The Cytostome of Trypanosoma cruzi and T. conorhini*

SYNOPSIS. A cytostome is described in culture forms and in intracellular stages of Trypanosoma cruzi and in culture forms of T. conorhini. The organelle opens into the flagellar pocket or close to it and runs deeply into the cell.     

The Fine Structure of Trypanosoma congolense in Its Bloodstream Phase

SYNOPSIS. The fine structure of 2 isolates of Trypanosoma congolense maintained in laboratory rodents has been studied from thin sections of osmium- and aldehyde-fixed flagellates. The pellicular complex, nucleus, and flagellar apparatus are all similar to those of other African trypanosomes. Aberrant intracellular differentiation of the flagellum is occasionally found. As in bloodstream forms of other salivarian trypanosomes the single mitochondrion forms an irregular canal running from one end of the body to the other, with a shallow bowl-shaped expansion forming a capsule for the fibrous kinetoplast (mitochondrial DNA). A connexion between the mitochondrial envelope of the kinetoplast and the basal body of the flagellum is not evident, and sometimes the flagellum base is not even apposed to the kinetoplast but lies behind it. Tubular cristae are present in the mitochondrial canal and, by light microscopy, this structure gives a positive reaction for NAD diaphorase suggesting at least some activity in electron transport, even tho at this stage in its life cycle respiration is doubtfully sensitive to cyanide and cytochrome pigments are in all probability absent. The region of the cytoplasm between the nucleus and the flagellar pocket has all the trappings associated with secretory cells in higher animals, or with the secretion of surface structures in phytoflagellates. just behind the nucleus a limb of granular reticulum subtends a Colgi stack of flattened saccules with attendant vesicles. Close to the distal pole of the Golgi complex is a network of smooth-membraned cisternae, termed here the agranular or secretory reticulum, which undergoes localized swelling with the accumulation of a secretory product to form large spherical sacs or vacuoles. These network-linked vacuoles probably correspond to the post nuclear vacuole complex visible by light microscopy. From its apparent secretory function this complex is regarded here as being possibly an extension or derivative of the Golgi complex, the smooth-membraned tubules lying alongside the 2 structures possibly representing a link between them. By analogy with phytoflagellates and the secretory cells of higher animals, it is suggested that the secretion is transported for discharge into the flagellar pocket by way of multivesicular bodies and smooth-walled tubules or vesicles. Spiny pits in the wall of the flagellar pocket, and similar-sized vesicles in the nearby cytoplasm, could be stages in either exocytosis of secretion or endocytosis (pinocytosis). It is tentatively suggested that the secretion may be the material from which the surface coat is formed. Neither a cytostome nor a contractile vacuole has been observed in T. congolense.     

Fine Structure of Trypanosoma cyclops in Noncellular Cultures*

The fine structure of the epimastigotes of Trypanosoma cyclops maintained in blood agar medium at 25 C is described. This organism was isolated from the Malaysian primates Macaca nemestrina and Macaca ira. A distinctive feature of T. cyclops is that it is pigmented when grown in the presence of hemoglobin. The pigment bodies apparently lack a substructure and are electron dense even in unstained sections. Most of the pigment is located posterior to the kinetoplast region but some is found adjacent and anterior to the kinetoplast. Cells from control cultures grown in medium lacking hemoglobin did not possess this type of pigment body. Similarly, pigment was not found in cells of an Indonesian trypanosome grown in medium containing hemoglobin. The cytoplasm of T. cyclops is bounded by a unit membrane which is specialized where it makes contact with the flagellum. A cytostome extends from the region of the flagellar pocket. The kinetoplast and nucleus are immediately posterior to the base of the flagellum. Transverse sections in the region of the flagellar pocket and flagellar base often reveal a group of 3 microtubules which are distinct from the pellicular microtubules.     

