Intracellular Parasitism group

Head of the group: Dr Haralabia Boleti,  hboleti@pasteur.gr, tel: +30 210 6478879

About

The Intracellular Parasitism group studies aspects of the molecular cell biology of Leishmania donovani, the aetiological agent of visceral leishmaniasis (kala azar), and of the parasite  interaction with the host cell macrophages.The main research interests of the group focus on:

a) molecular mechanisms of Leishmania donovani, phagocytosis in professional phagocytes and the role of putative parasite virulence factors in the maturation and remodeling of the Leishmaniophorous phagosome and

b) the structural and functional characterization of specific L. donovani phosphatases.

The above studies aim to contribute to the understanding of the initial steps of the infection,after the sand fly bite, and the parasite entry by phagocytosis into the phagocytic cells of the host immune system, as well as to the identification of new potential molecular targets for drug development.

Leishmaniasis is a disease of paramount importance for public health. Globally, over 350 million people in 88 counties are threatened to be afflicted with the different forms of the disease and an estimated 12 million people are infected with 2 million new cases per year (WHO). On the European continent, parasites of the genus Leishmania (the aetiological agents of Leishmaniases), while thought to be under control, have re-emerged in some southern Mediterranean countries including the Balkans and Greece. The symptoms of Leishmaniases range from disfiguring cutaneous and muco-cutaneous lesions that can cause widespread destruction of mucous membranes, to visceral disease (VL, kala-azar), affecting the haemopoetic organs, that if left untreated is fatal.

There are no protective human vaccines for leishmaniasis and used current therapies are ineffective. The reasons are 1) increasing resistant strains, 2) high toxicity and3) high drug prices. For these reasons the WHO considers as imperative need the development of new non-toxic drugs and a vaccine to control the growing number of  leishmaniasis cases.

Research projects
  • Study of the mechanisms of phagocytosis and survival of Leishmania in mammalian host phagocytes ( A.Papadaki, H.Boleti)

The Leishmania parasites, members of the Trypanosomatidae family of protozoans with more than 20 species described so far, are transmitted to the mammalian host upon the bloodmeal of the insect vector (sandflies of the genus Phlebotomus or Lutzomyia).The first step of Leishmania interaction with the mammalian host after the sand fly bite is the entry into the phagocytic cells (macrophages, neutrophils and dendritic) of the host immune system by phagocytosis.

 Upon their internalization by macrophages, Leishmania promastigotes (the parasite forms living in the sandfly), delay phagolysosome biogenesis, in order to establish infection in an environment devoid of oxidants. Once they differentiate into amastigotes the maturation of the parasitophorous vacuole proceeds to the phagolysosome stage. Survival of the parasite in the professional phagocytes is due to interference and subversion of host signaling pathways related to many aspects of cell physiology including the ones of phagosome maturation and apoptosis.

Our studies aim to:

  1. Contribute to the understanding of the molecular mechanisms of Leishmania phagocytosis in macrophages. More specifically we investigate the possibility the parasite  L.donovani subverges directly or indirectly the spatio-temporal distribution and the signaling activity of specific phosphoinositide (PI) species in the macrophages of the host during the maturation process of the parasitophorous phagosome.
  2. The study of putative parasite virulence factors, surface or secreted, that are potentially involved in the pathogen host recognition or the maturation and remodeling of the Leishmaniophorous phagosome and the survival and replication of the parasite within it.

In these studies we follow the steps of Leishmania phagocytosis in macrophages by microscopy techniques including time-lapse fluorescence microscopy.

We use genetically modified L. donovani expressing GFP or RFP proteins and cell culture models of macrophages (Raw264.7) expressing PI binding domains fused to GFP.  Putative virulence factors are cloned and expressed  in heterologous systems  for structural and functional studies. Additionally, we generate transgenic parasite strains that overexpress the putative virulence factors and evaluate their capacity to infect and survive in macrophages by FACS and microscopy techniques.

  • Structural and functional characterization of specific L. donovani phosphatases (A.Papadaki, A.Kotopouli, H.Boleti)

Focus of our work the last 3 years has been the L.dovovani histidine acid ecto-phosphatase designated as LdMacP (LdAcP3.1_AF149839.1) and two members of the atypical lipid like dual specificity group of Leishmania phosphatases designated as LdPIP22 ( LDBPK_220120) and LdPIP33 (LDBPK_332990).

