Trypanosomatids, Other Than Phytomonas spp., Isolated and Cultured from Fruit

Trypanosomatids were isolated from edible fruit. One of the isolates (from tangerine) presented a set of enzymes for the metabolism of arginine-ornithine similar to that of Leptomonas spp., and failed to be recognized by monoclonal antibodies specific for Phytomonas spp. The possibility that trypanosomatids other than Phytomonas spp. could infect fruit was further examined by inoculating tomatoes with species of Crithidia, Leptomonas and Herpetomonas. Some of these flagellates multiplied in tomatoes. Besides, house flies became infected with Crithidia sp. when fed on tomatoes experimentally inoculated with this flagellate. Therefore, isolation of a trypanosomatid from a plant should not constitute an absolute criterion for placing it in the genus Phytomonas.     

Enzymes of Ornithine-Arginine Metabolism in Trypanosomatids of the Genus Phytomonas1

Trypanosomatids recently isolated from the plants Euphorbia pinea, E. characias, E. hyssopifolia, Manihoi esculenta (cassava) and Lycopersicon sp. (tomato) plus the McGhee-Postell isolate of Phytomonas davidi have been examined for the presence of enzymes of ornithine-arginine metabolism. Arginase (EC 3.5.3.1) was not detected in the flagellates examined whereas arginine deiminase (EC 3.5.3.6) and citrullinehydrolase (EC 3.5.1.20) were present in all organisms. Phytomonas davidi and the isolate from E. hyssopifolia, besides these enzymes, also had ornithine carbamoyltransferase (EC 2.1.3.3). The enzymic constitution of these flagellates is compared from a taxonomic standpoint to that of previously studied trypanosomatids.     

Trypanosomatid parasites of plants (phytomonas)

Trypanosomatids of the genus Phytomonas have been known as parasites of lactiferous plants since the beginning of the century and have been the subject of renewed attention in the past decade, as they are now recognized to be pathogenic in plants of economic interest. Nevertheless, information about these flagellates is still scanty. Until recently they had not been cultured, or studied biochemically or ultrastructurally. Phytophagous insects are their putative vectors but exactly which species are involved remains to be established. There are many unanswered questions about the taxonomic identification, pathogenecity and transmission of Phytomonas spp as well as about their natural hosts and reservoirs; this article by Erney Camargo, Pieter Kastelein and Isaac Roitman highlights some of them.     

Characterization of Phytomonas isolated from fruits by electrophoretic isoenzymes and kinetoplast-DNA analysis

Abstract Two flagellates of the family trypanosomatidae were isolated from the fruits of Lycopersicon esculentum (tomato) and Annona cherimolia (cherimoya) in the southeastern region of Spain. The isolates were characterized by isoenzyme analysis using nine different isoenzymes and by analysis of kinetoplast DNA (kDNA) restriction fragment length polymorphism using four different restriction endonucleases. Most of the isoenzymes were unable to distinguish between the two fruit isolates, while they were all able to distinguish these two from four other Phytomonas isolates, three of which were from laticiferous plants i.e. Euphorbia characias E. hirta and E. hyssopifolia , and one was a phloem-restricted isolate associated with Hartrot disease. Only the enzyme Superoxide dismutase was able to differentiate between the two fruit isolates. Electrophoretic and restriction endonuclease analysis of kDNA minicircles, using four restriction enzymes, showed similar if not identical restriction cleavage patterns of the minicircles of the two isolates from fruits, while the patterns were different for the other isolates. These results confirm the hypothesis that the two isolates from fruits constitute a group of trypanosomatids that are the same or closely related and that this group can parasitize more than one host plant.     

Classification of trypanosomatids from fruits and seeds using morphological, biochemical and molecular markers revealed several genera among fruit isolates

Trypanosomatids are widespread in several plant families and although most isolates have been classified as Phytomonas, other trypanosomatid genera can also infect plants. In order to assess the natural occurrence of non-Phytomonas trypanosomatids in plants we characterized 21 new trypanosomatid cultures, 18 from fruits and three from seeds of 17 plant species. The trypanosomatids from fruit and seeds were compared in terms of morphological, growth, biochemical and molecular features. The high diversity among the isolates permitted the classification of the new flagellates into the genera Crithidia and Leptomonas as well as Phytomonas. The data showed that natural fruit infection with non-Phytomonas trypanosomatids is more common than usually thought, being detected in 43% of the fruit isolates.     

