In Vitro Production of Metacyclic Forms of Trypanosoma musculi and Their Infectivity in CBA Mice1

ABSTRACT. Bloodstream forms of Trypanosoma musculi, cultured in Schneider's drosophila medium at room temperature, multiply and differentiate through a series of developmental stages into infective metacyclic trypomastigotes in 10 days. Oral inoculation with these culture forms into CBA mice produced a parasitemia similar to that produced by intraperitoneal infection with bloodstream forms except for a three-day longer prepatent period. Attempts to induce parasitemia with bloodstream forms given orally were unsuccessful.     

Freeze-Fracture Study of the Bloodstream Form of Trypanosoma brucei gambiense

The ultrastructure of Trypanosoma brucei gambiense was investigated by the freeze-fracture method. Three different regions of the continuous plasma membrane; cell body proper, flagellar pocket, and flagellum were compared in density and distribution of the intramembranous particles (IMP's). The IMP-density was highest in the flagellar pocket membrane and lowest in flagellum. Intra membranous particles of the cell body membrane were distributed uniformly on both the protoplasmic (P) and exoplasmic (E) faces. On the P face of the flagellar membrane, a single row of IMP-clusters was seen along the juncture of the flagllum to the cell body. Since the spacing of the IMP-clusters was almost equal to the spacing of the paired rivet structures observed in thin section, these clusters likely are related to the junction of flagellum and cell body. At the neck of the flagellar pocket, several linear arrays of IMP's were found on the P face of the flagellar membrane, while on the E face rows of depressions were seen. At the flagellar base, the clusters of IMP's were only seen on the P face. On the flagellar pocket membrane, particle-rich depressions and linear particle arrays were also found on the P face, while on the E face such special particle arrangements were not recognized. These particle-rich depressions may correspond to the sites of pinocytosis of the bloodstream forms which have been demonstrated in thin sections.     

Cyclical Transmission of Trypanosoma brucei rhodesiense and Trypanosoma congolense by Tsetse Flies Infected with Culture-Form Procyclic Trypanosomes*

SYNOPSIS. Culture procyclic forms of Trypanosoma brucei rhodesiense and Trypanosoma congolense were fed to Glossina morsitans morsitans through artificial membranes. A very high percentage of the flies so fed produced established midgut infections, a proportion of which went on to develop into mature metacyclic trypanosomes capable of infecting mammalian hosts. The method offers a safe, clean way of infecting tsetse flies with African trypanosomes which reduces the need for trypanosome-infected animals in the laboratory.     

Cryopreservation of Pathogenic African Trypanosomes In Situ: Metacyclic and Bloodstream Forms

SYNOPSIS. Of several methods developed for cryopreservation of Trypanosoma vivax, Trypanosoma congolense , and Trypanosoma brucei metacyclic forms in tsetse fly organs, as well as bloodstream forms in host blood, one proved the most satisfactory. In this method, infected fly proboscises and salivary glands were placed in glass capillary tubes containing fetal calf serum with 8% (v/v) glycerol as the cryoprotectant. The method for bloodstream forms involved the addition of glycerol directly to infected blood, which was then dispensed into capillary tubes. Next the tubes were placed in paper containers inside a glass test tube with a 5 mm thick plasticine jacket. The insulated assembly was suspended in the liquid nitrogen vapor phase in an LR-35 (Union Carbide) refrigerator for 45 min. Under these conditions, the cooling rate was 2 C/min. The frozen samples were transferred to permanent storage. The viability and infectivity of the preserved organisms were found to be satisfactory upon testing, and no antigenic changes were observed. Laboratory and field applications of the method are discussed.     

Trypanosoma brucei: loss of variable antigens during transformation from bloodstream to procyclic forms in vitro.

