Targeting essential pathways in trypanosomatids gives insights into protozoan mechanisms of cell death

Apoptosis is a normal component of the development and health of multicellular organisms. However, apoptosis is now considered a prerogative of unicellular organisms, including the trypanosomatids of the genera Trypanosoma spp. and Leishmania spp., causative agents of some of the most important neglected human diseases. Trypanosomatids show typical hallmarks of apoptosis, although they lack some of the key molecules contributing to this process in metazoans, like caspase genes, Bcl-2 family genes and the TNF-related family of receptors. Despite the lack of these molecules, trypanosomatids appear to have the basic machinery to commit suicide. The components of the apoptotic execution machinery of these parasites are slowly coming into light, by targeting essential processes and pathways with different apoptogenic agents and inhibitors. This review will be confined to the events known to drive trypanosomatid parasites to apoptosis

Key role of the 3' untranslated region in the cell cycle regulated expression of the Leishmania infantum histone H2A genes: minor synergistic effect of the 5' untranslated region

<p>Abstract</p> <p>Background</p> <p>Histone synthesis in <it>Leishmania </it>is tightly coupled to DNA replication by a post-transcriptional mechanism operating at the level of translation.</p> <p>Results</p> <p>In this work we have analyzed the implication of the 5' and 3' untranslated regions (UTR) in the cell cycle regulated expression of the histone <it>H2A </it>in <it>Leishmania infantum</it>. For that purpose, <it>L. infantum </it>promastigotes were stably transfected with different plasmid constructs in which the <it>CAT </it>coding region used as a reporter was flanked by the 5' and 3' UTR regions of the different <it>H2A </it>genes. We report that in spite of their sequence differences, histone <it>H2A </it>5' and 3' UTRs conferred a cell cycle dependent pattern of expression on the CAT reporter since <it>de novo </it>synthesis of CAT increased when parasites enter the S phase. Using one established <it>L. infantum </it>cell line we showed that CAT expression is controlled by the same regulatory events that control the endogenous histone gene expression. Thus, although we did not detect changes in the level of <it>CAT </it>mRNAs during cell cycle progression, a drastic change in the polysome profiles of <it>CAT </it>mRNAs was observed during the progression from G1 to S phase. In the S phase <it>CAT </it>mRNAs were on polyribosomal fractions, but in the G1 phase the association of <it>CAT </it>transcripts with ribosomes was impaired. Furthermore, it was determined that the addition of just the <it>H2A </it>3' UTR to the <it>CAT </it>reporter gene is sufficient to achieve a similar pattern of post-transcriptional regulation indicating that this region contains the major regulatory sequences involved in the cell cycle dependent expression of the <it>H2A </it>genes. On the other hand, although CAT transcripts bearing the <it>H2A </it>5' alone were translated both in the G1 and S phase, higher percentages of transcripts were detected on polyribosomes in the S phase correlating with an increase in the <it>de novo </it>synthesis of CAT. Thus, it can be concluded that this region also contributes, although to a minor extent than the 3' UTR, in the enhancement of translation in the S phase relative to the G1 phase.</p> <p>Conclusion</p> <p>Our findings indicate that both, the 5' and the 3' UTRs contain sequence elements that contribute to the cell cycle expression of <it>L. infantum </it>H2A. The 3' UTR region is essential for cell cycle dependent translation of the <it>L. infantum </it>H2A transcripts whereas the 5' UTR has a minor contribution in their S phase dependent translation.</p

Evolutionary and geographical history of the Leishmania donovani complex with a revision of current taxonomy.

Leishmaniasis is a geographically widespread severe disease, with an increasing incidence of two million cases per year and 350 million people from 88 countries at risk. The causative agents are species of Leishmania, a protozoan flagellate. Visceral leishmaniasis, the most severe form of the disease, lethal if untreated, is caused by species of the Leishmania donovani complex. These species are morphologically indistinguishable but have been identified by molecular methods, predominantly multilocus enzyme electrophoresis. We have conducted a multifactorial genetic analysis that includes DNA sequences of protein-coding genes as well as noncoding segments, microsatellites, restriction-fragment length polymorphisms, and randomly amplified polymorphic DNAs, for a total of approximately 18,000 characters for each of 25 geographically representative strains. Genotype is strongly correlated with geographical (continental) origin, but not with current taxonomy or clinical outcome. We propose a new taxonomy, in which Leishmania infantum and L. donovani are the only recognized species of the L. donovani complex, and we present an evolutionary hypothesis for the origin and dispersal of the species. The genus Leishmania may have originated in South America, but diversified after migration into Asia. L. donovani and L. infantum diverged approximately 1 Mya, with further divergence of infraspecific genetic groups between 0.4 and 0.8 Mya. The prevailing mode of reproduction is clonal, but there is evidence of genetic exchange between strains, particularly in Africa

