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PubMed2025

Trypanosomatid histones: the building blocks of the epigenetic code of highly divergent eukaryotes.

Histones play a fundamental role in eukaryotic organisms not only as scaffolding proteins in DNA packaging but also in regulating gene expression. They constitute the protein reel around which DNA wraps forming nucleosomes. This initial packing gives rise to the chromatin fiber which is next folded into three-dimensional arrangements. Additionally, histones have expanded their functions through the emergence of histone variants which have specialized purposes and can deeply affect chromatin organization and dynamics. Moreover, both canonical histones and histone variants comprise the building blocks of the histone code by being targets of different post-translational modifications (PTMs) that occur in a highly regulated manner both in place and time. Most of the above-mentioned about chromatin organization is conserved among eukaryotes. However, trypanosomatid histones have many peculiarities that entail a special description. In this review, we compile the current knowledge of canonical core histones, histone variants, and their PTMs in trypanosomatids. We highlight the similarities and differences between histone variants and their canonical counterparts in trypanosomatids, and we compare them with those from model organisms. Finally, we discuss the crosstalk between different histone marks and their genomic distribution underlying the uniqueness of trypanosomatids.

Histones↗
PubMed1977

Presence and properties of thymidylate synthase in trypanosomatids.

High speed centrifugal supernatant fractions of homogenates of a number of trypanosomatids were assayed for thymidylate synthase (5,10-methylene-tetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) activity using the method of Lomax and Greenberg (1967) J. Biol. Chem. 242, 109-113). Similar activities were detected in Crithidia fasciculata, Crithidia oncopelti, the blood forms of Trypanosoma brucei, Trypansoma congolense and Trypanosoma lewisi and the blood, intracellular and culture forms of Trypanosoma cruzi, suggesting that all species synthesize at least some thymidylate de novo. The properties of the activities in C. fasciculata and the three forms of T. cruzi were compared with those of the isofunctional bacterial and mammalian enzymes. The trypanosotamid enzyme was inhibited by Mg2+, was much more sensitive to mercaptoethanol, had higher apparent Km values for substrate (dUMP) and cofactor (tetrahydrofolate), had a higher apparent molecular weight and was markedly more sensitive to inhibition by suramin. It is, therefore a possible target for chemotherapeutic attack, either on its own or in combination with a dihydrofolate reductase inhibitor. No evidence was obtained for the regulation of the trypanosomatid enzyme, either by its product, dTMP, or by dTDP or dTTp. This result agrees with previous studies which suggested that in trypanosomatids, the level of dTMP was regulated, at least in part, by a catabolic pathway consisting of a thymidylate phosphatase and a thymidine phosphorylase which degraded the excess of dTMP to thymine.

Animals↗
PubMed1977

Catabolism of deoxythymidylate in some trypanosomatids.

An initial observation concerning the failure of [3H]thymidine at high specific activity to be incorporated into the DNA of Crithidia fasciculata for more than a brief initial period has been correlated with the presence at high specific activity in the organism of a thymidine phosphorylase activity with an equilibrium in the direction of catabolism. This enzyme degrades thymidine to thymine which is not utilized by the organism. The enzyme has also been shown to be present in a number of other trypanosomatids, including the culture forms of Trypanosoma cruzi, where the specific activity was nearly as high as that in C. fasciculata. Evidence is presented that in C. fasciculata, the culture forms of T. cruzi and possibly other species of trypanosomatid, the thymidine phosphorylae, together with a thymidylate phosphatase, forms a catabolic pathway which degrades thymine nucleotides to thymine, which is then excreted. About 60% of the thymine nucleotides made by organisms appear to be metabolized through the pathway, suggesting that their synthesis is not subject to completely effective regulatory control.

Animals↗
PubMed1977

Problems in the comparative physiology of some trypanosomatid flagellates.

There is considerable evidence that trypanosomatid species vary in their metabolic characteristics, that they vary in the mechanisms by which they control these characteristics, and that a single species may vary metabolically without varying structurally. Studies to date from a number of laboratories also indicate there is still reason to believe that metabolic characteristics of trypanosomatid flagellates, as manifest in culture, are at least correlated (in some cases) with the behavior of the flagellate species in the metazoan host. It appears that our major tasks for the next several years are 1. to discover the extent to which these correlations are manifestations of characteristics required for life in the metazoan host, 2. to discover the extent to which these correlations are manifestations of characteristics which determine infection site within the metazoan host, 3. to discover which if any metabolic control mechanisms contribute to the multiplicity of clinical infections often seen in this group of protozoa, and 4. to discover the critical links between energy metabolism, the control of that metabolism, and life inside a host or host cell.