The loss of variable antigen from Trypansoma brucei during transformation from the bloodstream to the procyclic form in vitro has been monitored by agglutination and immunofluorescence reactions using antisera against both forms. Maximum agglutination of transforming trypanosomes with anti-culture form sera was obtained in 36–48 hr coinciding with loss of the surface coat as seen by electron microscopy. Agglutination with antisera against homologous bloodstream forms, however, reached a constant minimum but still positive level after 7–9 days: absorption of such antisera with culture or heterologous bloodstream forms reduced this period of persistent agglutinability to 72–84 hr, suggesting that the sera contained antibodies to “common” (surface membrane) antigens which became exposed when the surface coat was lost during transformation. The indirect immunofluorescence reaction provided a direct correlation of loss of antigen with loss of coat. The majority of trypanosomes lost detectable variable antigen by 36–48 hr, but a few flagellates, morphologically resembling bloodstream forms, retained the coat and capacity for labeling up to 84 hr; the numbers of such persistent bloodstream forms were shown to be sufficient to give a positive agglutination reaction for the population as a whole up to this time. Variable antigen appeared to be lost by dilution over the entire trypanosome surface rather than in patches or caps and the relevance of this observation to the process of antigenic variation is discussed.     

SAS-4 Protein in Trypanosoma brucei Controls Life Cycle Transitions by Modulating the Length of the Flagellum Attachment Zone Filament

The evolutionarily conserved centriole/basal body protein SAS-4 regulates centriole duplication in metazoa and basal body duplication in flagellated and ciliated organisms. Here, we report that the SAS-4 homolog in the flagellated protozoan Trypanosoma brucei, TbSAS-4, plays an unusual role in controlling life cycle transitions by regulating the length of the flagellum attachment zone (FAZ) filament, a specialized cytoskeletal structure required for flagellum adhesion and cell morphogenesis. TbSAS-4 is concentrated at the distal tip of the FAZ filament, and depletion of TbSAS-4 in the trypomastigote form disrupts the elongation of the new FAZ filament, generating cells with a shorter FAZ associated with a longer unattached flagellum and repositioned kinetoplast and basal body, reminiscent of epimastigote-like morphology. Further, we show that TbSAS-4 associates with six additional FAZ tip proteins, and depletion of TbSAS-4 disrupts the enrichment of these FAZ tip proteins at the new FAZ tip, suggesting a role of TbSAS-4 in maintaining the integrity of this FAZ tip protein complex. Together, these results uncover a novel function of TbSAS-4 in regulating the length of the FAZ filament to control basal body positioning and life cycle transitions in T. brucei.     

A study on the maturation of procyclic Trypanosoma brucei brucei in Glossina morsitans centralis and G. brevipalpis

Abstract. Teneral Glossina morsitans centralis and G. brevipalpis were fed in vitro upon medium containing procyclic Trypanosoma brucei brucei derived from the midguts of G. m. centralis or G. brevipalpis which had immature trypanosome infections. The tsetse were then maintained on rabbits and, on day 31, were dissected to determine the infection rates. In G. m. centralis the midgut and salivary gland infection rates by T. b. brucei were 46.0% and 27.0% with procyclic trypanosomes from G. m. centralis, and 45.4% and 24.7% with procyclic trypanosomes from G. brevipalpis, respectively. In G. brevipalpis the rates were 20.2% and 0.0% with procyclic trypanosomes from G. m. centralis, and 28.0% and 0.0% with procyclic trypanosomes from G. brevipalpis, respectively. Teneral G. m. centralis and G. brevipalpis were also fed similarly upon procyclic T. b. brucei derived from G.m.centralis or G. brevipalpis on day 31 of infection, the former tsetse species had mature infections while the latter were without infections in the salivary glands. In G.m.centralis the infection rates in the midgut and salivary glands were 48.9% and 17.0%, and 38.0% and 17.0% when fed on procyclic trypanosomes from G.m.centralis and G. brevipalpis, respectively. In G. brevipalpis the rates were 21.5% and 0.0%, and 10.7% and 0.0% with procyclic trypanosomes of G.m.centralis and G. brevipalpis origin, respectively. Thus, procyclic T. b. brucei from susceptible G.m.centralis could not complete cyclical development in refractory G. brevipalpis, whereas those from G. brevipalpis developed to metatrypanosomes in the salivary glands of G.m.centralis. Teneral and 15-day-old non-teneral G.m.centralis were fed in vitro upon heparinized goat's blood containing T. b. brucei bloodstream trypomastigotes, or upon medium containing procyclic T. b. brucei derived from G.m.centralis with mature infections. On day 31 their infection rates were determined. The infection rates by T. b. brucei in the midgut and salivary glands of G.m.centralis fed on the infected blood were 70.4% and 40.4% when fed as teneral tsetse, as against 15.3% and 4.0% when fed as non-teneral tsetse. Those tsetse which were fed on the medium containing procyclic trypanosomes showed rates of 50.0% and 25.6%, as against 11.6% and 2.5%, respectively. It would appear, therefore, that maturation of T. b. brucei in tsetse is probably not determined simply by an interaction between lectin and procyclic trypanosomes in the midgut of non-teneral tsetse, but it is the result of a complex interaction between many interrelated physiological factors of both the trypanosome and the tsetse vector.     

