Parasitology and Mycology Department Activity Report
DIRECTORS: Odile MERCEREAU-PUIJALON & Robert MENARD
The Department of Parasitology and Mycology dedicates its research activities to protozoan parasites, fungi and blood-feeding insect hosts/vectors. The parasites and fungi studied are responsible for human or livestock diseases of considerable public health impact. The Department’s scientific objectives are to dissect the relationships between these unicellular eukaryotic microorganisms, their hosts and their environment with the aim to develop novel approaches and tools to prevent, control and treat these infections and diseases. Importantly, the Department has a strong component on health-oriented research. Basic research on pathogen and blood-feeding insect vector biology is combined with malaria vaccine development, drug discovery in malaria, leishmaniasis and aspergillosis, and field-based work on parasite diversity and drug resistance.
A comprehensive research programme is conducted on malaria, embracing all phases of the biological cycle of the Plasmodium parasite in the Anopheles host(s) & vector(s) and the mammalian host, for both human and rodent malaria parasites. The programme involves four Units (MBG. BHPI. PMI, GGIV), one Laboratory (MV) and one Anopheles-dedicated Platform (CEPIA). Research on Trypanosomes is conducted in one Unit, focused on the biology of T. brucei during its entire life cycle (TCB). One Laboratory (IPI) and one G5 group (PV) work on Leishmania spp. in rodent hosts or in axenic culture systems. One Unit is dedicated to research on pathogenic fungi (Aspergillus).
The Department has a cutting edge expertise in the area of cellular and molecular biology of parasites, fungi and Anopheles. Rodent parasite species and human pathogens are studied, capitalizing on the on-campus availability of the entire life cycles of the parasites and state of the art imaging technology, including in vivo imaging. Multidisciplinary approaches are used to unravel the basis of the parasitic mode of life, the survival strategies of the various eukaryotic microbes, invasion of the host, as well as the factors governing transmission from and to the blood-feeding insect. Research aims at dissecting antigenic variation, erythrocyte membrane remodeling and proteolytic maturation of surface antigens of malaria parasites, cell wall biosynthesis in A. fumigatus, biogenesis of the flagellum in T. brucei, signaling underlying developmental programs of Leishmania parasites. Innovative methodologies for conditional mutagenesis of Plasmodium genes have been developed. Novel models have been established in order to explore host entry and dissemination of Plasmodium, Leishmania or Aspergillus. High-throughout content and live imaging are used to study the dynamic cellular interactions of the Plasmodium parasites with their host during invasion and establishment in the liver, the development of Leishmania amastigotes within host cells in vitro as well as in the skin and in the liver.
Natural vector and parasite populations are studied to identify genetic mechanisms of vector resistance against malaria parasites. Genetic mapping studies of wild Anopheles mosquitoes in Africa are used to identify the chromosomal loci that control mosquito infection by the parasites. Genomics, cell biology and biochemistry are used to understand the mechanisms that make mosquitoes susceptible or resistant to malaria parasite infection. Malaria-resistant mosquitoes are frequent in nature, and one control strategy could involve promoting the spread of naturally resistant mosquitoes in the natural population.
Malaria physiopathology is explored in vivo in a rodent model and using in vitro models for rosetting or cytoadhesion to chondroitin sulfate A. Mechanisms of parasite clearance are dissected using the unique ex vivo perfused human spleen experimental model as well as in a humanized mouse model. These studies are combined with sero-epidemiological studies in various endemic settings.
Several P. falciparum vaccine candidates have been discovered in the Department, some of them being currently in human phase 2b trial, others in human Phase 1 trial. Novel promising drug targets have been identified and drug-screening methodologies developed, allowing the establishment of drug discovery projects against Plasmodium, Leishmania and Aspergillus.
Overall 140 persons work in the Department, including 40 scientists, 54 postdoctoral fellows and PhD students and 44 technicians and administrative personnel. Collaborative studies are conducted with several units from the Institut Pasteur Network, in particular with scientists affiliated to the Department and working in the Instituts Pasteur from French Guiana, Senegal, Madagascar and Cambodia, as well as with Research Institutions in Mali and Burkina Faso.
