A Gene Encoding the Plant-Like Alternative Oxidase is Present in Phytomonas but Absent in Leishmania spp.

The constituents of the respiratory chain are believed to differ among the trypanosomatids; bloodstream stages of African trypanosomes and Phytomonas promastigotes oxidize ubiquinol by a ubiquinol:oxygen oxidoreductase, also known as alternative oxidase, whereas Leishmania spp. oxidize ubiquinol via a classic cytochrome-containing respiratory chain. The molecular basis for this elementary difference in ubiquinol oxidation by the mitochondrial electron-transport chain in distinct trypanosomatids was investigated. The presence of a gene encoding the plant-like alternative oxidase could be demonstrated in Phytomonas and Trypanosoma brucei , trypanosomatids that are known to contain alternative oxidase activity. Our results further demonstrated that Leishmania spp. lack a gene encoding the plant-like alternative oxidase, and therefore, all stages of Leishmania spp. will lack the alternative oxidase protein. The observed fundamental differences between the respiratory chains of distinct members of the trypanosomatid family are thus caused by the presence or absence of a gene encoding the plant-like alternative oxidase.     

Biogenesis of peroxisomes and glycosomes: trypanosomatid glycosome assembly is a promising new drug target

In trypanosomatids (Trypanosoma and Leishmania), protozoa responsible for serious diseases of mankind in tropical and subtropical countries, core carbohydrate metabolism including glycolysis is compartmentalized in peculiar peroxisomes called glycosomes. Proper biogenesis of these organelles and the correct sequestering of glycolytic enzymes are essential to these parasites. Biogenesis of glycosomes in trypanosomatids and that of peroxisomes in other eukaryotes, including the human host, occur via homologous processes involving proteins called peroxins, which exert their function through multiple, transient interactions with each other. Decreased expression of peroxins leads to death of trypanosomes. Peroxins show only a low level of sequence conservation. Therefore, it seems feasible to design compounds that will prevent interactions of proteins involved in biogenesis of trypanosomatid glycosomes without interfering with peroxisome formation in the human host cells. Such compounds would be suitable as lead drugs against trypanosomatid-borne diseases.     

Function and assembly of the Leishmania surface coat

Like many trypanosomatids, the cell surface coat of Leishmania spp. is responsible for mediating various host-parasite interactions as well as acting as a dense physical barrier. This confers protection to the parasites in the hostile environments of the sandfly midgut and the macrophage phagolysosome. The major components of the surface coat are tethered to the cell surface via glycosylphosphatidylinositol glycolipids, and the composition of this surface coat is exquisitely regulated during the course of the parasite life-cycle. In this paper, we review what is known about the composition, biosynthesis and function of these glycosylphosphatidylinositol-containing molecules found within the parasite surface coat.     

The more insect trypanosomatids under study-the more diverse Trypanosomatidae appears

From 10 trypanosomatids genera six comprise monogenetic parasites of insects and for the rest of four genera insects may serve as vectors. The invertebrate host is an essential element of trypanosomatids life cycle, but from more than 900 recognised vertebrate hosts only about 500 species of insects have been discovered to be the hosts of homoxenous trypanosomatids. Nothing or very little is known about insect trypanosomatids in many extensive areas such as South East Asia, Australia, Japan and some others. Each new region explored brings many new findings. Recently flagellates were found in new insect species and families. The border of parasites distribution was expanded till Central Asia, Far East and North over the Polar Circle. As paleogeographical events are now under contemplating in trypanosomatids phylogeny researches so northern insect trypanosomatids may attract some attention as the elements of postglacial fauna which is definitely young. Very broad host specificity of insect trypanosomatids and high probability to isolate non-specific parasite show causes that only the investigation of a culture may solve the question 'what parasite was really isolated?'. Examination of cell morphotypes in the host has clearly demonstrated that they are not sufficient for classification and may lead us to be mistaken. The number of insect trypanosomatid cultures is inadequate for characterisation of the diversity of insects trypanosomatids. Trypanosoma is actually the only trypanosomatid genus which is out of questions. Insect trypanosomatids comprise the most diversified part of trypanosomatids evolutionary tree. Recent ssrRNA phylogenetic analysis and morphological data show that three insect isolates represent new lineages on trypanosomatid evolutionary tree, as well as dendrograms derived from PCR data demonstrated some new groups of isolates. Therefore, the more insect trypanosomatids are involved in laboratory investigations--the more new clusters or/and new lineages are appearing on the tree.     