The Utilization of Glucose and Proline by Culture Forms of Trypanosoma brucei*

Exponentially dividing culture forms of Trypanosoma brucei did not utilize glucose provided in the culture medium. The inclusion of 2-deoxyglucose in the medium had no effect on the growth of the trypanosomes. Glucose could be replaced by proline in the liquid phase of biphasic medium without affecting the doubling time of the organisms. Proline added to the culture medium in this way disappeared during the log phase of growth. Glucose in the culture medium was used by the trypanosomes only when the stationary growth phase had been reached. Lipid accumulated in stationary phase trypanosomes grown in glucose-containing medium, but there was no lipid accumulation in log phase organisms or in those which had been grown in proline-containing medium. Bloodstream trypanosomes transferred to liquid medium rapidly utilized glucose over the first 12 hr of culture, and this was accompanied by an accumulation of free pyruvate in the medium. The rate of glucose utilization fell off over the next 36 hr; this was accompanied by a lowering of free pyruvate in the medium and a rise in the proline oxidase activity of the trypanosomes. The possible biologic significance of proline to trypanosomes developing in the midgut of the tsetse vector is discussed.     

Trypanosoma theileri: In vitro cultivation in tsetse fly and vertebrate cell culture systems

Epimastigote forms of Trypanosoma theileri were grown at 25°C in insect cell culture media and in Glossina tissue cultures for more than 6 months. Doubling times of 10–14 h during exponential growth were observed. In cell cultures which had been derived from pupal tsetse flies growth rates were higher than in cell free media; in a larval cell line, however, growth of T. theileri was inhibited. Ecdysteroids and juvenile hormone I reduced multiplication of T. theileri in cell free media. When T. theileri was incubated in different sera only fetal calf serum (FCS) supported growth. Epimastigote forms transformed into trypomastigote bloodforms when cultured at 37°C in FCS, vertebrate cell cultures, and Eagle's medium, but not in insect media or Glossina cell cultures. Oxygen uptake of epimastigotes could be inhibited by rotenone antimycin A and cyanide; trypomastigotes were not affected by these inhibitors.     

Protein Uptake and Digestion in Bloodstream and Culture Forms of Trypanosoma brucei*