Trypanosomatidae: Phytomonas detection in plants and phytophagous insects by PCR amplification of a genus-specific sequence of the spliced leader gene

In this paper we describe a method for the detection of Phytomonas spp. from plants and phytophagous insects using the PCR technique by targeting a genus-specific sequence of the spliced leader (SL) gene. PCR amplification of DNA from 48 plant and insect isolates previously classified as Phytomonas by morphological, biochemical, and molecular criteria resulted in all cases in a 100-bp fragment that hybridized with the Phytomonas-specific spliced leader-derived probe SL3'. Moreover, this Phytomonas-specific PCR could also detect Phytomonas spp. in crude preparations of naturally infected plants and insects. This method shows no reaction with any other trypanosomatid genera or with plant and insect host DNA, revealing it to be able to detect Phytomonas spp. from fruit, latex, or phloem of various host plants as well as from salivary glands and digestive tubes of several species of insect hosts. Results demonstrated that SLPCR is a simple, fast, specific, and sensitive method that can be applied to the diagnosis of Phytomonas among cultured trypanosomatids and directly in plants and putative vector insects. Therefore, the method was shown to be a very specific and sensitive tool for diagnosis of Phytomonas without the need for isolation, culture, and DNA extraction of flagellates, a feature that is very convenient for practical and epidemiological purposes.     

In Vitro Culture of Phytomonas Sp. Isolated from Euphorbia characias. Metabolic Studies by 1H NMR

ABSTRACT. We describe the in vitro culture of Phytomonas species isolated from Euphorbia characias . The best choice between tested media was SDM-79, in which promastigotes, after 6 days of culture, reached cell densities as high as 4 × 10⁷ cells/ml. Cells growing in LIT or MTL medium showed longer division times and lower cell densities. We succeeded in obtaining Phytomonas sp. amastigote and spheromastigote forms in modified GRACE's medium, yielding transformation rates of up to 70%. Electron microscopy studies were performed in order to characterize the ultrastructural features of these forms obtained in vitro. On the other hand, metabolic studies based on qualitative (nuclear magnetic resonance spectroscopy) and quantitative metabolic methods (enzymatic assays) showed that promastigote forms secreted mainly ethanol, acetate, glycine, glycerol, piruvate and succinate in SDM-79 medium, whereas the major metabolites found after transformation in modified Grace's medium were ethanol, acetate, glycine, piruvate and smaller amounts of glycerol.     

Identification of excreted iron superoxide dismutase for the diagnosis of Phtytomonas

An excreted iron superoxide dismutase (FeSODe) of pI 3.6 with a molecular weight of 28-30 kDa was detected in the in vitro culture of Phytomonas isolated from Euphorbia characias (SODeCHA) and from Lycopersicon esculentum (SODeTOM), in Grace's medium without serum. These FeSODe excreted into the medium had immunogenic capacity: the positivity of the anti-SODeCHA serum persisted to a dilution of 1/30,000, and for the anti-SODeTOM to 1/10,000 by Western blot. In addition, cross reaction was detected between the anti-SODe serum of Phytomonas isolated from E. characias against SODeTOM, and the anti-SODe serum from L. esculentum with SODeCHA. This characteristic offers the possibility of its use to diagnose plant trypanosomatids. The validation of the test was confirmed by experimental inoculation of tomato fruits with Phytomonas isolated from L. esculentum. At 7, 10, 15, and 21 days post infection, it was possible to detect the presence of the parasites with the anti-SODe serum of Phytomonas isolated from L. esculentum at a dilution of 1/250. These serological results were confirmed by visualization of the parasites by optical microscopy. The data of this study confirm that the SOD is sufficient to identify a trypanosomatid isolated from plants as belonging to the genus Phytomonas.     

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.     

Cytotoxicity of three new triazolo-pyrimidine derivatives against the plant trypanosomatid: Phytomonas sp. isolated from Euphorbia characias

There is no effective chemotherapy against diseases caused by Phytomonas sp., a plant trypanosomatid responsible for economic losses in major crops. We tested three triazolo-pyrimidine complexes [two with Pt(II), and another with Ru(III)] against promastigotes of Phytomonas sp. isolated from Euphorbia characias. The incorporation of radiolabelled precursors, ultrastructural alterations and changes in the pattern of metabolite excretion were examined. Different degrees of toxicity were found for each complex: the platinum compound showed an inhibition effect on nucleic acid synthesis, provoking alterations on the levels of mitochondria, nucleus and glycosomes. These results, together with others reported previously in our laboratory about the activity of pyrimidine derivatives, reflect the potential of these compounds as agents in the treatment of Phytomonas sp.     