Distribution of carbohydrates recognized by the lectins Euonymus europaeus and concanavalin A in monoxenic and heteroxenic trypanosomatids.

We observed a wide distribution of the carbohydrate epitopes galactosyl alpha(1-3)galactose (gal alpha1-3 gal), alpha-glucoside and alpha-mannoside in mono- and heteroxenic trypanosomatids by using fluorescein-labelled lectins of Euonymus europaeus (EE) and Concanavalin A (Con A) as well as sera from acute chagasic patients who have very high levels of anti-gal alpha(1-3)gal antibodies. The direct fluorescence test for gal alpha1-3 gal with EE was positive at minimum concentrations of 6 micrograms/ml for heteroxenic trypanosomatids and 0.7 micrograms/ml for monoxenic ones and for the plant parasite, Phytomonas. On the other hand, heteroxenic trypanosomatids that infect vertebrates bound ten-fold more Con A than monoxenic flagellates and Phytomonas. These data were confirmed in ELISA and Western Blot assays carried out with peroxidase-labelled EE and Con A. Euonymus europaeus recognized several glycoproteins in all trypanosomatids that we tested. Con A, however, recognized a glycoprotein cluster in heteroxenic protozoa, which ranging from 60-120 kDa, seemed to lack monoxenic parasites and Phytomonas. These findings suggest that alpha-D-mannose and alpha-D-glucose might play an important role in the interaction between trypanosomatids and vertebrate hosts.     

Distribution of Carbohydrates Recognized by the Lectins Euonymus europaeus and Concanavalin A in Monoxenic and Heteroxenic Trypanosomatids

We observed a wide distribution of the carbohydrate epitopes galactosylα(1–3) galactose (galα1–3 gal), α-glucoside, and α-mannoside in mono- and heteroxenic trypanosomatids by using fluorescein-labelled lectins of Euonymus europaeus (EE) and Concanavalin A (Con A) as well as sera from acute chagasic patients who have very high levels of anti-galα(1–3) gal antibodies. The direct fluorescence test for galα1–3 gal with EE was positive at minimum concentrations of 6 μg/ml for heteroxenic trypanosomatids and 0.7 μg/ml for monoxenic ones and for the plant parasite, Phytomonas. On the other hand, heteroxenic trypanosomatids that infect vertebrates bound ten-fold more Con A than monoxenic flagellates and Phytomonas. These data were confirmed in ELISA and Western Blot assays carried out with peroxidase-labelled EE and Con A. Euonymus europaeus recognized several glycoproteins in all trypanosomatids that we tested. Con A, however, recognized a glycoprotein cluster in heteroxenic protozoa, which ranging from 60–120 kDa, seemed to lack monoxenic parasites and Phytomonas. These findings suggest that α-D-mannose and α-D-glucose might play an important role in the interaction between trypanosomatids and vertebrate hosts.     

Discovery of Trypanosomatid Parasites in Globally Distributed Drosophila Species

Microbial parasites of animals include bacteria, viruses, and various unicellular eukaryotes. Because of the difficulty in studying these microorganisms in both humans and disease vectors, laboratory models are commonly used for experimental analysis of host-parasite interactions. Drosophila is one such model that has made significant contributions to our knowledge of bacterial, fungal, and viral infections. Despite this, less is known about other potential parasites associated with natural Drosophila populations. Here, we surveyed sixteen Drosophila populations comprising thirteen species from four continents and Hawaii and found that they are associated with an extensive diversity of trypanosomatids (Euglenozoa, Kinetoplastea). Phylogenetic analysis finds that Drosophila-associated trypanosomatids are closely related to taxa that are responsible for various types of leishmaniases and more distantly related to the taxa responsible for human African trypanosomiasis and Chagas disease. We suggest that Drosophila may provide a powerful system for studying the interactions between trypanosomatids and their hosts.     

