A Mensural Study of Division in Trypanosoma simiae and Trypanosoma congolense

SYNOPSIS. Dividing forms of Trypanosoma simiae and T. congolense in stained thin blood films taken from pigs infected by wild Glossina morsitans submorsitans were measured employing a technique which took account of the distance between the divided kinetoplasts, the positions of the nucleus or nuclei and the lengths of the original and developing flagella.
Analysis of these measurements showed that binary fission in these trypanosomes consisted of a gradual increase in the distance between the divided kinetoplasts along the long axis of the body; progressive outgrowth of a daughter flagellum from the blepharoplast associated with the posteriorly placed kinetoplast; migration of the nucleus toward the posterior end of the body; separation of the divided nuclei in the direction of the long axis of the body; and fission of the cytoplasm in an antero-posterior direction and finally separation into two individuals by a stepped, sliding motion.
No evidence to support syngamy or other type of germ cell reproduction was observed.     

Role of carbohydrates within variant surface glycoprotein of Trypanosoma congolense. Protection against proteolytic attack

The effects of tunicamycin on different aspects of structure and biosynthesis of variant surface glycoprotein from Trypanosoma congolense have been studied. Deglycosylated variant antigen becomes synthesized in vitro, is transported through the cell, and is deposited on the cell surface in equivalent amounts compared to the glycosylated species. In contrast to the glycosylated molecule only marginal amounts of high-molecular-mass fragments can be removed from the parasitic cell by externally added proteases in the case of tunicamycin-treated cells. Most of the material removed by proteases from the cell surface of tunicamycin-treated cells has a molecular mass lower than 2 kDa. Many additional proteolytic cleavage sites become accessible after removal of the glycan chains. There is no indication that in the deglycosylated molecule the same preferential protease-sensitive site exists as is found in the glycosylated species. These results suggest that glycosylation of variant surface glycoprotein could be important for the survival of the parasite within the host organism.     

Mechanisms of self-cure from Trypanosoma congolense infection in mice

The mechanism(s) of resistance to African trypanosomiasis caused by Trypanosoma congolense was investigated by using the Dinderesso/80/CRTA/3 isolate to which C57B1/6 are resistant (low parasitemia and self-cure) and BALB/c sensitive (high parasitemia and death). The resistance of C57B1/6 is similar to that found in some natural hosts of African trypanosomes such as certain indigenous West African cattle and wild Bovidae. The antibody response to epitopes exposed on the variant surface glycoprotein of a clone obtained from the Dinderesso/80/CRTA/3 isolate was measured by a complement-mediated lysis assay in C57B1/6 and BALB/c. After infections with 10(4), 10(5), or 10(7) motile organisms, antibody appeared in C57B1/6 4 to 8 days earlier than in BALB/c. Peak antibody titers were similar in both strains but were reached about 4 days earlier in C57B1/6. In this strain, antibody appeared during and controlled the first wave of parasitemia, whereas in BALB/c, parasitemia reached a plateau above 10(8) organisms per ml before antibody could be detected, and at this time the animals were dying. At peak antibody response, the proportion of immunoglobulin (Ig) M and IgG antibody was the same in both strains. The antibody response had the same kinetics in C57B1/6 and BALB/c after injection of 10(4), 10(5), and 10(7) lethally irradiated but intact parasites, but the peak titers were 10(3) to 10(4) times lower than after live challenge. The response to nonirradiated trypanosomes appeared to be T cell independent, because the antibody titers were the same in congenitally athymic nu/nu and normal C57B1/6, and no evidence for the induction of T cell activity could be demonstrated in the infected nude mice. A role for trypanolytic serum factors in resistance could not be demonstrated. The extent of immunosuppression after infection with nonirradiated organisms was compared in the two strains by measuring the in vitro response of their splenic lymphocytes to concanavalin A, pokeweed mitogen, and allogeneic cells and their ability to mount an in vivo response to an unrelated trypanosome challenge. Both strains were partially immunosuppressed during rising parasitemia, but as C57B1/6 controlled parasitemia, immunosuppression was gradually reversed, whereas in BALB/c it became worse. Several explanations might account for the resistance of C57B1/6 to the Dinderesso/80/CRTA/3 isolate of T. congolense. It appears that an early immune response is a decisive factor in this resistance.     

Characterisation of a cysteine protease from bloodstream forms of Trypanosoma congolense.

