Cyclical transmission of Trypanosoma brucei rhodesiense and Trypanosoma congolense by tsetse flies infected with culture-form procyclic trypanosomes

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.     

Non-specific induction of increased resistance in mice to Trypanosoma congolense and Trypanosoma brucei by immunostimulants.

Administration of the immunostimulants Corynebacterium parvum, Bacillus Calmette-Guérin (BCG) or Bordetella pertussis prior to, or at the same time as, challenge with Trypanosoma congolense significantly increased survival times in mice, both of trypano-susceptible (A/J) and trypano-resistant (C57Bl) strains. The increased survival time was associated with significant alterations in parasitaemia, which included lengthening of the pre-patent period, a delay in the time taken to reach the first peak of parasitaemia and a reduction in the level of parasitaemia. Similar results were obtained when these strains of mice were challenged with Trypanosoma brucei following pre-treatment with C. parvum. Thus, by the use of immunostimulants it was possible to reduce the susceptibility of mice to trypanosomiasis and the hope is that this can also be achieved with domestic livestock.     

Endocytosis by African trypanosomes. II. Occurrence in different life-cycle stages and intracellular sorting

Horseradish peroxidase (HRP) and colloidal gold-labeled proteins enter many of the endocytic organelles of bloodstream forms of Trypanosoma brucei and T. congolense. However, the colloidal gold markers were excluded from substantial parts of the pathway that contained HRP. Morphometric studies revealed that HRP entered organelles that accounted for approximately 5% of the total cell volume while transferrin-gold entered organelles that comprised approximately 2% of the total cell volume. In addition, large colloidal gold particles were excluded from organelles that contained smaller gold particles. Antibodies, raised against the variable surface glycoprotein, when applied to thawed cryosections were found to label structures from which endocytosed colloidal gold coupled to bovine serum albumin (BSA) was excluded. Endocytosis was shown to occur in two in vitro propagated forms of trypanosomes, similar to those found in the insect vector (Glossina spp.). The mammal-infective metacyclic forms were similar to bloodstream forms in that they endocytosed HRP and colloidal gold markers but excluded colloidal gold from approximately 3% of the endocytic organelles. Estimation of the flagellar pocket volumes of bloodstream form T. brucei showed that this organelle occupied 0.5% to 1.4% of the total cell volume. The flagellar pocket volume of T. congolense varied between life-cycle stages, with a fractional volume of 4.4% for bloodstream forms, 2.3% for metacyclic forms and 1.4% for procyclic forms. Endocytosis of HRP, but not of protein-gold markers, occurred in procyclic (uncoated) forms. Endocytosis by procyclic forms has heretofore not been reported.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptor-Mediated Endocytosis in the Bloodstream Form of Trypanosoma brucei1

The uptake of various host plasma proteins by the bloodstream form of Trypanosoma brucei was studied both biochemically, using radiolabeled proteins, and with the electron microscope, using colloidal gold particles as molecular tracers onto which plasma proteins had been adsorbed. Total plasma proteins and serum albumin were taken up by a mechanism of fluid endocytosis with low clearance (0.1 μ1 [mg cell protein]^-1 h^-1), while low-density lipoprotein (LDL) and transferrin were taken up by a receptor-mediated process with a clearance of two to three orders of magnitude higher than that of serum albumin. Binding prior to uptake of LDL and transferrin was saturable, depended on the presence of Ca²⁺, and the labeled ligand could be displaced by the homologous but not by heterologous protein. Binding of gold-labeled proteins was seen only to the membrane of the flagellar pocket and not elsewhere on the plasma membrane. After 1 h of incubation at 30°C with gold-labeled LDL and transferrin, labeled cellular structures represented respectively half and one-third of the total volume of all single-membrane bounded endocytotic and electron-dense vacuoles within the cell.     

