Haralabia Boleti | Intracellular Parasitism
My main interests lie in the research of pathogen-host cell interactions using molecular cellular biology and modern light microscopy imaging techniques.
Research in the “Intracellular parasitism group”, which I lead, focuses on aspects of the Molecular Cellular Biology of the flagellated protozoan parasite Leishmania donovani (L. donovani), and its interaction with the macrophages of the mammalian host. Aim of this research work is to contribute to the understanding of the molecular mechanisms underlying the initial stages of this parasitic infection as well as to identify and characterize new potential molecular targets for the development of new anti-leismanial drugs.
The single celled eukaryotic protozoan parasite Leishmania with more than 20 species described belongs to the Trypanosomatidae order of the kinetoplastid class of protists. Parasites of the Leishmania genus are dimorphic, living as flagellated forms (promastigotes) in the gut of their sandfly vector and as aflagellated forms (amastigotes) mainly inside phagolysosomes of mammalian host phagocytes. Leishmania promastigotes (metacyclics) are transmitted to the mammalian host upon the blood meal 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 (neutrophils, macrophages, and dendritic) of the host immune system by phagocytosis. Macrophages are the cells in which the parasite establishes infection and by which it is carried in organs distant from the site of infection as the liver, spleen and bone marrow in the case of visceral Leishmaniasis. Survival and propagation 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 phagolysosome biogenesis and apoptosis.
Infection of mammals with anthroponotic or anthropozoonotic Leishmania species causes a wide spectrum of human cutaneous (CL) and visceral (VL) diseases known as Leishmaniases. An estimated 300–500 000 cases of VL, the most severe form of the disease which is fatal if not treated, and over 20 000 deaths occur annually. Although several drugs against the different forms of Leishmaniasis are available, there is an urgent need to develop new ones due to severe side effects and high cost of the existing chemotherapeutics and the development of drug resistant parasite strains. It worths mentioning that no efficient vaccine against the different forms of Leismaniases exist.
Study of molecular mechanisms of Leishmania donovani (L. donovani) establishment of infection in professional phagocytes with focus on the role of parasite surface (i.e ecto-enzymes) or secreted molecules in the establishment of parasite life in the modified Leishmaniophorous phagolysosome.
With advanced microscopy methods we image the spatio-temporal distribution of phosphoinositides (PIs) and other molecular markers of phagosome maturation on the membrane of the Leishmaniophorous phagosome from the moment of promastigote engulfment up to 4 hrs after the initiation of parasite phagocytosis (Papadaki Α. PhD thesis 2015).
In parallel, we focus on studies of surface (ecto-phosphatases) or secreted parasitic molecules with aim to understand their possible role in the biogenesis of the Leishmaniophorous phagolysosome and the survival of Leishmania cells in it. We recently published a study on the characterization of an L. donovani membrane bound acid phosphatase (LdMAcP) that is localized at the external surface of the paraite’s plasma membrane and contributes to the parasitic virulence (Papadaki A. et al., Boleti H. 2015).
As secreted L. donovani molecules we study the structures and role of a protein from the family of sorting nexins discovered in a proteomics analysis of the promastigote secretome (Silverman JM et al. 2008) (Kourounis et al 2016, poster in the 67th conference of the HSBMB).
Development of advanced microscopy protocols for imaging (in vitro or in vivo) intracellular pathogens with the use of digital image/video processing/analysis software. In our studies we use genetically modified Leishmania strains expressing GFP or RFP fluorescent proteins and mammalian macrophage cell lines (mouse Raw264.7) expressing GFP or YFP hybrid polypeptides with PI binding domains (PX or PH domains) or hybrid fluorescent GTPases (i.e. Rab5, Rab7) molecular markers of phagolysosome biogenesis (Papadaki A. PhD thesis 2015).
