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.     

Characterization of the Target Antigens of Phytomonas-specific Monoclonal Antibodies

ABSTRACT. Seven Phytomonas -specific monoclonal antibodies produced against Phytomonas serpens and Phytomonas françai were further characterised in order to identify and localise their target antigens. Four monoclonal antibodies recognized carbohydrate surface epitopes, in three of the cases associated with surface glycoproteins with apparent molecular weight of 80 kDa. One monoclonal antibody apparently bound to a surface/internal protein epitope, whereas the two others recognized intra-cellular proteins. The cell surface epitopes recognized by monoclonal antibodies were detected specifically in the genus Phytomonas. These epitopes, which are detected in culture, plant and insect forms, may be useful as targets for Phytomonas identification.     

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.     

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.     

Three-dimensional reconstruction of glycosomes in trypanosomatids of the genus Phytomonas

Computer aided three dimensional (3-D) reconstruction of cells from two isolates of protozoa of the genus Phytomonas, trypanosomatids found in plants, were made from 0.3 mum thick sections, imaged on a Zeiss 902 electron microscope with a energy filter for inellastically scattered electrons, in order to obtain information about glycosomal shape diversity. Direct counts of peroxisomes (glycosomes) from Phytomonas sp. from Chamaesyce thymifolia indicated that there were fewer glycosomes per cell than the simple count of ultrathin section profiles would suggest and that these organelles could be long and branched. On the other hand, the stacked glycosomes observed in the isolate from Euphorbia characias were small individual structures and no connection was seen between them.     

Adenylyl cyclase and G-proteins in Phytomonas

Phytomonas sp. membranes have an adenylyl cyclase activity which is greater in the presence of Mn2+ than with Mg2+. The Mg2+ and Mn2+ activity ratio varies from one membrane preparation to another, suggesting that the adenylyl cyclase has a variable activation state. A[35S]GTP-gamma-S-binding activity with a Kd of 171 nM was detected in Phytomonas membranes. Incubation of these membranes with activated cholera or pertussis toxin and [adenylate 23P]NAD+ led to incorporation of radioactivity into bands of about 40-44 kDa. Crude membranes were electrophoresed on SDS-polyacrylamide gels and analyzed, by Western blotting, with the 9188 anti-alpha[s] antibody and the AS/7 antibody (anti-alpha[i], anti-alpha[i1], and anti-alpha[i2]. These procedures resulted in the identification of polypeptides of approximately 40-44 kDa. Phytomonas adenylyl cyclase could be activated by treatment of membrane preparations with cholera toxin, in the presence of NAD+, while similar treatment with pertussis toxin did not affect this enzyme activity. These studies indicate that in Phytomonas, adenylyl cyclase activity is coupled to an unknown receptor entity through G alpha[s] proteins.     

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.     

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.     

Life Cycle and Culturing of Phytomonas serpens (Gibbs), a Trypanosomatid Parasite of Tomatoes

Pure cultures of a trypanosomatid isolated from tomato fruits infected laboratory-raised tomatoes and nymphs of the hemipieran coreid Phthia picta . The flagellate could be transmitted back and forth from tomatoes to insects. Light and electron microscopy studies were done on culture, tomato and insect forms. Examination of enzymes of the ornithine-arginine metabolism revealed absence of arginase and presence of arginine deiminase and citrulline hydrolase. Monoclonal antibodies specific for Phytomonas spp. reacted positively with tomato and insect forms. Endonuclease digestion of the k-DNA of various Phytomonas spp. revealed a unique, distinctive pattern for the tomato flagellate. This flagellate thus seems to constitute a separate species of Phytomonas which we now call Phytomonas serpens (Gibbs).     

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.     

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.     

Comparative Analysis of the Fine Structure of Four Isolates of Trypanosomatids of the Genus Phytomonas1

The fine structure of trypanosomatids from the plants Euphorbia hyssopifolia, E. characias, E. pinea, and Manihot esculenta, cultivated under axenic conditions, was compared. All parasites had a structural organization characteristic of members of the Trypanosomatidae. The organization of the membranes of the endoplasmic reticulum (ER) varied according to the isolate. In Phytomonas francai (isolated from M. esculenta), the ER cisternae form parallel rows. In Phytomonas sp. from E. characias, a para-crystalline array of the ribosomes attached to the ER membranes was observed. The ER membranes found in Phytomonas sp. from E. pinea seemed to originate from the central portion of the protozoon, branching in all directions. The peroxisome-like organelle (glycosome) found in Phytomonas sp. from E. hyssopifolia and E. characias occasionally showed an organized array. Taken together, the morphological observations make it possible to distinguish the four isolates of Phytomonas.     