Differentiation and Gene Flow among European Populations of Leishmania infantum MON-1

Visceral leishmaniasis is caused by protozoan parasites of the genus Leishmania. This disease is a public health problem in countries bordering the Mediterranean, in China, and South America. Until now, isoenzyme analysis, a method with several advantages but also some limitations, is the gold standard for typing the causative agent L. infantum. We have developed a new method based on hypervariable DNA markers, the microsatellites. Its higher discriminatory power, genotype-based analysis, the possibility to use biological material instead of parasite cultures, and the fast analysis are the major improvements. We could demonstrate for the first time that there exist different geographically determined populations within the predominant zymodeme of L. infantum, which has important epidemiological implications. We also tested for relationships between genotype and clinical picture and/or host background. Leishmania is considered to reproduce mainly clonally; however, we found some indication for recombination in our study. Our work constitutes a solid basis for further population and epidemiological studies of L. infantum by completing the existing microsatellite database by analysing strains from other endemic foci

Multilocus Microsatellite Typing (MLMT) of Strains from Turkey and Cyprus Reveals a Novel Monophyletic L. donovani Sensu Lato Group

In eastern Mediterranean, leishmaniasis represents a major public health problem with considerable impact on morbidity and potential to spread. Cutaneous leishmaniasis (CL) caused by L. major or L. tropica accounts for most cases in this region although visceral leishmaniasis (VL) caused by L. infantum is also common. New foci of human CL caused by L. donovani complex strains were recently described in Cyprus and Turkey. Herein we analyzed Turkish strains from human CL foci in Çukurova region (north of Cyprus) and a human VL case in Kuşadasi. These were compared to Cypriot strains that were previously typed by Multilocus Enzyme Electrophoresis (MLEE) as L. donovani MON-37. Nevertheless, they were found genetically distinct from MON-37 strains of other regions and therefore their origin remained enigmatic. A population study was performed by Multilocus Microsatellite Typing (MLMT) and the profile of the Turkish strains was compared to previously analyzed L. donovani complex strains. Our results revealed close genetic relationship between Turkish and Cypriot strains, which form a genetically distinct L. infantum monophyletic group, suggesting that Cypriot strains may originate from Turkey. Our analysis indicates that the epidemiology of leishmaniasis in this region is more complicated than originally thought

Expression of a Novel Leishmania Gene Encoding a Histone H1-Like Protein in Leishmania major Modulates Parasite Infectivity In Vitro

We describe identification and characterization of a novel two-copy gene of the parasitic protozoan Leishmania that encodes a nuclear protein designated LNP18. This protein is highly conserved in the genus Leishmania, and it is developmentally regulated. It is an alanine- and lysine-rich protein with potential bipartite nuclear targeting sequence sites. LNP18 shows sequence similarity to H1 histones of trypanosomatids and of higher eukaryotes and in particular with histone H1 of Leishmania major. The nuclear localization of LNP18 was determined by indirect immunofluorescence and Western blot analysis of isolated nuclei by using antibodies raised against the recombinant protein as probes. The antibodies recognized predominantly a 18-kDa band or a 18-kDa-16-kDa doublet. Photochemical cross-linking of intact parasites followed by Western blot analysis provided evidence that LNP18 is indeed a DNA-binding protein. Generation of transfectants overexpressing LNP18 allowed us to determine the role of this protein in Leishmania infection of macrophages in vitro. These studies revealed that transfectants overexpressing LNP18 are significantly less infective than transfectants with the vector alone and suggested that the level of LNP18 expression modulates Leishmania infectivity, as assessed in vitro