2010 HIGHLIGHTS
Aspergillus
- GPI-anchored enzymes are essential for Aspergillus fumigatus
- Demonstration of specific O- and N-glycosylation pathways in A. fumigatus
- Characterization of the first fungal beta 1,3 branching enzymes in A. fumigatus
- Importance of the extracellular matrix during fungal lung invasion
- Characterizing the role of RNA silencing components in Cryptococcus neoformans
Malaria Biology and Genetics
- Demonstration of Plasmodium development in the skin of the host.
The existence of a pre-erythrocytic phase of Plasmodium infection was discovered in 1948. It was demonstrated at that time that sporozoites, the parasite stage injected by mosquitoes, transformed into merozoites, the parasite stage that invades red blood cells and causes the symptoms of malaria, inside hepatocytes. Since 1948, sporozoites of Plasmodium species that infect mammals were thought out develop (differentiate into merozoites) only inside hepatocytes. We have found that in species that infect rodents, sporozoites transform into merozoites not only in the liver but also in the skin, at the site of mosquito injection. These findings unveil a novel developmental stage in the parasite life cycle.
Biology of Host-Parasite Interactions
- Recombinant expression and structural analysis of the extracellular domain of the major virulence surface protein PfEMP1 (var2CSA) shows that inter DBL domain interactions are crucial for functional high affinity binding to the host ligand CSA.
- Spatial nuclear compartmentalisation is a major feature of gene expression. We have visualized for the first time the transcriptional active nuclear compartments in P. falciparum blood stages using BrUTP labeling combined with IF analysis. We show that the perinuclear space contains repressive but also permissive zones for differentially expressed virulence and rDNA genes
Parasite Molecular Immunology
- The Duffy blood group antigen is considered the obligate portal of entry of Plasmodium vivax into human red blood cells. This paradigm is severely questioned by our observation of common P. vivax infection and P.vivax malaria disease in Duffy-negative people in Madagascar, where human admixture has created a full range of Duffy erythrocyte expression phenotypes. Multiple P. vivax strains have the capacity to infect Duffy-negative people, suggesting that the parasite has evolved locally to bypass the Duffy infection hurdle. These findings current challenge vaccination strategies and raise concerns about potential spreading of such strains out of Madagascar into Duffy negative populations of Africa.
- Rosetting, the binding of uninfected red cells onto the surface of the infected red cell, is a cytoadherence phenotype associated with severe malaria in African children. Rosetting is mediated by a subset of variant adhesins. Little is known on the acquisition of antibodies to these adhesins and their serologic relationship. Development of monovariant cultures expressing an allele of a PfEMP1 adhesion domain showed that humans acquire an antibody repertoire to non-overlapping, variant-specific surface serotypes.
Malaria Vaccinology
- In recent years, we have successfully implemented seven vaccine trials in humans in phase I and phase IIb with two of our patented vaccine candidates and, by the end of 2010 we have initiated 3 additional trials with 3 of our patented vaccine candidates.
- The presence of IgG3 anti-MSP3 as anti-SR11.1 were found to have a major predictive effect against malaria infection in children.
- We have characterized a novel Ag, SR11.1, that combines the features of LSA3 and MSP3 and induces protection against mosquito challenges by P. yoelii in mice and by P. falciparum in primates.
- We have identified a novel hepatoma cell line fully susceptible to P.falciparum Liver Stage development, the least known phase of the Plasmodium life cycle.
Trypanosome Cell Biology
- Discovery of a novel differentiation step in the development of Trypanosoma brucei in salivary glands of the Tse tse fly. The parasites use a combination of divisions that are symetrical (to colonise the epithelium) and asymetrical (to generate infectious parasites) to ensure continuous production of parasites in the saliva and optimal transmission.
Parasite Virulence
- Publication of a series of papers revealing that parasite-specific phosphorylation of Leishmania stress proteins is essential for parasite survival
Immunophysiology and Intracellular Parasitism
- A novel integrative approach combining luciferase-expressing Leishmania imaging with a very sensitive quantitative analysis of both Leishmania and mouse transcripts, was developed. It allows to record in real time the Leishmania major and L. donovani amastigote population size fluctuations and the mouse transcript abundance accounting for these fluctuations.
