Publications by authors named "Pascale Pescher"

20 Publications

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Comparative transcriptomic analysis of antimony resistant and susceptible Leishmania infantum lines.

Parasit Vectors 2020 Nov 30;13(1):600. Epub 2020 Nov 30.

Genômica Funcional de Parasitos, Instituto René Rachou, Fiocruz Minas, Belo Horizonte, MG, Brazil.

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 (Sb)-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 Sb-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 Sb-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 Sb-resistant line are associated with protein phosphorylation, microtubule-based movement, ubiquitination, host-parasite interaction, cellular process and other categories. The downregulated transcripts in the Sb-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.
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http://dx.doi.org/10.1186/s13071-020-04486-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7706067PMC
November 2020

Dynamic imaging reveals surface exposure of virulent amastigotes during pyroptosis of infected macrophages.

J Cell Sci 2020 06 22;134(5). Epub 2020 Jun 22.

Institut Pasteur, Unité de Parasitologie Moléculaire et Signalisation, INSERM U1201, 75015 Paris, France

spp. are obligate intracellular parasites that infect phagocytes, notably macrophages. No information is available on how parasites respond to pyroptosis of their host cell, which is 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 amastigotes and sequentially treated with lipopolysaccharide and ATP to induce pyroptosis. 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 retained their full infectivity towards naïve macrophages suggesting that host cell pyroptosis may contribute to parasite dissemination.
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http://dx.doi.org/10.1242/jcs.242776DOI Listing
June 2020

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

J Proteomics 2020 02 14;213:103617. Epub 2019 Dec 14.

Institut Pasteur and Institut National de Santé et Recherche Médicale INSERM U1201, Unité de Parasitologie Moléculaire et Signalisation, Paris, France. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jprot.2019.103617DOI Listing
February 2020

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

mBio 2018 11 6;9(6). Epub 2018 Nov 6.

Unité de Parasitologiemoléculaire et Signalisation, Institut Pasteur, Paris, France

Protozoan parasites of the genus adapt to environmental change through chromosome and gene copy number variations. Only little is known about external or intrinsic factors that govern genomic adaptation. Here, by conducting longitudinal genome analyses of 10 new 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 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 isolates, which may represent a nonclassical mechanism of telomere maintenance that can preserve integrity of chromosome ends during selection for fast growth. Together our data draw a complex picture of 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. Protozoan parasites of the genus cause severe human and veterinary diseases worldwide, termed leishmaniases. A hallmark of 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 , , and 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.
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http://dx.doi.org/10.1128/mBio.01399-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222132PMC
November 2018

Reverse Epidemiology: An Experimental Framework to Drive Biomarker Discovery by Functional Genetic Screening Using Relevant Animal Models.

Front Cell Infect Microbiol 2018 19;8:325. Epub 2018 Sep 19.

Institut Pasteur, Unité de Parasitologie Moléculaire et Signalisation, INSERM U1201, Paris, France.

biomarker discovery remains an important challenge that needs to be revisited in light of our increasing knowledge on parasite-specific biology, notably its genome instability. In the absence of classical transcriptional regulation in these early-branching eukaryotes, fluctuations in transcript abundance can be generated by gene and chromosome amplifications, which have been linked to parasite phenotypic variability with respect to virulence, tissue tropism, and drug resistance. Conducting evolutionary experiments to study mechanisms of environmental adaptation, we recently validated the link between parasite genetic amplification and fitness gain, thus defining gene and chromosome copy number variations (CNVs) as important biomarkers. These experiments also demonstrated that long-term culture adaptation can strongly interfere with epidemiologically relevant, genetic signals, which challenges current protocols for biomarker discovery, all of which rely on expansion of clinical isolates. Here we propose an experimental framework independent of long-term culture termed "reverse" epidemiology, which applies established protocols for functional genetic screening of cosmid-transfected parasites in animal models for the identification of clinically relevant genetic loci that then inform targeted field studies for their validation as biomarkers.
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http://dx.doi.org/10.3389/fcimb.2018.00325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6157315PMC
August 2019

Haplotype selection as an adaptive mechanism in the protozoan pathogen Leishmania donovani.

Nat Ecol Evol 2017 Dec 6;1(12):1961-1969. Epub 2017 Nov 6.

Institut Pasteur, INSERM U1201, Unité de Parasitologie Moléculaire et Signalisation, 75015, Paris, France.