Structural and functional characterization of specific L. donovani phosphatases as candidate anti-Leishmanial drug targets. Main focus of our work is the study of the LdMacP (LdAcP3.1_AF149839.1) Histidine Acid ecto-phosphatase from L. donovani (Shakarian, A.M., et al.2002, Papadaki et al. 2015), and of two members from a family of phosphoinositide phosphatases expressed only in microorganisms, homologs of the MptpB virulence factor from Mycobacterium tuberculosis (Beresford, N.J. et al. 2010). The two L. donovani phosphatases under study are encoded by the Open Reading Frames (ORF) LDBPK_220120 and LDBPK_332990.
Validation of the Leishmania tarentolae (L. tarentolae) organism (non pathogenic to humans) as an alternative expression system for recombinant eukaryotic membrane proteins (Biotechnological applications) and as an in vitro system for the screening of anti-leismanial compounds.
We use the L.tarentolae promastigote cells a) for expression of mammalian nucleobase transporters (ΕVOTRAΝS project; b) expression of the human PRL3 phosphatase (IKYDA program, collaboration with the lab of Μaja Köhn EMBL, Heidelberg) and c) for the production of L. donovani phosphatases for structural and functional analysis.
For the screening of candidate anti-leismanial drugs we have generated genetically modified L. tarentolae strains that express the red fluorescent protein mcherry with which, according to published protocols our group is open for collaboration for the screening of compounds with antiparasitic properties.
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. ( PDF )2015
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. ( PDF )2013
Boleti H., Smirlis D., Dalagiorgou G., Meurs E., 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. Memb. Biol. 27(1):50-74. ( PDF )2010
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. 10;424(3):367-74. ( PDF )2009
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. ( PDF )2008
Vassilakι Ν, Boleti Η. and 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. ( PDF )2007
Boleti H. & Robotis, J.F (2004). Viral Hepatitis. xPharm 1.0, edited by S.J. Enna dn David, B. Bylund, Published by, Inc. Elsevier.
Boleti H. (2004). Chlamydia Infections. xPharm 1.0, edited by S.J. Enna dn David, B. Bylund, Published by, Inc. Elsevier. ( Link )
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. ( PDF )2003
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. ( PDF )
Poupel, O., Boleti H., Axisa, S., and Tardieux, I. (2000). Toxfilin, 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. ( PDF )
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. ( PDF )1999
Amalia Papadaki, Medical School, U. of Ioannina, defended in April 2015Title: Study of the molecular mechanisms underlying Leishmania spp. survival within the phagocytes of the mammalian host ( PDF )
Anastasia Kotopouli, Chemistry Dep., U. of Athens (MSc Program Biochemistry), defended in February 2014
Title: Cloning, molecular and functional characterization of the putative phosphoinositide phosphatase LdPIP22 from the parasitic protozoan Leishmania donovani ( PDF )
Anargyros Doukas, Biology Dept, U. of Athens (MSc program Microbial Biotechnology), defended in January 2015
Title: Construction and characterization of transgenic strains of the protozoan Leishmania tarentolae for heterologous expression of the red fluorescent protein mCherry and nucleobase transporter rSNBT1 from Rattus norvegicus. ( PDF )
Mathias Richard, Faculté de Sciences, U. of Montpellier, MSc BioPMBB-IMH, defended in June 2015
Title: Molecular Biology of the protozoan parasites Leishmania donovani: Cloning of an L. infantum putative phosphatase into plasmid vectors for expression of His-tagged or mCherry-tagged protein in bacteria, mammalian and Leishmania cells. ( PDF )
Olivia Paraskevi Tziouvara, Biology Dept, U. of Athens (MSc program Applications of Biology in Medicine) to be defended at the end of 2016/beginning of 2017
Title: Molecular and functional characterization of the Tyrosine and phosphoinositide phosphatase LdTyrPIP_22, from the protozoan parasite Leishmania donovani.