SYNOPSIS. The mechanisms of ferritin uptake and digestion differ in bloodstream and culture forms of Trypanosoma brucei. Ferritin enters bloodstream forms from the flagellar pocket by pinocytosis in large spiny-coated vesicles. These vesicles become continuous with straight tubular extensions of a complex, mostly tubular, collecting membrane system where ferritin is concentrated. From the collecting membrane system the tracer enters large digestive vacuoles. Small spiny-coated vesicles, which never contain ferritin, are found in the Golgi region, fusing with the collecting membrane system, and around the flagellar pocket. Acid phosphatase activity is present in some small spiny-coated vesicles which may represent primary lysosomes. This enzymic activity is also found in the flagellar pocket, pinocytotic vesicles, the collecting membrane system, the Golgi (mature face), and digestive vacuoles of bloodstream forms. About 50% of the acid phosphatase activity of blood forms is latent. The remaining nonlatent activity is firmly cell-associated and probably represents activity in the flagellar pocket. The structures involved in ferritin uptake and digestion are larger and more active in the short stumpy than in the long slender bloodstream forms. The short stumpy forms also have more autophagic vacuoles. No pinocytotic large, spiny-coated vesicles or Golgi-derived, small spiny-coated vesicles are seen in culture forms. Ferritin leaves the flagellar pocket of these forms and enters small smooth cisternae located just beneath bulges in the pocket membrane. The tracer then passes through a cisternal collecting membrane network, where it is concentrated, and then into multivesicular bodies. In the culture forms, acid phosphatase activity is localized in the cisternal system, multivesicular bodies, the Golgi (mature face), and small vesicles in the Golgi and cisternal regions. The flagellar pocket has no acid phosphatase activity, and almost all the activity is latent in these forms. The culture forms do not release acid phosphatase into culture medium during 4 days growth. Uptake of ferritin by all forms is almost completely inhibited by low temperature. These differences among the long slender and short stumpy bloodstream forms and culture forms are undoubtedly adaptive and reflect different needs of the parasite in different life cycle stages.     

Growth and Differentiation of Trypanosoma cruzi Cultivated with a Triatoma infestans Embryo Cell Line*

SYNOPSIS. Trypanosoma cruzi strain Peru was cultivated in the presence of an established cell line of Triatoma infestans embryo cells (TI-32). Bloodstream trypomastigotes differentiated into amastigote-like cells (first differentiation phase) which multiplied to form large clusters of cells. Because of their clustering nature, a new term, “staphylomastigotes,” has been proposed for this stage. After 10 days of cultivation, 90% of the staphylomastigotes underwent differentiation (2nd differentiation phase) to trypomastigotes (?98%) or epimastigotes (?2%). Bloodstream trypomastigotes cultivated without TI-32 cells underwent the first, but not the 2nd differentiation phase, although occasional epimastigotes were seen (< 1%). The evidence presented suggests that TI-32 cells produce a labile factor(s) important not only for initiation of the 2nd differentiation phase but also for maintaining the parasites in the trypomastigote stage. The pH of the culture medium was not the initiating factor for the 2nd differentiation phase. Infectivity studies indicated that staphylomastigotes were as infective as bloodstream trypomastigotes, but that metacyclic trypomastigotes isolated from culture after the 2nd differentiation phase were slightly more infective than bloodstream forms. Electromicrographs of styphylomastigotes do not provide any evidence of exchange of genetic material between cells.     

Analysis of the Antigenic Composition of Trypanosoma brucei brucei Bloodstream and Culture Forms by the Quantitative Direct Fluorescent Antibody Methods*

SYNOPSIS The quantitative direct fluorescent antibody (QDFA) methods were employed for the antigenic analysis of bloodstream forms and culture procyclics of 2 variants, TRUM (Trypanosome Research University of Massachusetts) 106 and TRUM 107, of Trypanosoma brucei brucei. Intact and trypsinized trypanosomes were studied. It was demonstrated that: (A) The specific variant antigens are localized in the surface coat of bloodstream trypomastigotes. (B) In addition to the common antigens shared by bloodstream forms and culture procyclics, there are also certain antigens unique to these latter stages. (C) Still another group of antigens. not found in the culture procyclics, appears to be shared by the bloodstream forms, irrespective of their variant-specific antigens. These antigens may be present in part in the coat or on the cell membrane and in part within the cytoplasm. (D) Irrespective of the bloodstream-form variant from which they are derived, the procyclics are antigenically the same. The QDFA results are analyzed statistically and discussed in the light of the available literature.     