Carbohydrate-Free Media for Crithidia

SYNOPSIS. The insect trypanosomatid Crithidia fasciculata grew well at pH 3.8–6.3 in defined carbohydrate-free media containing arginine (an essential amino acid) + proline + glutamic acid as substrates; glycerol was effective by itself. Precipitation of hemin in the acid media did not hinder growth. Further addition of succinic acid permitted growth matching that with carbohydrate. At pH 6.9–7.5 growth in this medium without carbohydrate with the aforementioned non-fermentable substrates was slight; added carbohydrate (as sucrose or sorbitol) permitted good growth. Utilization of non-carbohydrate substrates may contribute to Crithidia 's ability (and presumably to that of pathogenic trypanosomatids as well) to multiply in the insect gut and to the ability of some Trypanosoma species to multiply in the insect hemocoel and salivary gland.     

Oxidative stress and antioxidant defenses: a target for the treatment of diseases caused by parasitic protozoa

Parasitic protozoa cause several diseases, affecting hundreds of millions, particularly in underdeveloped countries. Although these organisms are eukaryotic cells, some of them present major differences with their mammalian host in selected metabolic pathways. These differences may be exploited as targets for developing better pharmacological agents for the treatment of specific parasitic diseases. This review describes some of the differences in terms of antioxidant defenses between these organisms and their mammalian host, which may provide useful targets for the treatment of these diseases. Some of the potential targets are: (i). iron metabolism in Plasmodium, (ii). the presence of a Fe-containing form of superoxide dismutase in trypanosomatids and malaria-causing parasites, (iii). the unique trypanothione-dependent antioxidant metabolism in trypanosomatids, (iv). the ascorbate peroxidase found in Trypanosoma cruzi and perhaps present in other trypanosomatids.     

Phylogenetic relationships of trypanosomatids parasitising true bugs (Insecta: Heteroptera) in sub-Saharan Africa

Three hundred and eighty-six heteropteran specimens belonging to more than 90 species captured in Ghana, Kenya and Ethiopia were examined for the presence of trypanosomatid flagellates. Of those, 100 (26%) specimens were positive for trypanosomatids and the spliced leader RNA gene sequence was obtained from 81 (80%) of the infected bugs. Its sequence-based analysis placed all examined flagellates in 28 typing units. Among 19 newly described typing units, 16 are restricted to sub-Saharan Africa, three belong to previously described species and six to typing units found on other continents. This result was corroborated by the analysis of the ssrRNA gene, sequenced for at least one representative of each major spliced leader RNA-based clade. In all trees obtained, flagellates originating from sub-Saharan Africa were intermingled with those isolated from American, Asian and European hosts, revealing a lack of geographic correlation. They are dispersed throughout most of the known diversity of monoxenous trypanosomatids. However, a complex picture emerged when co-evolution with their heteropteran hosts was taken into account, since some clades are specific for a single host clade, family or even species, whereas other flagellates display a very low host specificity, with a capacity to parasitise heteropteran bugs belonging to different genera/families. The family Reduviidae contains the widest spectrum of trypanosomatids, most likely a consequence of their predatory feeding behaviour, leading to an accumulation of a variety of flagellates from their prey. The plant pathogenic genus Phytomonas is reported here from Africa, to our knowledge for the first time. Finding the same typing units in hosts belonging to different heteropteran families and coming from different continents strongly indicates that the global diversity of the insect trypanosomatids is most likely lower than was predicted on the basis of the "one host-one parasite" paradigm. The analysis presented significantly extends the known diversity of monoxenous insect trypanosomatids and will be instrumental in building a new taxonomy that reflects their true phylogenetic relationships.     

Carbohydrate metabolism in adult schistosomes of different strains and species

In this comparative study the carbohydrate metabolism of Schistosoma japonicum, S. haematobium, S. intercalatum, S. bovis and three strains of S. mansoni was investigated. No large differences were found in the protein and glycogen contents of all species involved. In all species investigated, lactate was the main end product of carbohydrate breakdown. However, all parasites degraded part of the glucose to CO2 via the Krebs cycle. No difference was observed in the contribution of this aerobic process to energy production in the three strains of S. mansoni investigated. The differences in Krebs-cycle activity between the five schistosome species investigated were very small. Therefore, this study refutes the idea that significant differences exist in the carbohydrate metabolism of various schistosome species or strains.     