Culture Media↗
PubMed1979

A novel model for fluid secretion by the trypanosomatid contractile vacuole apparatus.

We have studied fluid secretion by the contractile vacuole apparatuss of the trypanosomatid flagellate Leptomonas collosoma with thin sections and freeze-fracture replicas of cells stabilized by ultrarapid freezing without prior fixation or cryoprotection. The ultrarapid freezing has revealed membrane specializations related to fluid segregation and transport as well as membrane rearrangements which may accompany water expulsion at systole. This osmoregulatory apparatu consists of the spongiome, the contractile vacuole, and the fluid discharge site. The coated tubules of the spongiome converge on the contractile vacuole from all directions. These 60- to 70-nm tubules contain characteristic double rows of 11-nm intramembrane particles in a helical configuration which fracture predominantly with the E face. Short double rows of similar particles are also frequently found on both faces of the contractile vacuole itself, in addition to many smaller particles on the P face. The spongiome tubules fuse with the vacuole during the filling stage of each cycle and then detach before secretion. The contractile vacuole membrane is permanently attached to the plasma membrane of the flagellar pocket by a dense adhesion plaque. In some ultrarapidly frozen cells, 20- to 40-nm perforations can be visualized within the plaque and the adjacent membranes during the presumptive time of discharge. The formation of the plaque perforations and the membrane channels occurs without fusion of the vacuole and the plasma membrane and does not require extracellular calcium. On the basis of our results, we have developed a model for water secretion which suggests that the adhesion plaque may induce pore formation in the adjoining lipid bilayers, thereby allowing bulk expulsion of the fluid.

Animals↗
PubMed1979

Cytochemical analysis at the fine-structural level of trypanosomatids stained with phosphotungstic acid.

The ethanolic phosphotungstic acid (PTA) technic was used to detect, at the fine-structural level, basic proteins in various developmental stages of pathogenic Trypanosoma cruzi, and nonpathogenic Herpetomonas samuelpessoai, Leptomonas samueli, and Crithidia deanei, trypanosomatids. Reactions were observed in the nucleus of all stages. In the kinetoplast of epimastigote and promastigote forms reactions were noted mainly at the periphery. In trypomastigotes and choanomastigotes forms, however, an intense reacion was observed thorughout the kinetoplast. Reactions were present in cytoplasmic vesicles related to protein storage in T. cruzi and in membrane-bounded peroxisome-like organelles of H. samuelpessoai, L. samueli and C. deanei. The network of filaments which forms the paraxial rod did not react. In the flagellum, reaction was noted only at the peripheral doublet microtubules. PTA reacts also with structures related to the junction between the flagellar and cell body membranes.

Animals↗
PubMed1979

Purine metabolism in trypanosomatids.

Purine nucleotide biosynthesis was studied in culture forms of Trypanosoma cruzi strain Y, Crithidia deanei (a reduviid trypanosomatid with an endosymbiote) and an aposymbiotic strain of C. deanei (obtained by curing C. deanei with chloramphenicol). Trypanosoma cruzi was found to synthesize purine nucleotides only fring incorporated into both adenine and guanine nucleotides. Similar results were obtained with guanine, indicating that this flagellate has a system for the interconversion of purine nucleotides. Crithidia deanei was able to synthesize purine and pyrimidine nucleotides from glycine ("de novo" pathway) and purine nucleotides from adenine and guanine ("salvage" pathway). Adenine was incorporated into both adenine and guanine nucleotides, while guanine was incorporated into guanine nucleotides only, indicating the presence of a metabolic block at the level of GMP reductase. The aposymbiotic C. deanei strain was unable to utilize glycine for the synthesis of purine nucleotides, although glycine was utilized for synthesizing pyrimidine nucleotides. These results suggest that the endosymbiote is implicated in the de novo purine nucleotide pathway of the C. deanei-endosymbiote complex. The incorporation of adenine and guanine by aposymbiotic C. deanei strain followed a pattern similar to that observed for C. deanei.