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.
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http://dx.doi.org/10.1038/s41559-017-0361-xDOI Listing
December 2017

New insights into experimental visceral leishmaniasis: Real-time in vivo imaging of Leishmania donovani virulence.

PLoS Negl Trop Dis 2017 09 25;11(9):e0005924. Epub 2017 Sep 25.

Institut Pasteur, Laboratoire des Processus Infectieux à Trypanosomatidés, Département Infection et Epidémiologie, 25-28 rue du Dr Roux, Paris, France.

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.
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http://dx.doi.org/10.1371/journal.pntd.0005924DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5629011PMC
September 2017

Phenotypic Characterization of a Leishmania donovani Cyclophilin 40 Null Mutant.

J Eukaryot Microbiol 2016 11 16;63(6):823-833. Epub 2016 Jun 16.

Unité de Parasitologie Moléculaire et Signalisation, Institut Pasteur and Institut National de la Santé et de la Recherche Médicale INSERM U1201, 25 rue du Dr Roux, F-75015, Paris, France.

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.
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http://dx.doi.org/10.1111/jeu.12329DOI Listing
November 2016

Phosphoproteomic analysis of wild-type and antimony-resistant Leishmania braziliensis lines by 2D-DIGE technology.

Proteomics 2015 Sep 17;15(17):2999-3019. Epub 2015 Jun 17.

Laboratório de Parasitologia Celular e Molecular, Centro de Pesquisas René Rachou CPqRR/Fiocruz, Belo Horizonte, MG, Brazil.

Protein phosphorylation is one of the most studied post-translational modifications that is involved in different cellular events in Leishmania. In this study, we performed a comparative phosphoproteomics analysis of potassium antimonyl tartrate (SbIII)-resistant and -susceptible lines of Leishmania braziliensis using a 2D-DIGE approach followed by MS. In order to investigate the differential phosphoprotein abundance associated with the drug-induced stress response and SbIII-resistance mechanisms, we compared nontreated and SbIII-treated samples of each line. Pair wise comparisons revealed a total of 116 spots that showed a statistically significant difference in phosphoprotein abundance, including 11 and 34 spots specifically correlated with drug treatment and resistance, respectively. We identified 48 different proteins distributed into seven biological process categories. The category "protein folding/chaperones and stress response" is mainly implicated in response to SbIII treatment, while the categories "antioxidant/detoxification," "metabolic process," "RNA/DNA processing," and "protein biosynthesis" are modulated in the case of antimony resistance. Multiple sequence alignments were performed to validate the conservation of phosphorylated residues in nine proteins identified here. Western blot assays were carried out to validate the quantitative phosphoproteome analysis. The results revealed differential expression level of three phosphoproteins in the lines analyzed. This novel study allowed us to profile the L. braziliensis phosphoproteome, identifying several potential candidates for biochemical or signaling networks associated with antimony resistance phenotype in this parasite.
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http://dx.doi.org/10.1002/pmic.201400611DOI Listing
September 2015

Probing druggability and biological function of essential proteins in Leishmania combining facilitated null mutant and plasmid shuffle analyses.

Mol Microbiol 2014 Jul 3;93(1):146-66. Epub 2014 Jun 3.

Institut Pasteur, CNRS URA 2581, Unité de Parasitologie moléculaire et Signalisation, Paris, France.

Leishmania parasites cause important human morbidity and mortality. Essential Leishmania genes escape genetic assessment by loss-of-function analyses due to lethal null mutant phenotypes, even though these genes and their products are biologically most significant and represent validated drug targets. Here we overcome this limitation using a facilitated null mutant approach applied for the functional genetic analysis of the MAP kinase LmaMPK4. This system relies on the episomal expression of the target gene from vector pXNG that expresses the Herpes simplex virus thymidine kinase gene thus rendering transgenic parasites susceptible for negative selection using the antiviral drug ganciclovir. Using this system we establish the genetic proof of LmaMPK4 as essential kinase in promastigotes. LmaMPK4 structure/function analysis by plasmid shuffle allowed us to identify regulatory kinase sequence elements relevant for chemotherapeutic intervention. A partial null mutant, expressing an MPK4 derivative with altered ATP-binding properties, showed defects in metacyclogenesis, establishing a first link of MPK4 function to parasite differentiation. The approaches presented here are broadly applicable to any essential gene in Leishmania thus overcoming major bottlenecks for their functional genetic analysis and their exploitation for structure-informed drug development.
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http://dx.doi.org/10.1111/mmi.12648DOI Listing
July 2014

The Leishmania donovani chaperone cyclophilin 40 is essential for intracellular infection independent of its stage-specific phosphorylation status.