A cysteine protease (trypanopain-Tc) with cathepsin-L-like properties has been purified from Trypanosoma congolense. The enzyme has an apparent molecular mass of 31-32 kDa by SDS/PAGE and 66 kDa by gel chromatography. It has a pI 7.4 and a high affinity for concanavalin A. Trypanopain-Tc catalyses the limited proteolysis of a variety of protein substrates such as fibrinogen, serum albumin and trypanosome variant-surface glycoprotein. It has minimal or no activity against casein or elastin. A variety of peptidyl amidomethylcoumarins and peptidyl diazomethanes were used to test the specificity of trypanopain-Tc. The better substrates had Arg or Lys in P1 and hydrophobic amino acids in P2 and P3. The best substrate found for trypanopain-Tc was Z-Phe-Arg-NHMec (Z, benzyloxycarbonyl; NHMec, 7-amido-4-methylcoumarin). The kinetic constants for the hydrolysis of Z-Phe-Arg-NHMec were kcat = 17.4 s-1, Km = 4.4 microM, kcat/Km = 4.0 microM-1.s-1, which are very similar to those of cathepsin L with this substrate. The specific substrates for cathepsin B (Z-Arg-Arg-NHMec) and cathepsin H (Arg-NHMec) were not hydrolysed by trypanopain-Tc under the conditions tested. The pH optimum of trypanopain-Tc against Z-Phe-Arg-NHMec was pH 6.0 but it showed a broad peak of activity extending well into the alkaline region. The enzyme was activated by low-molecular-mass thiol compounds and inhibited by cystatin, L-trans-epoxysuccinyl-4-guanidinobutane (E-64) and a variety of peptidyl diazomethanes. The most effective diazomethane inhibitors (Z-Leu-Leu-Met-CHN2, Z-Leu-Met-CHN2 and Z-Leu-Lys-CHN2, were inhibitory at nanomolar concentrations and were trypanocidal in vitro after 24-48 h incubation in greater than or equal to 20 microM [inhibitor]. However, it is not clear whether the trypanocidal activity of these inhibitors is a consequence of the inhibition of trypanopains or of some other essential proteolytic activities within the parasites.     

Characterization of epitopes on a variant surface glycoprotein from Trypanosoma congolense by six monoclonal antibodies

Monoclonal antibodies were isolated from mice immunized with variant surface glycoprotein of Trypanosoma congolense. Five out of the six monoclonals were able to detect epitopes at the cell surface in an indirect immunofluorescence analysis. One antibody did not react. Using protein-A-containing bacterial adsorbent all monoclonal antibodies precipitate glycosylated as well as non-glycosylated variant surface glycoprotein. Carbohydrate chains therefore do not appear to be part of the immunodeterminant structure recognized by the various monoclonal antibodies. Interaction of the monoclonal antibodies with protein fragments obtained by partial proteolysis with V8 protease from Staphylococcus aureus or papain allows the classification of the antibodies into three groups with different epitope specificity.     

Congopain from Trypanosoma congolense: drug target and vaccine candidate

Trypanosomes are the etiological agents of human sleeping sickness and livestock trypanosomosis (nagana), which are major diseases in Africa. Their cysteine proteases (CPs), which are members of the papain family, are expressed during the infective stages of the parasites' life cycle. They are suspected to act as pathogenic factors in the mammalian host, where they also trigger prominent immune responses. Trypanosoma congolense, a major pathogenic species in livestock, possesses at least two families of closely related CPs, named CP1 and CP2. Congopain, a CP2-type of enzyme, shares structural and functional resemblances with cruzipain from T. cruzi and with mammalian cathepsin L. Like CPs from other Trypanosomatids, congopain might be an attractive target for trypanocidal drugs. Here we summarise the current knowledge in the two main areas of research on congopain: first, the biochemical properties of congopain were characterised and its substrate specificity was determined, as a first step towards drug design; second, the possibility was being explored that inhibition of congopain by host-specific antibodies may mitigate the pathology associated with trypanosome infection.     

Phospholipase A2 from Trypanosoma congolense: Characterization and haematological properties

Phospholipase A₂ was isolated from Trypanosoma congolense and purified to electrophoretic homogeneity. The enzyme appeared to exist in a dimeric form with subunit molecular weights of 16 500 and 18 000. It had a pH optimum of 6·8. Kinetic analysis with different substrates, showed that the enzyme had exceptional specificity for 1,2,dimyristoyl-sn-phosphatidylcholine and 1,2,dioleoyl-sn-phosphatidylcholine with K_m values of 1·85 × 10^?3 M and 2·12 × 10^?3 M respectively. The Arrhenius plot was linear with an activation energy of 5·8 kcal mol^?1. Inhibition studies with parahydroxymercuribenzoate and tri-butyltinoxide were positive thus implicating a thiol group at the catalytic site of the enzyme. The enzyme was stable to heat treatment and possessed haemolytic and anticoagulating properties.     