Cell Surface Saccharides in Three Phytomonas Species Differing in Host Specificity

ABSTRACT. Cell surface carbohydrates of three phytoflagellates, Phytomonas francai. Phytomonas serpens and Phytomonas sp. from different hosts including cassava, coreid insect Phthia picta and the milkweed plant Euphorbia hyssopifolia, respectively, were analysed by agglutination assays employing a battery of highly purified lectins with affinity for receptor molecules containing N-acetylglucosamine (d-GlcNAc), N-acetylgalactosamine (D-GalNAc), galactose, mannose-like (D-Man-like) residues and fucose, and by binding assay using radiolabeled [¹²⁵I]-wheat germ agglutinin (WGA) and fluorescent WGA lectin, as well as glycosidases of known sugar specificity, Escherichia coli K with mannose-affinity fimbrial lectin was also used as an agglutination probe. In general, the presence of D-GlcNAc. D-GalNAc and D-Man-like residues was detected in the phytomonads' plasma membrane. These sugar moieties were confirmed in whole cell hydrolysates as assessed by gas-liquid chromatography (GLC) which in addition, also showed the presence of galactose and xylose. However, marked differences in cell surface carbohydrate structures were observed. Wheat germ agglutinin, which binds to sialic acid and/or d-GlcNAc-containing residues, shows selective agglutinin activities for P. francai and Phytomonas sp., while Bandeiraea simplicifolia II agglutinin (which recognizes d-GlcNAc units) specifically bound to Phytomonas sp. Helix pomatia agglutinin which binds to D-GalNAc-containing residues reacted preferentially with Phytomonas sp. and P. serpens. Con A, which recognizes D-Man-like receptors, agglutinates all the phytomonads; however, the higher interaction was observed with Phytomonas sp. P. francai was selectively agglutinated in the presence of E. coli fimbrial lectin. Fluorescence WGA binding was significantly decreased by N-acetylglucosaminidase activities and the cell agglutination was not altered by neuraminidase treatment, suggesting the presence of an exposed D-GlcNAc moiety on the P. francai and Phytomonas sp. surfaces. Binding studies with [¹²⁵I]-WGA essentially confirmed the fluorescence WGA binding and agglutination assays.     

Biology and Structure of Phytomonas staheli sp. n., a Trypanosomatid Located in Sieve Tubes of Coconut and Oil Palms

SYNOPSIS. Phytomonas staheli sp. n. is described from the oil palm ( Elaeis guineensis ) and the coconut palm ( Cocos nucifera ) of Surinam, South America. The phytomonad is the probable cause of "Hartrot" in the coconut palm and "Marchitez sopresiva" in oil palms. Parasites are confined to the sieve tubes in palms. Some success was obtained at cultivation of the organism from Elaeis.     

PCR amplification of the spliced leader gene for the diagnosis of trypanosomatid parasites of plants and insects in methanol-fixed smears

A PCR-based method was adapted for the amplification of DNA from methanol-fixed smears of insects and plants parasitized by trypanosomatids. The PCR target was the multicopy spliced leader (SL) gene. Amplicons were hybridized with an oligonucleotide probe (SL3') specific for Phytomonas. The method has the advantage of dispensing with the cultivation of parasites, many of which are very fastidious or non-cultivable. The technique was applied to archival glass slides and to newly collected material. It proved to specific for Phytomonas spp., enabling their detection in plants and insects. Sequence comparison of the amplicons obtained revealed the existence of different strains/species of Phytomonas circulating among diseased palsms and fruit.     

The multifunctional isopropyl alcohol dehydrogenase of Phytomonas sp. could be the result of a horizontal gene transfer from a bacterium to the trypanosomatid lineage

Isopropyl alcohol dehydrogenase (iPDH) is a dimeric mitochondrial alcohol dehydrogenase (ADH), so far detected within the Trypanosomatidae only in the genus Phytomonas. The cloning, sequencing, and heterologous expression of the two gene alleles of the enzyme revealed that it is a zinc-dependent medium-chain ADH. Both polypeptides have 361 amino acids. A mitochondrial targeting sequence was identified. The mature proteins each have 348 amino acids and a calculated molecular mass of 37 kDa. They differ only in one amino acid, which can explain the three isoenzymes and their respective isoelectric points previously found. A phylogenetic analysis locates iPDH within a cluster with fermentative ADHs from bacteria, sharing 74% similarity and 60% identity with Ralstonia eutropha ADH. The characterization of the two bacterially expressed Phytomonas enzymes and the comparison of their kinetic properties with those of the wild-type iPDH and of the R. eutropha ADH strongly support the idea of a horizontal gene transfer event from a bacterium to a trypanosomatid to explain the origin of the iPDH in Phytomonas. Phytomonas iPDH and R. eutropha ADH are able to use a wide range of substrates with similar Km values such as primary and secondary alcohols, diols, and aldehydes, as well as ketones such as acetone, diacetyl, and acetoin. We speculate that, as for R. eutropha ADH, Phytomonas iPDH acts as a safety valve for the release of excess reducing power.     