Departmental Platform: Centre for the Production and Infection of Anopheles
- Confirmation of our procedures for P. falciparum gametocyte culture and subsequent mosquito infection.
- Implementation of new procedures for in vitro production of P. falciparum ookinete
FUNDING AND NETWORKING
Research Projects of the Department were supported by the EU FP7 Life science Programme-funded Networks of excellence BioMalPar and EviMalar, and the grants PreMalStruct, ESF Fuminomics and LEISHDRUG, the Gates Foundation, the Howard Hughes Medical Institute, the National Institutes of Health, USA, the French Ministry of Foreign affairs, the Agence Nationale pour la Recherche, INSERM, CNRS, Institut Pasteur, the ANR-Carnot programme and the DIM Malinf-Région Ile-de-France.
2010 EVENTS
Organization of an EU conference on Neglected Protozoan Diseases hosting more than 300 scientists from industry, academia, and governments
HONORS AND AWARDS in 2010
ERC advanced grant to Artur Scherf on "Monoallelic expression in malaria parasites"
HFSP and EMBO post doc fellowship for Nicolai Siegel
Best poster prize at FASEB meeting “Biology of cilia and flagella”, Saxtons Rivers, VT, USA, to Daria Julkowska for her work on Trypanosome flagellum purification
2010 PUBLICATIONS
- Gastebois A, Mouyna I, Simenel C, Clavaud C, Coddeville B, Delepierre M, Latgé JP, Fontaine T. Characterization of a new beta(1-3)-glucan branching activity of Aspergillus fumigatus. J Biol Chem. 2010 Jan 22;285(4):2386-96. PMID: 19948732
- Mouyna I, Kniemeyer O, Jank T, Loussert C, Mellado E, Aimanianda V, Beauvais A, Wartenberg D, Sarfati J, Bayry J, Prévost MC, Brakhage AA, Strahl S, Huerre M, Latgé JP. Members of protein O-mannosyltransferase family in Aspergillus fumigatus differentially affect growth, morphogenesis and viability. Mol Microbiol. 2010 Jun 1;76(5):1205-21. PMID: 20398215
- Janbon G, Maeng S, Yang DH, Ko YJ, Jung KW, Moyrand F, Floyd A, Heitman J, Bahn YS. Characterizing the role of RNA silencing components in Cryptococcus neoformans. Fungal Genet Biol. 2010 Dec;47(12):1070-80. PMID: 21067947
- Gueirard P, Tavares J, Thiberge S, Bernex F, Ishino T, Milon G, Franke-Fayard B, Janse CJ, Ménard R, Amino R. Development of the malaria parasite in the skin of the mammalian host. Proc Natl Acad Sci U S A. 2010 Oct 26;107(43):18640-5. PMID: 20921402
- Zhang M, Fennell C, Ranford-Cartwright L, Sakthivel R, Gueirard P, Meister S, Caspi A, Doerig C, Nussenzweig RS, Tuteja R, Sullivan WJ Jr, Roos DS, Fontoura BM, Ménard R, Winzeler EA, Nussenzweig V. The Plasmodium eukaryotic initiation factor-2alpha kinase IK2 controls the latency of sporozoites in the mosquito salivary glands. J Exp Med. 2010 Jul 5;207(7):1465-74. PMID: 20584882
- Mancio-Silva L, Zhang Q, Scheidig-Benatar C, Scherf A. Clustering of dispersed ribosomal DNA and its role in gene regulation and chromosome-end associations in malaria parasites. Proc Natl Acad Sci U S A. 2010 Aug 24;107(34):15117-22. PMID: 20696894
- Srivastava A, Gangnard S, Round A, Dechavanne S, Juillerat A, Raynal B, Faure G, Baron B, Ramboarina S, Singh SK, Belrhali H, England P, Lewit-Bentley A, Scherf A, Bentley GA, Gamain B. Full-length extracellular region of the var2CSA variant of PfEMP1 is required for specific, high-affinity binding to CSA. Proc Natl Acad Sci U S A. 2010 Mar 16;107(11):4884-9. PMID: 20194779
- Ménard D, Barnadas C, Bouchier C, Henry-Halldin C, Gray R, Ratsimbasoa A, Thonier V, Carod J-F, Domarle O, Colin Y, Bertrand O, Picot J, King CL, Grimberg BT, Mercereau-Puijalon O, Zimmerman PA. Plasmodium vivax Clinical Malaria is Commonly Observed in Duffy-negative Malagasy People. Proc Natl Acad Sci U S A. 2010 Mar 30;107(13):5967-71. PMID: 20231434.