In vitro growth inhibition of bloodstream forms of Trypanosoma brucei and Trypanosoma congolense by iron chelators

African trypanosomes exert significant morbidity and mortality in man and livestock. Only a few drugs are available for the treatment of trypanosome infections and therefore, the development of new anti-trypanosomal agents is required. Previously it has been shown that bloodstream-form trypanosomes are sensitive to the iron chelator deferoxamine. In this study the effect of 13 iron chelators on the growth of Trypanosoma brucei, T. congolense and human HL-60 cells was tested in vitro. With the exception of 2 compounds, all chelators exhibited anti-trypanosomal activities, with 50% inhibitory concentration (IC₅₀) values ranging between 2.1 – 220 μM. However, the iron chelators also displayed cytotoxicity towards human HL-60 cells and therefore, only less favourable selectivity indices compared to commercially available drugs. Interfering with iron metabolism may be a new strategy in the treatment of trypanosome infections. More specifically, lipophilic iron-chelating agents may serve as lead compounds for novel anti-trypanosomal drug development.     

Rab11 mediates selective recycling and endocytic trafficking in Trypanosoma brucei

Trypanosoma brucei possesses a streamlined secretory system that guarantees efficient delivery to the cell surface of the critical glycosyl‐phosphatidylinositol (GPI)‐anchored virulence factors, variant surface glycoprotein (VSG) and transferrin receptor (TfR). Both are thought to be constitutively endocytosed and returned to the flagellar pocket via TbRab11+ recycling endosomes. We use conditional knockdown with established reporters to investigate the role of TbRab11 in specific endomembrane trafficking pathways in bloodstream trypanosomes. TbRab11 is essential. Ablation has a modest negative effect on general endocytosis, but does not affect turnover, steady state levels or surface localization of TfR. Nor are biosynthetic delivery to the cell surface and recycling of VSG affected. TbRab11 depletion also causes increased shedding of VSG into the media by formation of nanotubes and extracellular vesicles. In contrast to GPI‐anchored cargo, TbRab11 depletion reduces recycling of the transmembrane invariant surface protein, ISG65, leading to increased lysosomal turnover. Thus, TbRab11 plays a critical role in recycling of transmembrane, but not GPI‐anchored surface proteins. We proposed a two‐step model for VSG turnover involving release of VSG‐containing vesicles followed by GPI hydrolysis. Collectively, our results indicate a critical role of TbRab11 in the homeostatic maintenance of the secretory/endocytic system of bloodstream T. brucei.      

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.     

Relationships between protease activity, host blood and infection rates in Glossina morsitans sspp. infected with Trypanosoma congolense, T.brucei and T.simiae

Abstract. Midgut protease activity in Glossina morsitans centralis and G. m. morsitans , at 48h post bloodmeal averaged 1.8IU of trypsin-like activity. These two tsetse subspecies differ in their susceptibility to trypanosome infection. Except for low levels in flies fed on waterbuck blood (0.7IU), activity did not differ in flies fed a variety of host bloods (goat, pig, cow, buffalo, eland) and trypanosome species ( Trypanosoma congolense, T.brucei, T.simiae ). Protease activity was also not correlated with infection rates, despite large differences in infection rates among experiments. Nevertheless, addition of 0.06M D(+)-glucosamine to parasitaemic blood resulted in a three-fold reduction in protease activity, coincident with a large increase in infection rate. This effect did not occur when parasites or D(+)-glucosamine were added alone to the bloodmeal, suggesting that the effect was due to metabolism of D(+)-glucosamine by parasites.     

Trypanosoma congolense. I. Clinical observations of experimentally infected cattle

The course of disease was studied in 8 cattle infected with Trypanosoma congolense. Although the onset of patency was dependent on the numbers of infecting organisms, the duration of the infection was not. High fevers were present on the day of or the day after initial patency. Succeeding peaks of parasitemia, and a progressive weight loss of over 30% occurred. A decrease in packed cell volume (PCV) beginning the first week after infection was observed. Early in the course of the developing anemia, many polychromatophilic erythrocytes and occasional normoblasts were found in the blood. A leucopenia persisted for the duration of the disease. Total serum protein concentrations fell sharply during the first 5 weeks of infection, then gradually increased to low normal levels. Serum albumin levels followed a similar pattern for the first 5 weeks, and remained at a relatively low level. Although gamma globulin levels also declined during the first 5 weeks, their levels gradually surpassed those of preinfection samples. No marked changes in serum glucose were noted. A mild elevation of serum urea nitrogen values occurred early during infection, but subsided. The animals dying early after infection developed elevated total bilirubin levels.     