Developmental and Ultrastructural Characterization and Phylogenetic Analysis of Trypanosoma herthameyeri n. sp. of Brazilian Leptodactilydae Frogs

We described the phylogenetic affiliation, development in cultures and ultrastructural features of a trypanosome of Leptodacylus chaquensis from the Pantanal biome of Brazil. In the inferred phylogeny, this trypanosome nested into the Anura clade of the basal Aquatic clade of Trypanosoma, but was separate from all known species within this clade. This finding enabled us to describe it as Trypanosoma herthameyeri n. sp., which also infects other Leptodacylus species from the Pantanal and Caatinga biomes. Trypanosoma herthameyeri multiplies as small rounded forms clumped together and evolving into multiple‐fission forms and rosettes of epimastigotes released as long forms with long flagella; scarce trypomastigotes and glove‐like forms are common in stationary‐phase cultures. For the first time, a trypanosome from an amphibian was observed by field emission scanning electron microscopy, revealing a cytostome opening, well‐developed flagellar lamella, and many grooves in pumpkin‐like forms. Transmission electron microscopy showed highly developed Golgi complexes, relaxed catenation of KDNA, and a rich set of spongiome tubules in a regular parallel arrangement to the flagellar pocket as confirmed by electron tomography. Considering the basal position in the phylogenetic tree, developmental and ultrastructural data of T. herthameyeri are valuable for evolutionary studies of trypanosome architecture and cell biology.     

A cryptic cytostome is present inEuglena

Summary A short cylindrical pocket arises as an infolding from the ventral surface of the reservoir near the canal in several species ofEuglena (E. mutabilis, E. gracilis strain T,E. spec.). The structure is linked to a band of microtubules which is shown to be identical to the ventral flagellar root of the euglenoid flagellar root system. An absolute configuration analysis of the flagellar root system inE. mutabilis and a comparison with the flagellar apparatus of colourlessEuglenophyceae and the bodonids (Kinetoplastida) reveals structural and positional homology between the reservoir pocket ofEuglena and the cytostome of these organisms and strongly supports the phylogenetic derivation of theEuglenophyceae from theKinetoplastida and the evolution of greenEuglenophyceae from phagotrophic colourless taxa. The functional significance of the cryptic cytostome ofEuglena is discussed in relation to the occurrence of intracellular endosymbiotic bacteria.     

Unusual Structures in the Flagellar Apparatus of a Strain of Trypanosoma cruzi*

Certain structures, associated with the flagellum, and which had hitherto been described as appearing occasionally in some species of trypanosomes, were found very frequently in epimastigote forms of strain F of Trypanosoma cruzi: (a) a group of tubular elements in an electron-dense mass enclosed within a swelling of the flagellar membrane as the flagellum emerges from its reservoir; (b) an expansion of the flagellar membrane at the point of the above swelling, which in cross-sections appears as a ring; and (c) an electron dense band in the body of the organism alongside the border of the flagellar pocket. The possible significance of these structures and the fact that so far they have been found only in one strain of T. cruzi are discussed.     

The cytostome of Trypanosoma cruzi epimastigotes is associated with the flagellar complex.

Okuda, K., Esteva, M., Segura, E. L., and Bijovsky, A. T. 1999. The cytostome of Trypanosoma cruzi epimastigotes is associated with the flagellar complex. Experimental Parasitology 92, 223-231. Proliferative forms of Trypanosoma cruzi, amastigotes and epimastigotes, have a cytostome, a specialized structure formed by an invagination of the flagellar pocket's membrane surrounded by microtubules and frequently followed by a row of vesicles. All this assemblage penetrates deeply into the cytoplasm overpassing the nucleus. This structure, together with the flagellar pocket, appears to play an important role in the nutrition of the parasite. We demonstrated that the monoclonal antibody 2C4, made-up against isolated flagellar complex of T. cruzi epimastigotes, recognizes a protein doublet of 76 and 87 kDa in total epimastigotes homogenate. The 76-kDa polypeptide is enriched in the detergent-soluble fraction whereas the 87-kDa polypeptide is highly represented in the insoluble fractions and the purified flagella. Immuno-fluorescence assays show the antigen as a small spot at the flagellar pocket region. Immunogold labeling of ultrathin sections of epimastigote forms reveals gold particles at the opening of flagellar pocket, concentrated in the cytostome region. Immunocytochemistry of epimastigote whole-mount cytoskeletons reveals the labeling on an array of three to four microtubules that appears attached to flagellum, running in the direction of the nucleus. Ultrastructural observations have shown that the posterior region of isolated flagella, corresponding to the level of the flagellar pocket, possesses a microtubular structure compatible with that from the cytostome. The relationship between the cytostome, an endocytic organelle, and the flagellum is here described for the first time.     