The LdMacP ecto-phosphatase initially identified by molecular cloning (Shakarian, A.M., et al.2002), as a protein homologous to the secreted (LdSAcP1) histidine acid phosphatase (Shakarian, A.M., et al.1997) from L. donovani, is a membrane bound enzyme suggested to confer the tartate resistant acid ecto-phosphatase activity implicated in promastigote virulence. Our studies aim to complete its structural and functional characterization and define its role in parasite virulence.

We recently initiated studies on the two aDSPs LdPIPh22 and LdPIPh33 putative PI phosphatases as potential drug targets. These molecules belong to the Atypical lipid phosphatase, family described in bacteria and lower eucaryotes (Beresford, N.J. et al. 2010) and are homologues to the Mptb M.tuberculosis virulence factor. Bacterially expressed rLdPIPh22-8His protein, exhibits phosphatase activity in vitro, when using the synthetic p-nitrophenyl-phosphate [pNPP] as substrate. The substrate specificity of the enzyme is under investigation. A number of techniques are employed to study the functional and structural properties of the two proteins.

  • Evaluation of the Leishmania tarentolae LEXSY system as alternative recombinant protein expression system to express mammalian membrane transporters for structural and functional analysis.    (A.Doukas, H.Boleti)

We use the L. tarentolae pLexsy eukaryotic protein expression system (Breitling R., et al & Alexandrov K. 2002; Basile G, Peticca M . 2009) to express mammalian nucleobase membrane transporters. The L. tarentolae system is usable at biosafety S1 conditions being non pathogenic to humans, requires simple nutrients for growth in culture, allows efficient N-glycosylation of mammalian proteins (using a similar biantennary glycan pattern, which is absent in yeast or Drosophila) and displays physical auxotrophy on several amino acids, which facilitates potential structural studies. We also use this system to produce large quantities and purify from the L.tarentolae extracellular medium active enzymes for our studies.

Members

Head of the group

Post-doctoral fellows

PhD students

MSc students

Graduates and former members

  • Αmalia Papadaki, veterinerian, MSc epidemiology, University of Ioannina, PhD in Molecular Cellular Parasitology (2015)
  • Μathias Richard, microbiologist, University of Montpellier,  MSc thesis in Pathogen host-Interaction (2015)
  • Anargyros Doukas, chemist, University of Athens, MSc thesis in Microbial Biotechnology (2015)
  • Christos Karabellas, BSc Biological Sciences, London Metropolitan University, practical training (2014-2015)
  • Anastasia Kotopouli, Chemist, University of Athens, MSc thesis in Biochemistry (2014)
  • Maria P. Kotini, Biologist, Diploma work (2009)
Funding

Funding (2010-2015)

  • IKYDA 2014 PROGRAM for PROMOTION OF EXCHANGE AND SCIENTIFIC COOPERATION GREECE -GERMANY. ProjectTitle: LeishPhospho: Molecular characterization of specific phosphatases from the protozoan parasites Leishmania donovani and investigation of their potential as drug targets .(2014-2015)

German Partner: Dr Maja Kohn -EMBL, Heidelberg, Germany

Budget for ‘Intracellular parasitism team’:10,000 €

  • PLATON : BILATERAL R&T COLABORATION GREECE-FRANCE /GreekGeneral Secretariat for Research and Technology

ProjectTitle: LeishPhosphoTox: Molecular and functional characterization of specific phosphatases from the anthropozoonotic intracellular protozoan parasites Leishmania donovani and Toxoplasma gondii and investigation of their role in the parasite interaction with host cells; Exploration of their potential as drug targets. (2013-15)

French partner:  Dr Isabelle Tardieux /Co-Head of the «Barriers and Pathogens» Institut Cochin, INSERM U-1016.

Budget for ‘Intracellular parasitism team’:30,000 €

  • KRIPIS : DEVELOPMENT GRANTS FOR RESEARCH INSTITUTIONS/ GreekGeneral Secretariat for Research and Technology

Project Title: Infectious Diseases and Neurodegenerative in the 21st century. From the study of basic mechanisms in the development of translational research and cutting edge methodologies targeting diagnosis, prevention and treatment. Subproject EE2.5: Mechanisms of L.donovani parasite survival in host macrophages and identification of virulence factors. Subproject EE 4.5:  Identification, validation characterization of potential molecular targets for anti-leishmanial drug development. (2013-15)

Coordinator: Hellenic Pasteur Institute.