In Vitro Development of Exoerythrocytic Forms of Plasmodium Gallinaceum Sporozoites In Avian Macrophages

ABSTRACT Exoerythrocytic forms of Plasmodium gallinaceum were cultured in vitro using salivary gland sporozoites extracted from experimentally infected Aedes fluviatilis mosquitoes. the host cells were macrophage precursors from chicken bone marrow. At various times after introduction of Sporozoites, the cultures were stained by Giemsa or by immunofluorescence assay (IFA) using anti-sporozoite-specific monoclonal antibodies (MAb). the time to complete parasite development in vitro was 50-70 h. By 70 h, ruptured segmenters and free merozoites were visible within the cells. Inoculation of normal chickens with infected cultures induced parasitemia after a pre-patent period of 10-11 days. In vitro young exoerythrocytic forms, late schizonts that include the matured segmenters, and free merozoites shared common antigens with the sporozoites as revealed by IFA using anti-sporozoite-specific MAbs. Our data indicate that macrophages support development of P. gallinaceum sporozoites and that the circumsporozoite proteins are present until Ac end of the primary exoerythrocytic schizogony.     

Hydroxyurea: Effect On Growth, Structure, and [3H]Thymidine Uptake of Trypanosoma Brucei Procyclic Culture Forms

SYNOPSIS Hydroxyurea (HU) concentrations above 50 μg/ml reversibly inhibited cell division of Trypanosoma brucei brucei stock STIB 366 procyclic culture forms, but not growth of individual cells. the volume of the nucleus and of the cytoplasm increased in the presence of the drug as did the percentage of cells with 2 kinetoplasts. Electron microscopy revelaed that the nuclear membrane was extended forming protrusions which surrounded areas of the cytoplasm. Replication of the kinetoplast DNA did not seem to be affected by HU. the uptake of [³H]thymidine increased in the presence of 25 μ/ml HU 3-fold compared to control cells. the nuclear volume and the dry weight of the culture forms increased proportionally to the amount of label incorporated.     

Revisiting the Central Metabolism of the Bloodstream Forms of Trypanosoma brucei: Production of Acetate in the Mitochondrion Is Essential for Parasite Viability

Background

The bloodstream forms of Trypanosoma brucei, the causative agent of sleeping sickness, rely solely on glycolysis for ATP production. It is generally accepted that pyruvate is the major end-product excreted from glucose metabolism by the proliferative long-slender bloodstream forms of the parasite, with virtually no production of succinate and acetate, the main end-products excreted from glycolysis by all the other trypanosomatid adaptative forms, including the procyclic insect form of T. brucei.

Methodology/Principal Findings

A comparative NMR analysis showed that the bloodstream long-slender and procyclic trypanosomes excreted equivalent amounts of acetate and succinate from glucose metabolism. Key enzymes of acetate production from glucose-derived pyruvate and threonine are expressed in the mitochondrion of the long-slender forms, which produces 1.4-times more acetate from glucose than from threonine in the presence of an equal amount of both carbon sources. By using a combination of reverse genetics and NMR analyses, we showed that mitochondrial production of acetate is essential for the long-slender forms, since blocking of acetate biosynthesis from both carbon sources induces cell death. This was confirmed in the absence of threonine by the lethal phenotype of RNAi-mediated depletion of the pyruvate dehydrogenase, which is involved in glucose-derived acetate production. In addition, we showed that de novo fatty acid biosynthesis from acetate is essential for this parasite, as demonstrated by a lethal phenotype and metabolic analyses of RNAi-mediated depletion of acetyl-CoA synthetase, catalyzing the first cytosolic step of this pathway.

Conclusions/Significance

Acetate produced in the mitochondrion from glucose and threonine is synthetically essential for the long-slender mammalian forms of T. brucei to feed the essential fatty acid biosynthesis through the “acetate shuttle” that was recently described in the procyclic insect form of the parasite. Consequently, key enzymatic steps of this pathway, particularly acetyl-CoA synthetase, constitute new attractive drug targets against trypanosomiasis.     