Comparative Aspects of Energy Metabolism in Plant Trypanosomatids

ABSTRACT. The energy metabolism was compared among four different representatives of the genus Phytomonas isolated from different plants and localities: the sieve tubes of the hartrot-infected coconut palm in French Guyana, the latex fluid of Euphorbia hyssopifolia in French Guyana and the fruits of tomato and cherimoya in Spain. All four isolates produced acetate, ethanol, glycerol and glycine as metabolic end-products. In addition, small amount of succinate and pyruvate were excreted. Only minor quantitative differences were observed in the four isolates. Glycosomes, harboring the glycolytic enzymes, were present in all isolates. No evidence was found for an active involvement of the mitochondrion in metabolism. Respiration was insensitive to the classical inhibitors of the respiratory chain, such as antimycin and potassium cyanide, but inhibited by salicylhydroxamic acid. No evidence was found for the functioning of a citric-acid cycle. It is concluded that representative of this genus share the same highly active carbohydrate metabolism combined with a complete suppression of mitochondrial activity.     

Termite gut symbiotic archaezoa are becoming living metabolic fossils

Over the course of several million years, the eukaryotic gut symbionts of lower termites have become adapted to a cellulolytic environment. Up to now it has been believed that they produce nutriments using their own cellulolytic enzymes for the benefit of their termite host. However, we have now isolated two endoglucanases with similar apparent molecular masses of approximately 36 kDa from the not yet culturable symbiotic Archaezoa living in the hindgut of the most primitive Australian termite, Mastotermes darwiniensis. The N-terminal sequences of these cellulases exhibited significant homology to cellulases of termite origin, which belong to glycosyl hydrolase family 9. The corresponding genes were detected not in the mRNA pool of the flagellates but in the salivary glands of M. darwiniensis. This showed that cellulases isolated from the flagellate cells originated from the termite host. By use of a PCR-based approach, DNAs encoding cellulases belonging to glycosyl hydrolase family 45 were obtained from micromanipulated nuclei of the flagellates Koruga bonita and Deltotrichonympha nana. These results indicated that the intestinal flagellates of M. darwiniensis take up the termite's cellulases from gut contents. K. bonita and D. nana possess at least their own endoglucanase genes, which are still expressed, but without significant enzyme activity in the nutritive vacuole. These findings give the impression that the gut Archaezoa are heading toward a secondary loss of their own endoglucanases and that they use exclusively termite cellulases.     

Sera of chagasic patients react with antigens from the tomato parasite Phytomonas serpens

The genus Phytomonas comprises trypanosomatids that can parasitize a broad range of plant species. These flagellates can cause diseases in some plant families with a wide geographic distribution, which can result in great economic losses. We have demonstrated previously that Phytomonas serpens 15T, a tomato trypanosomatid, shares antigens with Trypanosoma cruzi, the agent of human Chagas disease. Herein, two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to identify proteins of P. serpens 15T that are recognized by sera from patients with Chagas disease. After 2D-electrophoresis of whole-cell lysates, 31 peptides were selected and analyzed by tandem mass spectrometry. Twenty-eight polypeptides were identified, resulting in 22 different putative proteins. The identified proteins were classified into 8 groups according to biological process, most of which were clustered into a cellular metabolic process category. These results generated a collection of proteins that can provide a starting point to obtain insights into antigenic cross reactivity among trypanosomatids and to explore P. serpens antigens as candidates for vaccine and immunologic diagnosis studies.     

Ultrastructural Studies on the Trypanosomatid Phytomonas serpens in the Salivary Glands of a Phytophagous Hemipteran

Electron microscopy of salivary glands of the phytophagous hemipteran Phihia picta infected with Phytomonas serpens revealed the presence of flagellates in the gland periphery beneath the gland envelope, in the gland central lumen, between gland cells in the intercellular space and inside the gland cells. In the latter case, flagellates were found in the cytoplasm whether or not it was surrounded by a vacuolar membrane. Flagellates were always of the promastigote type, sometimes displaying a large twisted body. Morphological peculiarities of flagellates in different gland locations are recorded.     

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.     

Fatty acid and sterol composition of three phytomonas species.

Fatty acid and sterol analysis were performed on Phytomonas serpens and Phytomonas sp. grown in chemically defined and complex medium, and P. fran?ai cultivated in complex medium. The three species of the genus Phytomonas had qualitatively identical fatty acid patterns. Oleic, linoleic, and linolenic were the major unsaturated fatty acids. Miristic and stearic were the major saturated fatty acids. Ergosterol was the only sterol isolated from Phytmonas sp. and P. serpens grown in a sterol-free medium, indicating that it was synthesized de novo. When P. fran?ai that does not grow in defined medium was cultivated in a complex medium, cholesterol was the only sterol detected. The fatty acids and sterol isolated from Phytomonas sp. and P. serpens grown in a chemically defined lipid-free medium indicated that they were able to biosynthesize fatty acids and ergosterol from acetate or from acetate precursors such as glucose or threonine.     

