradient important for mitotic spindle assembly, is speculative in Leishmania spp., not only because this parasite undergoes a closed mitosis but also because the main localisation of LdRan is different from that of core histone H3. Interaction of Ran with the leishmanial linker histone H1 (LeishH1), suggests that this association maybe involved in modulation of other pathways than those documented for the metazoan Ran-core histone association

Soteriadou K: Leishmania donovani Ran-GTPase interacts at the nuclear rim with linker histone H1. Biochem J 2009, 424(3):367–374.Submit your manuscript at www.biomedcentral.com/submit

Ran-GTPase regulates multiple cellular processes such as nucleocytoplasmic transport, mitotic spindle assembly, nuclear envelope assembly, cell-cycle progression and the mitotic checkpoint. The leishmanial Ran protein, in contrast with its mammalian counterpart which is predominately nucleoplasmic, is localized at the nuclear rim. The aim of the present study was to characterize the LdRan (Leishmania donovani Ran) orthologue with an emphasis on the Ran-histone association. LdRan was found to be developmentally regulated, expressed 3-fold less in the amastigote stage. LdRan overexpression caused a growth defect linked to a delayed S-phase progression in promastigotes as for its mammalian counterpart. We report for the first time that Ran interacts with a linker histone, histone H1, in vitro and that the two proteins co-localize at the parasite nuclear rim. Interaction of Ran with core histones H3 and H4, creating in metazoans a chromosomal Ran-GTP gradient important for mitotic spindle assembly, is speculative in Leishmania spp., not only because this parasite undergoes a closed mitosis, but also because the main localization of LdRan is different from that of core histone H3. Interaction of Ran with the leishmanial linker histone H1 (LeishH1) suggests that this association maybe involved in modulation of pathways other than those documented for the metazoan Ran-core histone association

ER targeting and retention of the HCV NS4B protein relies on the concerted action of multiple structural features including its transmembrane domains.

International audienceThe Hepatitis C virus (HCV) NS4B protein, a multispanning endoplasmic reticulum (ER) membrane protein, generates intracellular rearrangements of ER-derived membranes, essential for HCV replication. In this study, we characterized NS4B elements involved in the process of targeting, association and retention in the ER membrane. We investigated the localization and membrane association of a number of C- or N-terminal NS4B deletions expressed as GFP chimeras by biochemical and fluorescence microscopy techniques. A second set of GFP-NS4B chimeras containing the plasma membrane ecto-ATPase CD39 at the C-terminus of each NS4B deletion mutant was used to further examine the role of N-terminal NS4B sequences in ER retention. Several structural elements, besides the first two transmembrane domains (TMs), within the NS4B N-terminal half (residues 1-130) were found to mediate association of the NS4B-GFP chimeras with ER membranes. Both TM1 and TM2 are required for ER anchoring and retention but are not sufficient for ER retention. Sequences upstream of TM1 are also required. These include two putative amphipathic alpha-helices and a Leucine Rich Repeat-like motif, a sequence highly conserved in all HCV genotypes. The N-terminal 55peptidic sequence, containing the 1st amphipathic helix, mediates association of the 55N-GFP chimera with cellular membranes including the ER, but is dispensable for ER targeting of the entire NS4B molecule. Importantly, the C-terminal 70peptidic sequence can associate with membranes positive for ER markers in the absence of any predicted TMs. In conclusion, HCV NS4B targeting and retention in the ER results from the concerted action of several NS4B structural elements

SILAC-based quantitative proteomics reveals pleiotropic, phenotypic modulation in primary murine macrophages infected with the protozoan pathogen Leishmania donovani

International audienceLeishmaniases 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

Leishmania dual specificity tyrosine regulated kinase 1 (DYRK1) is required for sustaining Leishmania stationary phase phenotype

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.PostprintPeer reviewe