Mol Microbiol 2014 Jul 23;93(1):80-97. Epub 2014 May 23.

Institut Pasteur and Centre National de la Recherche Scientifique URA 2581, Unité de Parasitologie Moléculaire et Signalisation, 25 rue du Dr Roux, F-75015, Paris, France; Clos Group (Leishmanasis), Bernhard-Nocht-Institut für Tropenmedizin, Bernhard-Nocht-Str. 74, D-20359, Hamburg, Germany.

During its life cycle, the protozoan pathogen Leishmania donovani is exposed to contrasting environments inside insect vector and vertebrate host, to which the parasite must adapt for extra- and intracellular survival. Combining null mutant analysis with phosphorylation site-specific mutagenesis and functional complementation we genetically tested the requirement of the L. donovani chaperone cyclophilin 40 (LdCyP40) for infection. Targeted replacement of LdCyP40 had no effect on parasite viability, axenic amastigote differentiation, and resistance to various forms of environmental stress in culture, suggesting important functional redundancy to other parasite chaperones. However, ultrastructural analyses and video microscopy of cyp40-/- promastigotes uncovered important defects in cell shape, organization of the subpellicular tubulin network and motility at stationary growth phase. More importantly, cyp40-/- parasites were unable to establish intracellular infection in murine macrophages and were eliminated during the first 24 h post infection. Surprisingly, cyp40-/- infectivity was restored in complemented parasites expressing a CyP40 mutant of the unique S274 phosphorylation site. Together our data reveal non-redundant CyP40 functions in parasite cytoskeletal remodelling relevant for the development of infectious parasites in vitro independent of its phosphorylation status, and provide a framework for the genetic analysis of Leishmania-specific phosphorylation sites and their role in regulating parasite protein function.
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http://dx.doi.org/10.1111/mmi.12639DOI Listing
July 2014

Pharmacological assessment defines Leishmania donovani casein kinase 1 as a drug target and reveals important functions in parasite viability and intracellular infection.

Antimicrob Agents Chemother 2014 23;58(3):1501-15. Epub 2013 Dec 23.

Institut Pasteur and CNRS URA2581, Unité de Parasitologie Moléculaire et Signalisation, Paris, France.

Protein kinase inhibitors have emerged as new drugs in various therapeutic areas, including leishmaniasis, an important parasitic disease. Members of the Leishmania casein kinase 1 (CK1) family represent promising therapeutic targets. Leishmania casein kinase 1 isoform 2 (CK1.2) has been identified as an exokinase capable of phosphorylating host proteins, thus exerting a potential immune-suppressive action on infected host cells. Moreover, its inhibition reduces promastigote growth. Despite these important properties, its requirement for intracellular infection and its chemical validation as a therapeutic target in the disease-relevant amastigote stage remain to be established. In this study, we used a multidisciplinary approach combining bioinformatics, biochemical, and pharmacological analyses with a macrophage infection assay to characterize and define Leishmania CK1.2 as a valid drug target. We show that recombinant and transgenic Leishmania CK1.2 (i) can phosphorylate CK1-specific substrates, (ii) is sensitive to temperature, and (iii) is susceptible to CK1-specific inhibitors. CK1.2 is constitutively expressed at both the promastigote insect stage and the vertebrate amastigote stage. We further demonstrated that reduction of CK1 activity by specific inhibitors, such as D4476, blocks promastigote growth, strongly compromises axenic amastigote viability, and decreases the number of intracellular Leishmania donovani and L. amazonensis amastigotes in infected macrophages. These results underline the potential role of CK1 kinases in intracellular survival. The identification of differences in structure and inhibition profiles compared to those of mammalian CK1 kinases opens new opportunities for Leishmania CK1.2 antileishmanial drug development. Our report provides the first chemical validation of Leishmania CK1 protein kinases, required for amastigote intracellular survival, as therapeutic targets.
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http://dx.doi.org/10.1128/AAC.02022-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3957854PMC
October 2014

Alba-domain proteins of Trypanosoma brucei are cytoplasmic RNA-binding proteins that interact with the translation machinery.

PLoS One 2011 21;6(7):e22463. Epub 2011 Jul 21.

