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The leishmaniases are a group of diseases transmitted by the sand fly and caused by various species of the protozoan parasite Leishmania. A broad spectrum of clinical manifestations with varying severity can be observed. Visceral leishmaniasis (VL, kala-azar) can even cause death if left untreated. The leishmaniases are considered as a major public health problem with great social and economic impact. In our country approximately 50 VL cases are reported each year. The prevalence in dogs, the reservoir host, is up to 34 % in endemic areas like Attiki. However, it is very difficult to provide realistic estimates since official reporting of cases although obligatory is not systematic.
The World Health Organization emphasizes the need for the development of new non-toxic drugs as well as of an effective vaccine, in order to control the continuously increasing number of cases. Additionally the development of reliable and sensitive diagnostic tools/ methods for early detection of the parasite is of major importance. In order to contribute to the fight against this major health issue we focus our research in the following research fields:
1. THE UNDERSTANDING OF THE MOLECULAR AND CELLULAR BASIS OF PATHOGENESIS: STUDY OF PATHOGEN-HOST INTERACTION CRITICAL FOR PATHOGEN EVASION
Delineation of pathogen factors involved in infectivity
In particular we study molecules playing an important role in the parasite life-cycle and infectivity such as linker histone H1 and the leishmanial Ran protein, which belongs to the Ras superfamily. To this end we either generate transgenic parasites that over-express the protein of interest or parasites, where the gene of interest has been knocked out by homologous recombination. The transgenic parasites are being used in in vivo and in vitro experiments for establishing the role for the proteins under study in parasite infectivity.
By using this approach we have shown that overexpression of Leishmania histone H1 (LeishH1) in Leishmania spp. significantly reduces infectivity in macrophages, assessed in vitro, whereas in vivo parasites that overexpress histone H1 are not infectious. Overexpression of LeishH1 causes a delay in cell-cycle progression and more specifically in the G1/S transition and S phase completion. These parasites also delay to differentiate to the amastigote stage, resulting in not being able to survive within the macrophage environment (Smirlis et al, 2006).
Current research interests involve the determination of differences in the “proteome” between control (bearing plasmid alone) and parasites over-expressing LeishH1 by 2 dimensional polyacrylamide gel electrophoresis followed by mass spectroscopy.
We are also interested in the mechanism regulating the Leishmania histones expression. The histone protein levels in metazoans are regulated according to the cell-cycle. The protein SLBP (stem-loop binding protein) is a major factor of this mechanism. SLBP interacts with a stem-loop structure present in the histone transcripts. The regulation of histone expression in the Leishmania parasite seems to have some common characteristics with the metazoan mechanism. We have characterized a similar stem-loop structure in the Leishmania histone transcripts, which seems to be involved in the regulation of their expression. The SLBP protein has been cloned and we are studying its interaction with the stem-loop structure, as well as its biological role in the parasite.
Ran GTPase belongs to the Ras superfamily of monomeric G proteins and in mammalian cells is involved in the regulation of nucleocytoplasmic trafficking, nuclear envelope assembly, mitotic spindle assembly, DNA replication and mitotic checkpoint. RanGTP is a chromatin signal that stimulates spindle assembly. We have cloned the leishmanial Ran gene and we are currently studying its role in the parasitic life-cycle.
Subversion of phagocytes (neutrophils and macrophages) by Leishmania (Responsible scientist: Dr H. Boleti)
A main objective of this project is to set up in vitro cellular systems in order to study the interaction of strains of the L. donovani complex with neutrophils and their silent dissemination in macrophages through phagocytosis of apoptotic-Leishmania-loaded neutrophils. These systems will be further used for the identification i) of host genes implicated in the subversion of neutrophils and macrophages by Leishmania and ii) of species specific Leishmania features that affect these processes.
Our studies focus on:
• the establishment of in vitro cellular systems to study phagocytosis of Leishmania metacyclic promastigotes by neutrophils
• the investigation of the mechanisms of survival of the Leishmania metacyclic promastigotes in neutrophils with
emphasis on the parasitophorous vacuole remodelling
• the establishment of in vitro cellular systems to study phagocytosis of Leishmania-loaded apoptotic neutrophils
by macrophages
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J744 macrophages phagocytosing GFP-expressing
L.donovani (DIC and fluorescence microscopy) |
2. STUDY OF THE LEISHMANIAL GSK-3 AND OF THE ANTILEISHMANIAL ACTIVITY OF NATURAL PRODUCTS WITH THE AIM TO IDENTIFY DRUG TARGETS AND TO DEVELOP SELECTIVE INHIBITORS.
In particular we study the antileishmanial activity of indirubins known to target mammalian cyclin-dependent kinases (CDKs) and/or glycogen synthase kinase (GSK-3). We have cloned the leishmanial GSK-3 and we investigate whether this kinase could be a target of indirubins. We have also studied the antileishmanial activity of the taxoid 10-deacetylbaccatin III, isolated from dried needles and small branches of the European yew tree (Taxus baccata). Interestingly 10-deacetylbaccatin III was found to selectively inhibit the growth of L. donovani intracellular amastigotes within J774 murine macrophages in vitro at nanomolar concentrations
The susceptibility of clinical isolates to first-line drugs used for treating leishmaniasis is also within our research interests.
3. DEVELOPMENT OF MOLECULAR METHODS FOR THE DIAGNOSIS AND TYPING OF PARASITIC SPECIES FOR USAGE IN EPIDEMIOLOGICAL STUDIES
Efforts are being made for the development of a molecular tool that would offer a user-friendly, rapid and sensitive diagnostic method for the diagnosis of leishmaniasis. The initial step of this method requires the PCR amplification of chosen DNA regions, which are present in the genome of Leishmania parasites. A further step involving the addition of a specific DNA probe is required for the detection of the PCR product on the molecular strip.
Even though Leishmaniasis is endemic in Greece, studies dealing with its epidemiology are limited. L. tropica is the species responsible for cutaneous leishmaniases in Greece and L. infantum is the causative agent of visceral leishmaniasis. Besides the predominant MON-1 zymodeme, typing of isolates from human and canine host from Athens and Crete, with the use of Multilocus Enzyme Electrophoresis (MLEE), considered as the gold standard for typing Leishmania, revealed a high percentage of MON-98 (29.3%) which is the highest percentage that has been reported worldwide.
Typing and population structure analysis of isolates is carried out using molecular methods with high discriminatory power. In particular, we have developed a typing method which is based on the amplification of the K26 gene and the subsequent differentiation of the strains based on the size/sequence polymorphism of the gene. The K26-PCR based assay is specific for the Leishmania donovani complex and discriminates L. donovani / L. infantum zymodemes. Analysis of the population structure and dynamics of the isolates is carried out by multilocus microsatellite typing (MLMT) using a set of 14 microsatellite markers which are polymorphic within MON-1 strains.
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