Production of Serine Proteases by the Oyster Pathogen Perkinsus marinus (Apicomplexa) In Vitro

ABSTRACT. Analysis of the cell-free supernatants of Perkinsus marinus cultures by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining revealed the presence of as many as 17 bands ranging in molecular weight from 239 to 32 kDa. These bands were not present in un-inoculated medium. Moreover, P. marinus produces extracellular proteins that possess proteolytic activities; the cell-free supernatants of P. marinus cultures could digest a variety of proteins including gelatin, casein, fibronectin and laminin. Oyster plasma was also digested by cell-free culture supernatants. The proteolytic activity in cell-free culture supernatants was detected 24 h post-inoculation, while no proteolytic activity could be detected in cell lysates. The proteolytic activities were characterized using substrate-impregnated sodium dodecylsulfate-polyacrylamide gels and had approximate molecular weights ranging from 55 to 35 kDa. The proteolytic activity of cell-free culture supernatants was inhibited by the serine protease inhibitors phenylmethylsulphonyl fluoride, 3,4-dichloroisocoumarin and soybean trypsin inhibitor. In contrast, inhibitors (i.e. trans-epoxysuccinyll-leucylamido(4-guanidino)-butane, 1, 10-phenanthroline, captopril, ethylenediaminetetracetic acid, pepstatin A or diazoacetyl-DL-norleucine methyl ester) from the other three classes of proteases had no effect. It was concluded that the P. marinus proteases in cell-free culture supernatants are serine proteases.     

Trypanosoma brucei: differential requirement of membrane potential for import of proteins into mitochondria in two developmental stages

Trypanosome alternative oxidase (TAO) and the cytochrome oxidase (COX) are two developmentally regulated terminal oxidases of the mitochondrial electron transport chain in Trypanosoma brucei. Here, we have compared the import of TAO and cytochrome oxidase subunit IV (COIV), two stage-specific nuclear encoded mitochondrial proteins, into the bloodstream and procyclic form mitochondria of T. brucei to understand the import processes in two different developmental stages. Under in vitro conditions TAO and COIV were imported and processed into isolated mitochondria from both the bloodstream and procyclic forms. With mitochondria isolated from the procyclic form, the import of TAO and COIV was dependent on the mitochondrial inner membrane potential (delta psi) and required protein(s) on the outer membrane. Import of these proteins also depended on the presence of both internal and external ATP. However, import of TAO and COIV into isolated mitochondria from the bloodstream form was not inhibited after the mitochondrial delta psi was dissipated by valinomycin, CCCP, or valinomycin and oligomycin in combination. In contrast, import of these proteins into bloodstream mitochondria was abolished after the hydrolysis of ATP by apyrase or removal of the ATP and ATP-generating system, suggesting that import is dependent on the presence of external ATP. Together, these data suggest that nuclear encoded proteins such as TAO and COIV are imported in the mitochondria of the bloodstream and the procyclic forms via different mechanism. Differential import conditions of nuclear encoded mitochondrial proteins of T. brucei possibly help it to adapt to different life forms.     

Gametocytogenesis of Leucocytozoon caulleryi in In Vitro Culture: Effect of Human Red Blood Cells on the Development of Second-Generation Merozoites

For investigating development of second-generation merozoites of Leucocytozoon caulleryi into mature gametocytes, infected erythrocytes from chickens at 15 d after sporozoites inoculation were cultured in RPMI-1640 modified medium supplemented with 10% horse serum and 0.5 ml of human erythrocytes (type O). When culture was carried out at 37°C in a humidified atmosphere of 5% CO₂ for 7 d, the very small number of second-generation merozoites developed into morphologically mature gametocytes. However, in the high carbon dioxide and low oxygen condition, mature gametocytes weren't observed in cluture. The role of human erythrocytes added has not been clarified yet.