Institute of Cell Biology, University of Bern, Bern, Switzerland.

Trypanosoma brucei and related pathogens transcribe most genes as polycistronic arrays that are subsequently processed into monocistronic mRNAs. Expression is frequently regulated post-transcriptionally by cis-acting elements in the untranslated regions (UTRs). GPEET and EP procyclins are the major surface proteins of procyclic (insect midgut) forms of T. brucei. Three regulatory elements common to the 3' UTRs of both mRNAs regulate mRNA turnover and translation. The glycerol-responsive element (GRE) is unique to the GPEET 3' UTR and regulates its expression independently from EP. A synthetic RNA encompassing the GRE showed robust sequence-specific interactions with cytoplasmic proteins in electromobility shift assays. This, combined with column chromatography, led to the identification of 3 Alba-domain proteins. RNAi against Alba3 caused a growth phenotype and reduced the levels of Alba1 and Alba2 proteins, indicative of interactions between family members. Tandem-affinity purification and co-immunoprecipitation verified these interactions and also identified Alba4 in sub-stoichiometric amounts. Alba proteins are cytoplasmic and are recruited to starvation granules together with poly(A) RNA. Concomitant depletion of all four Alba proteins by RNAi specifically reduced translation of a reporter transcript flanked by the GPEET 3' UTR. Pulldown of tagged Alba proteins confirmed interactions with poly(A) binding proteins, ribosomal protein P0 and, in the case of Alba3, the cap-binding protein eIF4E4. In addition, Alba2 and Alba3 partially cosediment with polyribosomes in sucrose gradients. Alba-domain proteins seem to have exhibited great functional plasticity in the course of evolution. First identified as DNA-binding proteins in Archaea, then in association with nuclear RNase MRP/P in yeast and mammalian cells, they were recently described as components of a translationally silent complex containing stage-regulated mRNAs in Plasmodium. Our results are also consistent with stage-specific regulation of translation in trypanosomes, but most likely in the context of initiation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0022463PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3141063PMC
December 2011

Imaging host cell-Leishmania interaction dynamics implicates parasite motility, lysosome recruitment, and host cell wounding in the infection process.

Cell Host Microbe 2011 Apr;9(4):319-30

Institut Pasteur, CNRS URA 2581, INSERM AVENIR Program, Paris, France.

Leishmania donovani causes human visceral leishmaniasis. The parasite infectious cycle comprises extracellular flagellated promastigotes that proliferate inside the insect vector, and intracellular nonmotile amastigotes that multiply within infected host cells. Using primary macrophages infected with virulent metacyclic promastigotes and high spatiotemporal resolution microscopy, we dissect the dynamics of the early infection process. We find that motile promastigotes enter macrophages in a polarized manner through their flagellar tip and are engulfed into host lysosomal compartments. Persistent intracellular flagellar activity leads to reorientation of the parasite flagellum toward the host cell periphery and results in oscillatory parasite movement. The latter is associated with local lysosomal exocytosis and host cell plasma membrane wounding. These findings implicate lysosome recruitment followed by lysosome exocytosis, consistent with parasite-driven host cell injury, as key cellular events in Leishmania host cell infection. This work highlights the role of promastigote polarity and motility during parasite entry.
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http://dx.doi.org/10.1016/j.chom.2011.03.011DOI Listing
April 2011

Quantitative proteome profiling informs on phenotypic traits that adapt Leishmania donovani for axenic and intracellular proliferation.

Cell Microbiol 2011 Jul 26;13(7):978-91. Epub 2011 Apr 26.

Laboratory of Parasite Virulence Trypanosome Cell Biology Unit, Institut Pasteur, CNRS URA 2581/Institut National de la Santé et de la Recherche Médicale AVENIR Program, Paris, France.