Measure of molecular diversity within the Trypanosoma brucei subspecies Trypanosoma brucei brucei and Trypanosoma brucei gambiense as revealed by genotypic characterization

We have evaluated whether sequence polymorphisms in the rRNA intergenic spacer region can be used to study the relatedness of two subspecies of Trypanosoma brucei. Thirteen T. brucei isolates made up of 6 T. b. brucei and 7 T. b. gambiense were analyzed using restriction fragment length polymorphism (RFLP). By PCR-based restriction mapping of the ITS1-5.8S-ITS2 ribosomal repeat unit, we found a fingerprint pattern that separately identifies each of the two subspecies analyzed, with unique restriction fragments observed in all but 1 of the T. b. gambiense "human" isolates. Interestingly, the restriction profile for a virulent group 2 T. b. gambiense human isolate revealed an unusual RFLP pattern different from the profile of other human isolates. Sequencing data from four representatives of each of the two subspecies indicated that the intergenic spacer region had a conserved ITS-1 and a variable 5.8S with unique transversions, insertions, or deletions. The ITS-2 regions contained a single repeated element at similar positions in all isolates examined, but not in 2 of the human isolates. A unique 4-bp [C(3)A] sequence was found within the 5.8S region of human T. b. gambiense isolates. Phylogenetic analysis of the data suggests that their common ancestor was a nonhuman animal pathogen and that human pathogenicity might have evolved secondarily. Our data show that cryptic species within the T. brucei group can be distinguished by differences in the PCR-RFLP profile of the rDNA repeat.     

Kinetics of methionine transport and metabolism by Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

Methionine is an essential amino acid for both prokaryotic and eukaryotic organisms; however, little is known concerning its utilization in African trypanosomes, protozoa of the Trypanosoma brucei group. This study explored the Michaelis-Menten kinetic constants for transport and pool formation as well as metabolic utilization of methionine by two divergent strains of African trypanosomes, Trypanosoma brucei brucei (a veterinary pathogen), highly sensitive to trypanocidal agents, and Trypanosoma brucei rhodesiense (a human pathogenic isolate), highly refractory to trypanocidal arsenicals. The Michaelis-Menten constants derived by Hanes-Woolf analysis for transport of methionine for T. b. brucei and T. b. rhodesiense, respectively, were as follows: K(M) values, 1. 15 and 1.75 mM; V(max) values, 3.97 x 10(-5) and 4.86 x 10(-5) mol/L/min. Very similar values were obtained by Lineweaver-Burk analysis (K(M), 0.25 and 1.0 mM; V(max), 1 x 10(-5) and 2.0 x 10(-5) mol/L/min, T. b. brucei and T. b. rhodesiense, respectively). Cooperativity analyses by Hill (log-log) plot gave Hill coefficients (n) of 6 and 2 for T. b. brucei and T. b. rhodesiense, respectively. Cytosolic accumulation of methionine after 10-min incubation with 25 mM exogenous methionine was 1.8-fold greater in T. b. rhodesiense than T. b. brucei (2.1 vs 1.1 mM, respectively). In African trypanosomes as in their mammalian host, S-adenosylmethionine (AdoMet) is the major product of methionine metabolism. Accumulation of AdoMet was measured by HPLC analysis of cytosolic extracts incubated in the presence of increasing cytosolic methionine. In trypanosomes incubated for 10 min with saturating methionine, both organisms accumulated similar amounts of AdoMet (approximately 23 microM), but the level of trans-sulfuration products (cystathionine and cysteine) in T. b. rhodesiense was double that of T. b. brucei. Methionine incorporation during protein synthesis in T. b. brucei was 2.5 times that of T. b. rhodesiense. These results further confirm our belief that the major pathways of methionine utilization, for polyamine synthesis, protein transmethylation and the trans-sulfuration pathway, are excellent targets for chemotherapeutic intervention against African trypanosomes.