Phytomonas: analysis of polymorphism and genetic relatedness between isolates from plants and phytophagous insects from different geographic regions by RAPD fingerprints and synapomorphic markers

The random amplification of polymorphic DNA was used for easy, quick and sensitive assessment of genetic polymorphism within Phytomonas to discriminate isolates and determine genetic relationships within the genus. We examined 48 Phytomonas spp., 31 isolates from plants and 17 from insects, from different geographic regions. Topology of the dendrogram based on randomly amplified polymorphic DNA fingerprints segregated the Phytomonas spp. into 5 main clusters, despite the high genetic variability within this genus. Similar clustering could also be obtained by both visual and cross-hybridization analysis of randomly amplified synapomorphic DNA fragments. There was some concordance between the genetic relationship of isolates and their plant tissue tropism. Moreover, Phytomonas spp. from plants and insects were grouped according to geographic origin, thus revealing a complex structure of this taxon comprising several clusters of very closely related organisms.     

Ribosomal DNA restriction analysis and synthetic oligonucleotide probing in the identification of genera of lower trypanosomatids.

Fifty-four species or isolates of insect trypanosomatids were examined for the presence of selected restriction enzyme sites in the small (SSU) and large (LSU) rRNA coding units of ribosomal genes. In the SSU, sites for Eco RI, Bgl II, Pst I, and Hind III were found to occur at the same location for all species examined, thus displaying a universal distribution among trypanosomatids. In the LSU, a site for Bgl II in the 24S-alpha sequence and sites for Hind III and Pst I in the 24S-beta sequence were found in all species examined. In contrast, a site for Pvu II in the SSU exhibited a genus-related distribution, being present in Crithidia and Herpetomonas but absent in Phytomonas. A site for Hind III in the 24S-alpha sequence of the LSU also exhibited genus-restricted distribution. The site was present in Crithidia but absent in Phytomonas and Herpetomonas. These findings were confirmed by dot hybridization with a synthetic oligonucleotide complementary to the 18S rRNA sequence containing the Pvu II site. Results point to the usefulness of restriction markers as diagnostic tools for distinguishing the lower trypanosomatid genera Crithidia, Herpetomonas, and Phytomonas at the same time revealing a marked complexity within the genus Leptomonas.     

Culture and Generic Identification of Trypanosomatids of Phytophagous Hemiptera in Brazil

Among the 372 phytophagous Hemiptera examined, 133 insects of 28 species (Coreidae 18, Pentatomidae 7, Pyrrhocoridae 2, Lygaeidae 1) were infected with trypanosomatids. Gut infections only were found in 68.4%, gut and salivary gland infections in 29.3% and salivary infections alone in 2.3%. Fifty-one cultures were isolated from 38 insects. Cultures were characterized by assay of certain ornithinc-arginine metabolism enzymes and by indirect immunofluorescence against monoclonal antibodies specific for Phytomonas spp. Ten cultures were identified as either Crithidia or Leptomonas. Twenty-one promastigote cultures had an enzyme pattern hitherto recorded only for Leishmania and 16 cultures were identified as Phytomonas.     

A family of highly conserved glycosomal 2-hydroxyacid dehydrogenases from Phytomonas sp

Phytomonas sp. contains two malate dehydrogenase isoforms, a mitochondrial isoenzyme with a high specificity for oxaloacetate and a glycosomal isozyme that acts on a broad range of substrates (Uttaro, A. D., and Opperdoes, F.R. (1997) Mol. Biochem. Parasitol. 89, 51-59). Here, we show that the low specificity of the latter isoenzyme is the result of a number of recent gene duplications that gave rise to a family of glycosomal 2-hydroxyacid dehydrogenase genes. Two of these genes were cloned, sequenced, and overexpressed in Escherichia coli. Although both gene products have 322 amino acids, share 90.4% identical residues, and have a similar hydrophobicity profile and net charge, their kinetic properties were strikingly different. One isoform behaved as a real malate dehydrogenase with a high specificity for oxaloacetate, whereas the other showed no activity with oxaloacetate but was able to reduce other oxoacids, such as phenyl pyruvate, 2-oxoisocaproate, 2-oxovalerate, 2-oxobutyrate, 2-oxo-4-methiolbutyrate, and pyruvate.