Protozoan parasites of the genus Leishmania are important human pathogens that differentiate inside host macrophages into an amastigote life cycle stage. Although this stage causes the pathogenesis of leishmaniasis, only few proteins have been implicated in amastigote intracellular survival. Here we compare morphology, infectivity and protein expression of L. donovani LD1S grown in host free (axenic) culture, or exclusively propagated in infected hamsters, with the aim to reveal parasite traits absent in axenic but selected for in hamster-derived amastigotes through leishmanicidal host activities. Axenic and splenic amastigotes showed a striking difference in virulence and the ability to cause experimental hepato-splenomegaly in infected hamsters. 2D-DIGE analysis revealed statistically significant differences in abundance for 152 spots, with 14 spots showing fivefold or higher abundance in splenic amastigotes. Proteins identified by MS analysis include the anti-oxidant enzyme tryparedoxin peroxidase, and enzymes implicated in protein and amino acid metabolism. Analysis of parasite growth in vitro in minimal medium demonstrated increased survival of hamster-derived compared with axenic parasites under conditions that mimic the nutrient poor, cytotoxic phagolysosome. Thus, our comparative proteomics analysis sheds important new light on the biochemistry of bona fide amastigotes and informs on survival factors relevant for intracellular L. donovani infection.
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http://dx.doi.org/10.1111/j.1462-5822.2011.01593.xDOI Listing
July 2011

Identification of Leishmania-specific protein phosphorylation sites by LC-ESI-MS/MS and comparative genomics analyses.

Proteomics 2010 Nov;10(21):3868-83

Institut Pasteur, Plate-forme de Protéomique and CNRS URA 2185, Department of Structural Biology and Chemistry, Pasteur-Genopole Ile-de-France, Paris, France.

Human pathogenic protozoa of the genus Leishmania undergo various developmental transitions during the infectious cycle that are triggered by changes in the host environment. How these parasites sense, transduce, and respond to these signals is only poorly understood. Here we used phosphoproteomic approaches to monitor signaling events in L. donovani axenic amastigotes, which may be important for intracellular parasite survival. LC-ESI-MS/MS analysis of IMAC-enriched phosphoprotein extracts identified 445 putative phosphoproteins in two independent biological experiments. Functional enrichment analysis allowed us to gain insight into parasite pathways that are regulated by protein phosphorylation and revealed significant enrichment in our data set of proteins whose biological functions are associated with protein turn-over, stress response, and signal transduction. LC-ESI-MS/MS analysis of TiO(2)-enriched phosphopeptides confirmed these results and identified 157 unique phosphopeptides covering 181 unique phosphorylation sites in 126 distinct proteins. Investigation of phosphorylation site conservation across related trypanosomatids and higher eukaryotes by multiple sequence alignment and cluster analysis revealed L. donovani-specific phosphoresidues in highly conserved proteins that share significant sequence homology to orthologs of the human host. These unique phosphorylation sites reveal important differences between host and parasite biology and post-translational protein regulation, which may be exploited for the design of novel anti-parasitic interventions.
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http://dx.doi.org/10.1002/pmic.201000305DOI Listing
November 2010

Leishmania major MPK7 protein kinase activity inhibits intracellular growth of the pathogenic amastigote stage.

Eukaryot Cell 2010 Jan 2;9(1):22-30. Epub 2009 Oct 2.

Institut Pasteur, CNRS URA 2581, Laboratory of Parasite Virulence, and the Institut National de la Santé et de la Recherche Médicale Avenir Program, 25 rue du Dr. Roux, 75015 Paris, France.

During the infectious cycle, protozoan parasites of the genus Leishmania undergo several adaptive differentiation steps that are induced by environmental factors and crucial for parasite infectivity. Genetic analyses of signaling proteins underlying Leishmania stage differentiation are often rendered difficult due to lethal null mutant phenotypes. Here we used a transgenic strategy to gain insight into the functions of the mitogen-activated Leishmania major protein kinases LmaMPK7 and LmaMPK10 in parasite virulence. We established L. major and Leishmania donovani lines expressing episomal green fluorescent protein (GFP)-LmaMPK7 and GFP-LmaMPK10 fusion proteins. The transgenic lines were normal in promastigote morphology, growth, and the ability to differentiate into metacyclic and amastigote stages. While parasites expressing GFP-LmaMPK10 showed normal infectivity by mouse footpad analysis and macrophage infection assays, GFP-LmaMPK7 transgenic parasites displayed a strong delay in lesion formation and reduced intracellular parasite growth. Significantly, the effects of GFP-LmaMPK7 on virulence and proliferation were due exclusively to protein kinase activity, as the overexpression of two kinase-dead mutants had no effect on parasite infectivity. GFP-LmaMPK7 transgenic L. donovani cells revealed a reversible, stage-specific growth defect in axenic amastigotes that was independent of cell death but linked to nonsynchronous growth arrest and a significant reduction of de novo protein biosynthesis. Our data suggest that LmaMPK7 protein kinase activity may be implicated in parasite growth control and thus relevant for the development of nonproliferating stages during the infectious cycle.
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http://dx.doi.org/10.1128/EC.00196-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2805286PMC
January 2010

A dynamic map of antigen recognition by CD4 T cells at the site of Leishmania major infection.