Drosos Kourounis, Biology Dept, U. of Athens (MSc program Applications of Biology in Medicine) to be defended at the end of 2016/beginning of 2017
Title: Cloning and molecular characterization of the secreted Phosphoinositide Binding nexin-like protein LdPIΒPnex from the protozoan Leishmania donovani
Maria Kotini, Diploma thesis, Biology Dept, of Athens, Defended in Nov. 2009.
Title: Cosntruction and characterization of trasngenic parasites Leishmania donovani expressing the red fluorescent protein mRFP1and of anti-mRFP1 polyclonal antibody.
2011-2017-Lectures «Leishmania tarantolae, a eucaryotic cell system with important biotechnological applications». MSc program «MICROBIAL BIOTECHNOLOGY», Department of Biology, University of Athens
2015 – Lectures «Host-Cell mechanisms that become targets of intracellular pathogens» MSc program «APPLICATIONS OF BIOLOGY IN MEDICINE» Biology Department, University of Athens
2011-2014 – Lectures «Study of Cellular Organization with modern Fluorescene Microscopy techniques» MSc program “BIOCHEMISTRY”, Department of Chemistry, University of Athens
Cutting edge technologies and their contribution to the Biomedical Sciences»
3-day practical courses for Biology Teachers of Secondary School education. An activity for diffusion of Advanced Scientific Knowledge to selected groups of society. ( Poster )
IKYDA (2014-2015) – PROGRAM of PROMOTION OF EXCHANGE AND SCIENTIFIC COOPERATION GREECE – GERMANY. Project title: LeishPhospho: Molecular characterization of specific phosphatases from the intracellular protozoan parasite Leishmania donovani and exploration of their potential as drug targets. Scientific Responsible of Greek team: Dr Haralabia Boleti, Scientific Responsible of the German team: Dr Μaja Köhn
Sponsorship τof the Pharmaceutical company Abbvie to the research team of Intracellular parasitism for research on Leishmaniasis. Scientific Responsible: Dr Haralabia Boleti. Budget: 5,000 €
PLATON-BILATERAL S & T COOPERATION GREECE –FRANCE (2013-2015).
Project title: 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.
Scientific Responsible of Greek team: Dr Haralabia Boleti,
Scientific Responsible of French team: Dr Isabelle Tardieux
Budget: 30,000 €
ΚΡΗΠΙΣ Ι (2013-2015): GREEK GENERAL SECREATARIAT of RESEARCH and TECHNOLOGY
Project title: Infectious and Neurodegenerative diseases in the 21st century. From the study of basic mechanisms to development of translational research and cutting edge technologies with aim to improve preventive diagnosis and treatment. Subproject EE2.5: Survival mechanisms of the L. donovani parasites in the macrophage host cell and identification of virulence factors. Subproject EE 4.5: Identification, evaluation and characterization of candidate molecular targets for the development of anti-leismanial drugs. (coordinations: Hellenic Pasteur Insitute).
Budget for Intracellular parasitism group: 42,900 €
THALES (2012-2015): GREEK MINISTRY OF EDUCATION
Project title: EVOTRANS-Membrane Transport: Sturcture-Function relationships and Evolution. Project coordiantor: E. Frilliggos. University of Ioannina. Subproject Π.Ε.1-4 scientific responsible: H. Boleti
Budget for Intracellular Parasitism group: 40,000 €
Sponsorship by the pharmaceutical company Genesis Pharma: Project titel: Study of the molecular mechanisms by which the protozoan parasite Leishmania donovani infects humans and domestic animals to cause the disease of Kala azar. Scientific responsible: Dr Haralabia Boleti
Budget: 15.000 €
Poster award at the 67ο Panhellenic conference of the Hellenic Society of Biochemistry and Molecular Biology, Ioannina, (25-27 Nov. 2017). Title: LdPIΒPnex, a secreted nexin-like protein from the protozoan parasite Leishmania donovani. Authors:Drosos Kourounis, Amalia Papadaki, Olympia Tziouvara, Haralabia Boleti, Intracellular parasitism group, Hellenic Pasteur Institute