Attachment of flagellum to the cell body is important to the kinetics of transferrin uptake by Trypanosoma cruzi

The flagellar pocket and the cytostome are surface domains of Trypanosoma cruzi epimastigote involved in acquisition of nutrients. The cytostome is physically connected to the flagellar complex. To investigate if this association plays a role in endocytosis in T. cruzi, the endocytic activity in wild type and gp72 null mutant (flagellum-cell body attachment region is absent) epimastigotes was compared. Both wild type and mutant cells were incubated with transferrin conjugated with Alexa 543 or gold particles over different time periods and thereafter qualitatively and quantitatively analyzed by flow cytometry and transmission electron microscopy. Flow cytometry analysis showed a reduction in transferrin uptake by null mutant after 30 min of incubation. In addition, at this time period, signals detected by fluorescence microscopy were slightly lower in null mutant cells. At lower incubation times, no differences between wild type and mutant epimastigotes could be observed. Quantitative data obtained by morphometric and flow cytometry analysis suggested that the speed of the endocytic process in the null mutant was similar to wild type cells, although null mutants were not able to bind cargo and therefore internalize as much as wild type epimastigotes. Our observations suggest that the physical association between cytostome and the flagellar complex plays a role in endocytosis efficiency by epimastigotes of T. cruzi.     

Trypanosoma cruzi: ultrastructural, cytochemical and freeze-fracture studies of protein uptake.

Epimastigotes and trypomastigotes of Trypanosoma cruzi, obtained from liquid cultures, have vesicles and multivesicular structures in their cytoplasm. Horseradish peroxidase (HRP) was used as a tracer to study the uptake of protein by these two forms. In epimastogotes HRP is ingested by a process of pinocytosis which occurs through the cytostome. Trypomastigotes do not have a cytostome, and pinocytosis occurs through the flagellar pocket region. The pinocytotic vesicles can fuse with each other to form large multivesicular structures that are more abundant in epimastigotes than in trypomastigotes. The cell membrane as well as the membranes of the pinocytotic vesicles and the large multivesicular structure have carbohydrates, as detected by the periodic acid-thiosemicarbazide-silver proteinate technique. Intramembranous particles were observed by using the freeze-fracture technique. The cell membrane has many particles, whereas the membranes of the vesicles and multivesicular structure have few or no particles.     

Trypanosoma cruzi: location of a specific antigen on the surface of bloodstream trypomastigote and culture epimastigote forms.

The nature of surface antigens of culture epimastigote and bloodstream trypomastigote forms of Trypanosoma cruzi was investigated by light and electron microscopy using indirect immunofluorescence and peroxidase labeling techniques and antisera against unique, common, and contaminant antigens. A specific antigen, identified by monospecific rabbit antiserum (anti-component 5 antiserum), is the major constituent of the cell surface and flagellar membrane of both the culture epimastigote and bloodstream trypomastigote forms. Antigens of heterologous stercorarian trypanosomes (Trypanosoma rangeli) and of culture medium proteins could not be detected on the cell surface of culture epimastigote forms and bloodstream trypomastigote forms.     

The Form and Function of the Cytostome-Cytopharynx of the Culture Forms of the Elasmobranch Haemoflagellate Trypanosoma raiae Laveran & Mesnil

SYNOPSIS. In the culture forms of the elasmobranch trypanosome Trypanosoma raiae is found a prominent cytopharyngeal complex. This consists of a group of 5 or 6 microtubules associated with a deep invagination of the cell membrane which arises from a cytostome near the opening of the flagellar pocket. This structure is a constant feature of the various epimastigote and trypomastigote forms that this flagellate has in culture. Replication of the cytopharyngeal apparatus is completed before cytokinesis.
Experiments using ferritin as an electron dense tracer show that endocytosis occurs from the blind ending of the cytopharynx both in the exponential and stationary phases of growth in vitro. Ferritin is transported from the cytopharynx by endocytotic vesicles to large, membrane-bound vacuoles in the posterior region of the cell. Ultrastructural location of non-specific acid phosphatase within these digestive vacuoles and also within the Golgi apparatus is reported.
Coated vesicles found in association with the flagellar pocket are another route of uptake of ferritin by T. raiae.     