Morphological, biochemical and molecular characterization of Herpetomonas samuelpessoai camargoi n. subsp., a trypanosomatid isolated from the flower of the squash Cucurbita moschata

We report the morphological, biochemical and molecular characteristics of a trypanosomatid isolated from the flower of Cucurbita moschata. Although the trypanosomatid was isolated from a plant, the lack of recognition of Phytomonas-specific molecular markers based on spliced-leader and ribosomal genes as well as by monoclonal antibodies specific for Phytomonas argues against assigning it to this genus. Because the isolate displayed typical opisthomastigote forms in culture, it is assigned to the genus Herpetomonas. Analysis of randomly amplified polymorphic DNA (RAPD) patterns and characterization of ribosomal SSU and ITS markers suggest that it is more closely related to H. samuelpessoai than to any other species. However, the presence of spined flagellates in culture (displaying lateral expansions of the plasma membrane originating near the flagellar pocket) and isolate-specific RAPD fingerprints argue strongly that the trypanosomatid belongs to a new subspecies, for which the name Herpetomonas samuelpessoai camargoi n. subsp. is proposed.     

Mechanisms of growth inhibition of Phytomonas serpens by the alkaloids tomatine and tomatidine

Phytomonas serpens are flagellates in the family Trypanosomatidae that parasitise the tomato plant (Solanum lycopersicum L.), which results in fruits with low commercial value. The tomato glycoalkaloid tomatine and its aglycone tomatidine inhibit the growth of P. serpens in axenic cultures. Tomatine, like many other saponins, induces permeabilisation of the cell membrane and a loss of cell content, including the cytosolic enzyme pyruvate kinase. In contrast, tomatidine does not cause permeabilisation of membranes, but instead provokes morphological changes, including vacuolisation. Phytomonas treated with tomatidine show an increased accumulation of labelled neutral lipids (BODYPY-palmitic), a notable decrease in the amount of C₂₄-alkylated sterols and an increase in zymosterol content. These results are consistent with the inhibition of 24-sterol methyltransferase (SMT), which is an important enzyme that is responsible for the methylation of sterols at the 24 position. We propose that the main target of tomatidine is the sterols biosynthetic pathway, specifically, inhibition of the 24-SMT. Altogether, the results obtained in the present paper suggest a more general effect of alkaloids in trypanosomatids, which opens potential therapeutic possibilities for the treatment of the diseases caused by these pathogens.     

An Ultrastructural Study of Phytomonas davidi Lafont (Trypanosomatidae)1

Phytomonas davidi (Trypanosomatidae) possesses typical trypanosomatid organelles: subpellicular microtubules, kinetoplast-mitochondrial complex, K-DNA, and four subflagellar pocket microtubules. A greater concentration of subpellicular microtubules was observed in the latex forms than in those found in the salivary glands of its insect vector. Only in the latex flagellates (the stage with postnuclear torsion) were subpellicular microtubules interconnected by crossbridges observed. Morphology and development of mitochondrial aristae varied according to the source of the flagellates. Organisms taken from culture medium had extensively developed plate-like cristae; sparse tubular cristae were observed in the latex forms; and highly developed tubular cristae were seen in flagellates from the lumen of the vector's salivary glands, though organisms in the salivary gland channels had few or none.     

Heterogeneous production of metallo-type peptidases in parasites belonging to the family Trypanosomatidae

Proteolytic enzymes play a central role in the physiology of all living organisms, participating in several metabolic pathways and in different phases of parasite-host interactions. We have identified cell-associated peptidase activities in 33 distinct flagellates, including representatives of almost all known trypanosomatid genera parasitizing insects (Herpetomonas, Crithidia, Leishmania, Trypanosoma, Leptomonas, Phytomonas, Blastocrithidia and Endotrypanum) as well as the biflagellate kinetoplastid Bodo, by using SDS-PAGE containing gelatin as co-polymerized substrate and proteolytic inhibitors. Under the alkaline pH (9.0) conditions employed, all the flagellates presented at least one peptidase, with the exception of Crithidia acanthocephali and Phytomonas serpens, which did not display any detectable proteolytic enzyme activity. All the proteolytic activities were completely inhibited by 1,10-phenanthroline, a zinc-chelating agent, putatively identifying these activities as metallo-type peptidases. EDTA and EGTA, two other metallopeptidase inhibitors, E-64 (a cysteine peptidase inhibitor), pepstatin A (an aspartyl peptidase inhibitor) and PMSF (a serine peptidase inhibitor) did not interfere with the metallopeptidase activities detected in the studied trypanosomatids. Conversely, Bodo-derived peptidases were resistant to 1,10-phenanthroline and only partially inhibited by EDTA, showing a distinct inhibition profile. Together, our data demonstrated great heterogeneity of expression of metallopeptidases in a wide range of parasites belonging to the family Trypanosomatidae.