Adenine↗
PubMed1979

Dyskinetoplasty in two species of trypanosomatids.

Dyskinetoplastic cells from both Crithidia fasciculata and Trypanosoma equiperdum lack detectable kinetoplast DNA (kDNA) by conventional staining techniques. Two dyskinetoplastic strains of T. equiperdum, either acriflavine-induced or spontaneously occurring, show normal amounts of kDNA (p = 1.692 g/cm3) in analytical caesium chloride, ultracentrifugation. Electron and fluorescence microscopy of the dyskinetoplastic strains of T. equiperdum suggest that the kDNA network is fragmented and dispersed throughout the mitochondrion. The fragmentation and dispersion of the kDNA, rather than a reduction in the amount of kDNA, is the cause of the lack of kinetoplast staining in the dyskinetoplastic strains of T. equiperdum. Acriflavine-treated cultures of C. fasciculata show a decrease in the amount of kDNA (p = 1.703 g/cm3) corresponding to the percentage of dyskinetoplastic cells in the cultures. Electron and fluorescence microscopy of acriflavine-treated cultures of C. fasciculata show the loss of the kDNA network in cells which lack Giemsa and Feulgen staining, confirming the hypothesis that the kDNA is lost in dyskinetoplastic trypanosomatids from insects. Possible modes of acriflavine action are considered and a proposed mechanism for acriflavine action in trypanosomes from mammals is presented.

Acriflavine↗
PubMed1979

Acid hydrolases of trypanosomatid flagellates.

Homogenates from 5 species of Trypanosomatids were screened for the presence of a series of acid hydrolases. The insect flagellae, Crithidia sp., contains 5 enzymes reminiscent of plant parasitism, which were absent from or of very low specific activity in parasites of the genera, Trypanosoma and Leishmania. The latter mammalian parasites, on the other hand, exhibited higher acid proteinase and alpha-D-mannosidase activity levels.

Animals↗
PubMed2025

Analysis of Leishbuviridae from Trypanosomatids.

Over the last decade, considerable progress has been made in unraveling RNA virus diversity. This has contributed to our understanding of the evolution of these viruses, which include emerging zoonotic human pathogens. Current success has been greatly facilitated by the development of next-generation sequencing platforms instrumental for meta-transcriptomic studies. However, due to the rapid evolution of RNA viruses, there are numerous "blind spots" waiting to be explored; one of those is the RNA virome of unicellular eukaryotes. Here, we present the pipeline, which has been successfully used to characterize various types of RNA viruses, including Leishbuviridae (Bunyaviricetes, Hareavirales) in the parasitic flagellates of the family Trypanosomatidae. The pipeline relies on axenic in vitro cell culture and double-stranded RNA enrichment, followed by direct RNA-sequencing. A detailed procedure description starting from the initial total RNA preparation to the final assembly of the viral segments is provided.

High-Throughput Nucleotide Sequencing↗
PubMed1977

Fixation of trypanosomatids for electron microscopy with the glutaraldehyde-tannic acid method.

Epimastigotes from Trypanosoma cruzi and promastigotes from Herpetomonas samuelpessoai were fixed with glutaraldehyde-tannic acid. Different concentrations of tannic acid were tested. With this technique the cellular membranes appear in negative contrast offering the same aspect as seen in cells fixed in glutaraldehyde only without post-fixation in osmium tetraoxide. An electron-dense deposit appears on the surface which possibly represents positively charged groups. The structure of the sub-pellicular microtubules appears well defined and it was possible to distinguish the 13 protofilaments which compose the microtubule wall.

Animals↗
PubMed1977

The genetic system of kinetoplasts in trypanosomatides.