The phylogeny of the Trypanosomatidae: the molecular and morphological approaches

The results of comparative analysis of two phylogenetic trees of the trypanosomatids based on morphological and molecular characters are discussed. The morphological dendrogram was based on 33 ultrastructural characters, 6 light microscope characteristics and 8 biological characters. Molecular UPGMA dendrogram depicting differences (Dice distance) between examined trypanosomatids is based on the universally primed PCR polymorphisms. The general topology of both dendrograms are similar, with the Trypanosoma at the base. The genus Wallaceina appears to be monophyletic. In a contrary, the genera Leptomonas, Crithidia and Herpetomonas look like artificial groups according to both methods used. The cyst-forming homoxenous trypanosomatids from insects represent a monophyletic clade, which seems to be a separate genus. Two species of within genus Wallaceina are arranged as a separate subgenus.     

Cyclic AMP signaling in trypanosomatids

Curative interference with signal transduction pathways is a spectacularly successful concept in many domains of modern pharmacology; indeed, the 'wonder drug' Viagra is but a humble inhibitor of a cyclic GMP (cGMP)-specific phosphodiesterase and, thus, interferes with cGMP-signaling in a strategic organ. In fact, about half of the 100 most successful drugs currently on the market act through modulating cellular signal transduction. Despite these encouraging findings, signal transduction pathways as potential drug targets in trypanosomatids have remained largely unexplored. However, what little is known indicates that adenylyl cyclases of trypanosomatids, and probably other enzymes of the cyclic nucleotide signaling pathways, are significantly different from their mammalian counterparts. Here, Christina Naula and Thomas Seebeck summarize what is known about cAMP signal transduction in trypanosomatids.     

Sugars, signalling, and plant development

Like all organisms, plants require energy for growth. They achieve this by absorbing light and fixing it into a usable, chemical form via photosynthesis. The resulting carbohydrate (sugar) energy is then utilized as substrates for growth, or stored as reserves. It is therefore not surprising that modulation of carbohydrate metabolism can have profound effects on plant growth, particularly cell division and expansion. However, recent studies on mutants such as stimpy or ramosa3 have also suggested that sugars can act as signalling molecules that control distinct aspects of plant development. This review will focus on these more specific roles of sugars in development, and will concentrate on two major areas: (i) cross-talk between sugar and hormonal signalling; and (ii) potential direct developmental effects of sugars. In the latter, developmental mutant phenotypes that are modulated by sugars as well as a putative role for trehalose-6-phosphate in inflorescence development are discussed. Because plant growth and development are plastic, and are greatly affected by environmental and nutritional conditions, the distinction between purely metabolic and specific developmental effects is somewhat blurred, but the focus will be on clear examples where sugar-related processes or molecules have been linked to known developmental mechanisms.     

Electrophoretic analysis of endonuclease-generated fragments of k-DNA, of esterase isoenzymes, and of surface proteins as aids for species identification of insect trypanosomatids

In order to verify the applicability of biochemical methods for species identification of Trypanosomatidae, 13 species of monoxenic trypanosomatids plus the heteroxenous Trypanosoma cruzi were comparatively analyzed by three different biochemical methods. Insect trypanosomatids examined were: Crithidia acanthocephali, C. fasciculata (three varieties), C. luciliae luciliae, C. luciliae thermophila, C. deanei, C. oncopelti, Herpetomonas muscarum muscarum, H. megaseliae, H. samuelpessoai, H. mariadeanei, Leptomonas seymouri, L. collosoma, L. samueli, and Blastocrithidia culicis. Also included in the survey were aposymbiotic strains of C. deanei and C. oncopelti. Methods used were: electrophoretic profiling of endonuclease-generated fragments of k-DNA, esterase isoenzymes profiling, and polyacrylamide-gel electrophoresis (SDS-PAGE) of radioiodinated cell surface proteins. Interspecific but not intraspecific differences were detected by all three methods among the 13 monoxenic species examined. Thus, it is concluded that these methods can be successfully used, in addition to classical criteria, for species identification of insect trypanosomatids.     