Antigens of Subcellular Fractions of Trypanosoma cruzi. III. Humoral immune response and histopathology of immunized mice*

SYNOPSIS. The relation of humoral antibody response to protection was evaluated in mice immunized with whole homogenates of Trypanosoma cruzi or with its flagellar fraction by direct agglutination and indirect fluorescent antibody test as well as by lytic and neutralizing activity against blood trypomastigotes. The results indicated that lytic antibodies were not implicated directly in protection against these trypanosomes. It was evident from histopathologic examination that the higher the degree of protection achieved, the lower the tissue damage observed in the challenged mice. Serum-neutralizing activity was highest in the groups protected most effectively.     

Trypanosoma brucei brucei and T. b. gambiense: Stumpy Bloodstream Forms Express More CB1 Epitope in Endosomes and Lysosomes than Slender Forms

CB1-glycoprotein is a component of flagellar pocket, endosome, and lysosome membranes of long, slender bloodstream forms of the Trypanosoma brucei subgroup of African trypanosomes. We have used immunoblotting, immunofluorescence, and cryoimmunoelectron microscopy to study CB1-glycoprotein expression as long, slender bloodstream forms of pleomorphic T. b. brucei and T. b. gambiense transform through intermediate stages into short, stumpy forms. Intermediate and stumpy forms express more CB1-glycoprotein than long, slender forms. These results, coupled with previous work showing that procyclic forms do not express CB1-glycoprotein, show that the expression of lysosomal membrane glycoproteins is regulated coordinately with other aspects of lysosome and endosome function as these trypanosomes go through their life cycle.     

Evidence for a sliding-resistance at the tip of the trypanosome flagellum

Motility in trypanosomes is achieved through the undulating behaviour of a single "9 + 2" flagellum; normally the flagellar waves begin at the flagellar tip and propagate towards the base. For flagella in general, however, propagation is from base-to-tip and it is believed that bend formation, and sustained regular oscillation, depend upon a localised resistance to inter-doublet sliding - which is normally conferred by structures at the flagellar base, typically the basal body. We therefore predicted that in trypanosomes there must be a resistive structure at the flagellar tip. Electron micrographs of Crithidia deanei, Herpetomonas megaseliae, Trypanosoma brucei and Leishmania major have confirmed that such attachments are present. Thus, it can be assumed that in trypanosomes microtubule sliding at the flagellar tip is resisted sufficiently to permit bend formation.     

The in vivo distribution of Cr-labeled Trypanosoma cruzi in mice

The optimal conditions for labeling Trypanosoma cruzi culture forms with ⁵¹CrO₄²⁻ were determined. Labeled trypanosomes or labeled human red blood cells (RBCs) were injected intravenously into normal C3H(He) female mice and the rate of clearance and organ distribution of the isotope were observed over a 30 h period. It was found that trypanosomes and xenogeneic RBCs were cleared rapidly from the peripheral blood and accumulated primarily in the liver, spleen, lungs and kidneys. A difference was noted in accumulation of trypanosomes and RBCs in these mice.     

Trypanosoma lewisi: ultrastructural observations of surface antigen movement induced by antibody.

Cellular antigens of Trypanosoma lewisi have been located with ferritin-conjugated antibody (FCA) at the ultrastructural level. Incubation of live T. lewisi with antibodies from infected rats and ferritin-labeled rabbit anti-rat γ-globulin induced aggregation of surface antigens in the flagellar pockets, the desmosomal regions, and the flagellar membranes of parasites. Aggregation of surface antigens was not observed when the trypanosomes were incubated with γ-globulin and FCA at low temperatures (0–4 C). Sections of trypanosomes incubated at 37 C for 15 or 30 min after incubation at 0 C with FCA showed internalization of FCA in membrane-bound vesicles. Studying the movement and aggregation of these parasites' surface antigens may give information about the molecular dynamics of the plasma membrane and provide insights into the trypanosomes' antigenic modulation and the hosts' immunological responses.     