Trypanosoma congolense: calf erythrocyte survival.

Hereford calves infected with Trypanosoma congolense developed an anemia which was most severe 10 weeks after infection when packed cell volumes (PCV) averaged 21.1 ± 2.5% (±2 SE) as compared to 33.1 ± 2.1% for controls. At the termination of the study, at 28 weeks postinfection PCVs of infected animals had risen to 27.5 ± 1.0% as compared to 34.0 ± 1.7% for controls. In parallel with PCVs the apparent half-lives of ⁵¹Cr-labeled erythrocytes were reduced as early as the first 2 weeks postinfection. The greatest difference in erythrocyte half-lives between infected and control animals was found during the fourth to sixth weeks of infection when volumes for infected animals averaged 128 ± 46 hr as compared to 321 ± 30 hr for controls. During this period the parasitemia was at its highest level. At 28 weeks postinfection the average apparent half-life of infected animals was 243 ± 43 hr compared with 304 ± 11 hr for controls. No differences were observed in gastrointestinal loss of ⁵¹Cr between infected and control animals; however, urinary excretion of isotope was greatly increased in infected animals when compared to controls. No significant changes in total blood volumes were observed between infected and control animals but total plasma volumes increased and total erythrocyte volumes decreased significantly in infected animals.     

Trypanosoma congolense: isolation and purification.

Yields of Trypanosoma congolense grown in rats may be increased by placing the rats in a 37 °C environment for 1 hr prior to sacrifice. A further increase in the number of parasites recovered per rat may be achieved by replacement of blood removed by a lactated Ringer's solution with 5% glucose as the rat is being bled from the abdominal aorta. The Ringer's solution serves to maintain intravascular volume during the bleeding procedure and thereby prevents premature cardiac arrest. Erythrocytes in infected blood may be then lysed by raising and rapidly lowering the osmolarity of the blood. This permits separation of the trypanosomes from 95% of the erythrocytes by differential centrifugation. The remaining blood cell contamination may then be removed on a small DEAE-cellulose column. The purified trypanosomes are motile, infective, and intact as judged by electron microscopy. More than 10¹⁰ purified T. congolense can be obtained from three adult rats by these methods.     

Susceptibility and resistance to Trypanosoma congolense infections

We have put emphasis on recent findings in experimental Trypanosoma congolense infections in highly susceptible BALB/c and relatively resistant C57Bl/6 mice. Based on various analyses, it has been shown that a major difference in resistance to T. congolense infections is expressed early in infection at the macrophage level. A novel plastic-adherent Thy1.2(+) suppressor lymphocyte, which in absolute synergy with a Thy 1.2(-) cell exerts its suppression via interleukin-10 and interferon-gamma opens up an exciting new field of research.     

Pathways of glucose catabolism in procyclic Trypanosoma congolense

Studies of respiration on glucose in procyclic Trypanosoma congolense in the presence of rotenone, antimycin, cyanide, salicylhydroxamic acid and malonate have indicated the presence of NADH dehydrogenase, cytochrome b-c1, cytochrome aa3, trypanosome alternate oxidase and NADH fumarate reductase/succinate dehydrogenase pathway that contributes electrons to coenzyme Q of the respiratory chain. The rotenone sensitive NADH dehydrogenase, the trypanosome alternate oxidase, and cytochrome aa3 accounted for 24.5 +/- 6.5, 36.2 +/- 4.2 and 54.1 +/- 5.5% respectively of the total respiration. Activities of lactate dehydrogenase, NAD(+)-linked malic enzyme and pyruvate kinase were less than 6 nanomoles/min/mg protein suggesting that they play a minor role in energy metabolism of the parasite. Phosphoenolpyruvate carboxykinase, pyruvate dehydrogenase, succinate dehydrogenase, NADP(+)-linked malic enzyme, NADH fumarate reductase, malate dehydrogenase, and alpha-ketoglutarate dehydrogenase and glycerol kinase on the other hand had specific activities greater than 60 nanomoles/min/mg protein. These enzyme activities could account for the production of pyruvate, acetate, succinate and glycerol. The results further show that the amount of glycerol produced was 35-48% of the combined total of pyruvate, acetate and succinate produced. It is apparent that some of the glycerol 3-phosphate produced in glycolysis in the presence of salicylhydroxamic acid is dephosphorylated to form glycerol while the rest is oxidised via cytochrome aa3 to form acetate, succinate and pyruvate.