Budget for ‘Intracellular parasitism team’: 42,900 €

  • THALES: Ministry of Education Lifelong Learning and Religion / Project code no 80805

Project Title: EVOTRANS “Membrane transport: structure function and evolutionary relationships” Subproject: “Study of the family of NAT/NCS2 transporters to understand mechanisms of development of different specificities between homologous transporters with high evolutionary conservation: 1.4, NAT Expression of homologous NAT mammalian transporters in Leishmania tarentolae».  (2012-2015)

Coordinator: Prof. E.Friligos,  University of Ioannina.

Budget for ‘Intracellular parasitism team’: 40,000 €

  • Sponsorship Genesis Pharma: Project Title: Study of molecular mechanisms by which the protozoan parasite Leishmania donovani infectes humans and animals. Intracellular parasitism group. (2011-2012).

Budget: 15,000 €

Selected Publications

Selected publications

  • Papadaki A, Politou AS, Smirlis D, Kotini MP, Kourou K, Papamarcaki T, Boleti H. (2015). The Leishmania donovani histidine acid ecto-phosphatase LdMAcP: insight into its structure and function. Biochem J. 467(3):473-86
  • Athanasopoulos A, Boleti H, Scazzocchio C, Sophianopoulou V. (2013). Eisosome distribution and localization in the meiotic progeny of Aspergillus nidulans. Fungal Genet Biol. 53:84-96.
  • Boleti H, Smirlis D, Dalagiorgou G, Meurs EF, Christoforidis S, Mavromara P. (2010). ER targeting and retention of the HCV NS4B protein relies on the concerted action of multiple structural features including its transmembrane domains. Mol Membr Biol. 27(1):50-74
  • Smirlis D, Boleti H, Gaitanou M, Soto M, Soteriadou K. (2009). Leishmania donovani Ran-GTPase interacts at the nuclear rim with linker histone H1. Biochem J. 424(3):367-74.
  • Vassilaki N, Boleti H, Mavromara P. (2008). Expression studies of the HCV-1a core+1 open reading frame in mammalian cells.Virus Res. 133(2):123-35.
  • Vassilaki N, Boleti H, Mavromara P. (2007).Expression studies of the core+1 protein of the hepatitis C virus 1a in mammalian cells. The influence of the core protein and proteasomes on the intracellular levels of core+1. FEBS J. 274(16): 4057-74
  • Ibrahim-Granet, O., Philippe,B., Boleti, H., Boisvieux-Ulrich, E.,  Prévost, MC., Grenet, D., Stern, M., and JP Latgé. (2003). Phagocytosis and intracellular fate of Aspergillus fumigatus conidia in alveolar macrophages. Infection and Immunity, 71(2):891-903
  • Boleti, H., Ojcius, D. and Dautry-Varsat, A. (2000). Fluorescent labelling of  intra-cellular bacteria in living host cells. J. Microbiological Methods, 1;40 (3): 265-274
  • Poupel, O., Boleti H., Axisa, S., and Tardieux, I. (2000). Toxofilin, a novel actin binding protein from Toxoplasma gondii, sequesters actin monomers and caps actin filaments. J. Mol. Biol. of the Cell, 11(1): 355-68
  • Boleti, H., Benmerah, A., Ojcius, D., Cerf-Bensussan,N., Dautry-Varsat, A. (1999). Chlamydia infection of epithelial cells expressing dynamin and Eps15 mutants:clathrin-independent entry into cells and dynamin-dependent productive growth. J. of Cell Science, 112:1487-1496
  • Wittmann T., Boleti H., Antony  C., Karsenti E, and Vernos I. (1998). A leucine zipper targets Xklp2 to a dynein/dynactin containing complex that accumulates at microtubule minus ends during mitosis. J. Cell Biol. 143 (3), 673-685
  • Boleti H., Karsenti E., Vernos I. (1996). Xklp2, a new Xenopus centrosomal Kinesin Like Protein Required for centrosome separation during mitosis. Cell, 84: 49-59