Trypanosoma cruzi glycoprotein 72: Immunological analysis and cellular localization

Two monoclonal antibodies were used to biochemically characterize glycoprotein 72 (GP72) from Trypanosoma cruzi and to localize the protein in live and fixed parasites by indirect immunofluorescence and in thin section of parasites by immunogold electron microscopy. GP72 was shown in immunoblots to be specific for the epimastigote stage; the protein could not be detected in trypomastigotes. Each antibody reacted with a different epitope on the glycoprotein and deglycosylation of GP72 ablated reactivity with one of the antibodies. Indirect immunofluorescence and electron microscopic evaluation of parasite associated gold particles showed the presence of GP72 in the cell surface membrane including the flagellar pocket and the cytostome. In addition, cytoplasmic membrane vesicles of the endosomal-lysosomal system stained intensely.     

Branched Electron Transport Chain in <Emphasis Type="Italic">Trypanosoma mega</Emphasis>

THE life cycle of certain pathogenic African trypanosomes is characterized by a striking change in the mechanism of oxidative reactions on which the aerobic metabolism of the organism depends. Vertebrate bloodstream forms of Trypanosoma brucei group trypanosomes apparently depend on a non-mitochondrial, cyanide-insensitive α-glycerophosphate oxidase system¹. Cells which become established when trypanosomes are grown in culture resemble those found in the insect vector. Early reports on the metabolism of these culture forms described a cyanide-sensitive terminal respiration², associated with the presence of a complex mitochondrial network^3,4 and cytochrome pigments^2,5,6. The only reports of cyanide-insensitive respiration in culture forms have been for recently transformed Trypanosoma brucei^7,8 and Trypanosoma mega⁹.     

Transmission of Megatrypanum Trypanosomes to Cervus dama by Tabanidae1

Four fallow deer, Cervus dama, became infected with Trypanosoma (Megatrypanum) sp. by oral application of triturated guts from tabanids collected in an area with deer but without any cattle; four control calves remained negative. Upon challenge with triturated guts from tabanids from an area with pastured cattle, the four calves became infected with Trypanosoma (M.) theileri. The prepatent period in deer was five days or less. Haematopota spp. and Tabanus spp. were identified as vectors of the deer trypanosomes. It is concluded that the trypanosomes of C. dama belong to a Megatrypanum species that is not identical with T. theileri.     

A nested PCR for the ssrRNA gene detects Trypanosoma binneyi in the platypus and Trypanosoma sp. in wombats and kangaroos in Australia

Trypanosome infections in their natural hosts are frequently difficult to detect by microscopy, and culture methods are unreliable and not suitable for all species of Trypanosoma. A nested PCR strategy for detecting and identifying Trypanosoma species, suitable for detecting both known and unknown trypanosomes, is presented. Thirty-two blood samples from 23 species of Australian birds and mammals were screened by a nested PCR for the presence of Trypanosoma sp. ssrRNA. Three infections were detected, one in an eastern grey kangaroo (Macropus giganteus), one in a common wombat (Vombatus ursinus) and one in a platypus (Ornithorhynchus anatinus). The kangaroo and wombat are new host records for Trypanosoma sp.; the platypus parasite was Trypanosoma hinneyi. The three parasites could be distinguished by restriction fragment length polymorphisms of the amplified fragment of the ssrRNA gene. The kangaroo and wombat parasites were also isolated in a semi-solid blood agar medium. The culture forms of the kangaroo trypanosome had an expanded flagellar sheath in which structures similar to hemidesmosomes were detected by EM. The nested PCR was at least as sensitive as culture, and analysis of the PCR products gave parasite-specific fingerprints. Therefore this method could be suitable for rapidly screening host animals for the presence of trypanosomes and identifying the infecting strain.