Proteomic analysis of the Trypanosoma cruzi ribosomal proteins

Trypanosoma cruzi is a parasite responsible for Chagas disease. The identification of new targets for chemotherapy is a major challenge for the control of this disease. Several lines of evidences suggest that the translational system in trypanosomatids show important differences compared to other eukaryotes. However, there little is known information about this. We have performed a detailed data mining search for ribosomal protein genes in T. cruzi genome data base combined with mass spectrometry analysis of purified T. cruzi ribosomes. Our results show that T. cruzi ribosomal proteins have ∼50% sequence identity to yeast ones. Nevertheless, some parasite proteins are longer due to the presence of several N- or C-terminal extensions, which are exclusive of trypanosomatids. In particular, L19 and S21 show C-terminal extensions of 168 and 164 amino acids, respectively. In addition, we detected two 60S subunit proteins that had not been previously detected in the T. cruzi total proteome; namely, L22 and L42.     

Hepatocyte heterogeneity in the metabolism of carbohydrates.

Periportal and perivenous hepatocytes possess different amounts and activities of the rate-generating enzymes of carbohydrate and oxidative energy metabolism and thus different metabolic capacities. This is the basis of the model of metabolic zonation, according to which periportal cells catalyze predominantly the oxidative catabolism of fatty and amino acids as well as glucose release and glycogen formation via gluconeogenesis, and perivenous cells carry out preferentially glucose uptake for glycogen synthesis and glycolysis coupled to liponeogenesis. The input of humoral and nervous signals into the periportal and perivenous zones is different; gradients of oxygen, substrates and products, hormones and mediators and nerve densities exist which are important not only for the short-term regulation of carbohydrate metabolism but also for the long-term regulation of zonal gene expression. The specialization of periportal and perivenous hepatocytes in carbohydrate metabolism has been well characterized. In vivo evidence is provided by the complex metabolic situation termed the 'glucose paradox' and by zonal flux differences calculated on the basis of the distribution of enzymes and metabolites. In vitro evidence is given by the different flux rates determined with classical invasive techniques, e.g. in periportal-like and perivenous-like hepatocytes in cell culture, in periportal- and perivenous-enriched hepatocyte populations and in perfused livers during orthograde and retrograde flow, as well as with noninvasive techniques using miniature oxygen electrodes, e.g. in livers perfused in either direction. Differences of opinion in the interpretation of studies with invasive and noninvasive techniques by the authors are discussed. The declining gradient in oxygen concentrations, the decreasing glucagon/insulin ratio and the different innervation could be important factors in the zonal expression of the genes of carbohydrate-metabolizing enzymes. While it is clear that the hepatocytes sense the glucagon/insulin gradients via the respective hormone receptors, it is not known how they sense different oxygen tensions; the O2 sensor may be an oxygen-binding heme protein. The zonal separation of glucose release and uptake appears to be important for the liver to operate as a 'glucostat'. Thus, zonation of carbohydrate metabolism develops gradually during the first weeks of life, in part before and in part with weaning, when (in rat and mouse) the fat- and protein-rich but carbohydrate-poor nutrition via milk is replaced by carbohydrate-rich food. Similarly, zonation of carbohydrate metabolism adapts to longer lasting alterations in the need of a 'glucostat', such as starvation, diabetes, portocaval anastomoses or partial hepatectomy.     

Monoclonal Antibodies for the Identification of Trypanosomatids of the Genus Phytomonas

Monoclonal antibodies have been produced against culture forms of Phytomonas francai and Phytomonas serpens parasites, respectively, in cassava roots and tomato fruits. These monoclonal antibodies have been tested against 5 other Phytomonas spp. isolated from plants and 14 species of trypanosomatids of various genera. Monoclonal antibodies were found to react exclusively with Phytomonas spp., always giving negative results with other trypanosomatid genera. Thus, these monoclonal antibodies seem to be an effective tool for the identification of phytomonads among insect trypanosomatids.     

Cellular trafficking in trypanosomatids: a new target for therapies?

Pathogenic trypanosomatids cause a plethora of diseases marked by the lack of efficient vaccines and therapies. As a consequence, studies are being conducted that are geared towards the understanding of basic mechanisms and various biological aspects of these parasites that might be used as targets for new developments in these areas. One such aspect is the understanding of specific cellular trafficking mechanisms that might be attacked with the intention of disease control. In this paper, we give an overview of our current knowledge of cellular targeting mechanisms in trypanosomatids, with special emphasis on our data related to lysosomal targeting of cysteine proteinases in Leishmania.