In the present report, the genetic system of Crithidia oncopelti kinetoplast is used as a model for investigation of kinetoplast DNA (kDNA) structure, its transcription, protein synthesizing apparatus of the kinetoplast and the protein synthesis controlled genetically by kDNA. It was shown that kDNA of C. oncopelti can be isolated from cells or from kinetoplast fraction in the form of a network complex structure consisting of a lot of circular molecules. These minicircles have a contour length of about 0.83 micronm and molecular weight of 1.6 X 10(6). The kDNA was demonstrated to be of higher AT content type than nuclear DNA. Besides, kDNA is characterized by a lesser degree of clustering of pyrimidines as compared with the nuclear one. The isolated kinetoplasts of C. oncopelti were shown to exhibit activity of DNA dependent RNA polymerase. The effect of some antibiotics and intercalating substances on RNA synthesis in kinetoplasts and mitochondria appears to be identical. Kinetoplasts of C. oncopelti have their own protein synthesizing system, whose components (ribosomes, rRNA, proteins, factors of incorporation) differ from those of the cytoplasm. Inhibition of translation by some antibiotics and of transcription by acriflavin allowed the suggestion that several proteins of kinetoplast ribosomes may be synthesized within this organoid. It was shown then that kDNA may be involved in the formation of the protein synthesizing apparatus in the kinetoplast.

Animals↗
PubMed1975

Leishmaniasis in Brazil: X. Some observations of intradermal reactions to different trypanosomatid antigens of patients suffering from cutaneous and mucocutaneous leishmaniasis.

In spite of the difficulty of antigen standardization, paired intradermal skin tests demonstrated qualitative differences between leishmanin and in vitro exo-antigen. Some of these differences may be due to the destruction of certain antigens by phenol, while others seem to reflect basic antigenic differences. Leishmania mexicana amazonensis in vitro exo-antigen produced immediate anaphylactic reponses in 74.4% of the patients with parasitologically proven cutaneous leishmaniasis. Although more individuals responded to leishmanin, 82.4% as compared to 73.9%, the leishmanial in vitro exo-antigen gave significantly larger delayed responses, Trypanosoma cruzi trypamosomin elicited delayed reactions, while trypanosomal in vitro exo-antigen only produced immediate anaphylactic reactions in persons with active cutaneous leishmaniasis.

Animals↗
PubMed1977

Isolation and properties of flagella of trypanosomatids.

A procedufe is described for the isolation of flagella of Crithidia fasciculata, Herpetomonas samuelpessoai and Leishmania tarentolae in a highly purified state and giving reasonably good yield. The 3 types of flagella give a similar electrophoretic pattern of proteins. It is shown that H. samuelpessoai and, to a lesser extent, C. fasciculata flagella confer protection against Trypanosoma cruzi infection.

Animals↗
PubMed1978

Effects of methylglyoxal bis(ganylhydrazone) on trypanosomatid flagellates: inhibition of growth and nucleoside incorporation in Trypanosoma brucei.

Methyglyoxal bis (guanylhydrazone) (MGBG) at 0.5 mM had little effect in vitro on Blastocrithidia culicis, Crithidia oncopelti, and Leishmania spp., but completely inhibited growth of Trypanosoma brucei. Inhibition became irreversible after a 3-h exposure of T. brucei culture procyclics. Treated organisms remained motile, but failed to divide. Polyamines, spermidine, and spermine, did not reverse the anti-trypanosome action of MGBG (preloading of cells or concurrent administration). Two intraperitoneal injections of the drug at a concentration of 50 mg/kg body weight at a 1-day interval greatly reduced the parasitemia of T. brucei and T. congolense in rats. Trypanosome infections, however, relapsed and killed the animals in 6 days after treatment. It was evident from the results of tracer experiments with T brucei that MGBF significantly lowered incorporation of [3H]thymidine by culture pocyclics and of [3H]uridine by bloodstream forms; in both stages [3H]leucine incorporation was only slightly inhibited. It is suggested that MGBG interferes with nucleoside incorporation by Trypanosoma and that its mode of action is different in bloodstream and culture procyclics.

Animals↗
PubMed1979

Five trypanosomatid species of insects distinguished by isoenzymes.

Blastocrithidia culicis, Crithidia deanei, Crithidia fasciculata, Herpetomonas samuelpessoai, Leptomonas seymouri and Leishmania tarentolae grown in cultures were compared by electrophoretic mobility for isoenzymes in 6 enzymes. All species were found distinct in these characteristics. Endosymbiotic C. deanei, which was identical to the aposymbiotic C. deanei in 5 enzymes, had an extra band in aspartate aminotransferase. No differences in isoenzymes were found between members of one species maintained in 2 different culture media.

Alanine Transaminase↗