- Vigan-Womas I, Guillotte M, Juillerat A, Vallières C, Lewit-Bentley A, Tall A, Baril L, Bentley GA, Mercereau-Puijalon O. Allelic diversity of the Plasmodium falciparum erythrocyte membrane protein 1 entails variant-specific red cell surface epitopes. PLoSONE 6(1):e16544/doi:10.1371/ journal.pone.0016544
- Arnold L, Tyagi RK, Mejia P, Van Rooijen N, Pérignon JL, Druilhe P. Analysis of innate defences against Plasmodium falciparum in immunodeficient mice. Malar J. 2010 Jul 9;9:197. PMID: 20618960
- Rotureau B, Subota I, Bastin P. Molecular bases of cytoskeleton plasticity during the Trypanosoma brucei parasite cycle. Cell Microbiol. 2010 Dec 16. doi: 10.1111/PMID: 21159115.
- Molla-Herman A, Ghossoub R, Blisnick T, Meunier A, Serres C, Silbermann F, Emmerson C, Romeo K, Bourdoncle P, Schmitt A, Saunier S, Spassky N, Bastin P, Benmerah A. The ciliary pocket: an endocytic membrane domain at the base of primary and motile cilia. J Cell Sci. 2010 May 15;123(Pt 10):1785-95. PMID: 20427320 (4ème article le plus téléchargé de l’année).
- Hem S, Gherardini PF, Osorio y Fortéa J, Hourdel V, Morales MA, Watanabe R, Pescher P, Kuzyk MA, Smith D, Borchers CH, Zilberstein D, Helmer-Citterich M, Namane A, Späth GF. Identification of Leishmania-specific protein phosphorylation sites by LC-ESI-MS/MS and comparative genomics analyses. Proteomics. 2010 Nov;10(21):3868-83. PMID:20960452
- Morales MA, Watanabe R, Dacher M, Chafey P, Osorio y Fortéa J, Scott DA, Beverley SM, Ommen G, Clos J, Hem S, Lenormand P, Rousselle JC, Namane A, Späth GF. Phosphoproteome dynamics reveal heat-shock protein complexes specific to the Leishmania donovani infectious stage. Proc Natl Acad Sci U S A. 2010 May 4;107(18):8381-6. PMID:20404152
- Lecoeur H, Buffet PA, Milon G, Lang T.2010. Early curative applications of the aminoglycoside WR279396 on an experimental Leishmania major-loaded cutaneous site do not impair the acquisition of immunity. Antimicrob Agents Chemother. 2010 Mar; 54:984-90. PMID:20038619
- De La Llave E, Lecoeur H, Besse A, Milon G, Prina E, Lang T.2010.A combined luciferase imaging and reverse transcription polymerase chain reaction assay for the study of Leishmania amastigote burden and correlated mouse tissue transcript fluctuations.Cell Microbiol. 2010 Sep 16. doi: [Epub ahead of print] Cell Microbiol. 2011 Jan;13(1):81-91PMID:20846338
- Toty C, Barré H, Le Goff G, Larget-Thiéry I, Rahola N, Couret D, Fontenille D. Malaria risk in Corsica, former hot spot of malaria in France. Malar J. 2010 Aug 12;9:231. PubMed PMID: 20704707
- Chertemps T, Mitri C, Perrot S, Sautereau J, Jacques JC, Thiery I, Bourgouin C, Rosinski-Chupin I. Anopheles gambiae PRS1 modulates Plasmodium development at both midgut and salivary gland steps. PLoS One. 2010 Jul 12;5(7):e11538. PubMed PMID: 2063494.