Cell Host Microbe 2009 Jul;6(1):23-33

Institut Pasteur, G5 Dynamiques des Réponses Immunes, Inserm U668, Equipe Avenir, Paris F-75724, France.

CD4 T helper cells play a central role in the control of infection by intracellular parasites. How efficiently pathogen-specific CD4 T cells detect infected cells in vivo is unclear. Here, we employed intravital two-photon imaging to examine the behavior of pathogen-specific CD4 T cells at the site of Leishmania major infection. While activated CD4 T cells enter the inflamed tissue irrespective of their antigen specificity, pathogen-specific T cells preferentially decelerated and accumulated in infected regions of the dermis. Antigen recognition by CD4 T cells was heterogeneous, involving both stable and dynamic contacts with infected phagocytes. However, not all infected cells induced arrest or deceleration of pathogen-specific T cells, and dense clusters of infected cells were poorly accessible to migrating T cells. Thus, disparities in the dynamics of T cell contacts with infected cells and local variation in T cell access to infected cells are important elements of the host-pathogen interplay.
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http://dx.doi.org/10.1016/j.chom.2009.04.014DOI Listing
July 2009

Enhanced cloning efficiency of mouse bone marrow macrophage progenitors correlates with increased content of CSF-1 receptor of their progeny at low oxygen tension.

Microbes Infect 2003 Oct;5(12):1064-9

Hôpital Necker-Enfants-Malades, Inserm U393, Tour Lavoisier, 2e étage, 149, rue de Sèvres, 75015 Paris, France.

Mononuclear phagocytes are located in every tissue of metazoan organisms. In this extravascular space, they are designated as macrophages and are known to sense and process many signals including the local oxygen tension (PO2), which ranges from 150 mmHg at the lung apices to around 40 mmHg in mixed venous blood and most organs, and to less than 10 mmHg in tissues where long-term and dynamic remodeling processes occur. Most tissue macrophages survive and maintain their differentiated status within an environment bathed by colony-stimulating factor (CSF)-1 through the CSF-1 receptor, encoded by the Csf1r gene. In order to investigate the mRNA expression profile of macrophages as a function of PO2, we developed an in vitro model in which monocyte-derived macrophages were generated from mouse bone marrow progenitor cells grown and maintained under low (36 mmHg) or atmospheric (142 mmHg) PO2, in the presence of L929-conditioned medium (L-CM) as a source of CSF-1. We show that CSF-1-reactive C57BL/6 bone marrow cells displayed an increased cloning efficiency under a PO2 of 36, compared with 142 mmHg. Furthermore, we provide evidence of the overexpression of both CSF-1 receptor protein and mRNA by mouse monocyte-derived macrophages generated from bone marrow under low PO2.
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http://dx.doi.org/10.1016/j.micinf.2003.07.005DOI Listing
October 2003

Characterization of a putative type IV aminophospholipid transporter P-type ATPase.

Mamm Genome 2003 Jan;14(1):21-30

Hôpital Necker-Enfants Malades, INSERM U393, Tour Lavoisier, 2 ème étage, 149 rue de Sèvres, 75015 Paris, France.

The P-type ATPases comprise a well-studied family of proteins involved in the active transport of charged substrates across biological membranes. Starting from a mouse bone marrow-derived macrophage cDNA library and using a signal peptide trapping strategy, we identified a new P-type ATPase family member. We characterized the genomic structure of this gene, named Atp10d, as well as its human counterpart. The presence of P-type ATPase consensus motifs and phylogenetic analysis showed that this gene is a member of the type IV, putative amphipath transporters subfamily. We showed that this gene is expressed in kidney and placenta. We also found that the C57BL/6 strain carries a constitutive stop codon in the sequence of Atp10d exon 12, whereas 14 other inbred mouse strains show an uninterrupted reading frame at this location. This mutation in C57BL/6 should lead to a non-functional protein, suggesting that this gene may not be essential. We discuss the involvement of the Atp10d gene in the fat-prone phenotype of the C57BL/6 strain and its physical mapping within a QTL associated with HDL-cholesterol levels.
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http://dx.doi.org/10.1007/s00335-002-3032-3DOI Listing
January 2003
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