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.     

Comparative positions of kinetoplasts in Trypanosoma musculi and Trypanosoma lewisi during development in vitro

Abstract. The development of Trypanosoma musculi and Trypanosoma lewisi were studied in vitro in the presence of adherent splenic cells. Both parasites developed only when attached by their flagellar tips to adherent splenic cells. During the proliferation of T . musculi , the kinetoplast migrated towards the nucleus, and once in the vicinity of the nucleus, the nuclear division was triggered. The kinetoplast of T . lewisi did not migrate towards the nucleus, but remained at its original location. The nucleus and kinetoplast divided at the same time in both parasites, and parasites started dividing from their flagellar ends and T . musculi and T . lewisi daughter cells were formed within 48 h. The unavailability of the adherent splenic cells in vitro led the parasites to transform into round/oval nonviable forms.     

Generation of constitutive and inducible trans-sialylation dominant-negative phenotypes in Trypanosoma brucei and Trypanosoma cruzi

Trans-sialylation is a unique enzymatic process that is restrictedto some trypanosome species. By expressing developmentally regulatedtrans-sialidases, these protozoan parasites cleave sialic acidsfrom host glycoconjugates and transfer them to acceptors ontheir own cell surfaces. The biological function of this processis not understood, but trans-sialylation is expected to be importantin the invasion of mammalian cells by Trypanosoma cruzi andthe survival of Trypanosoma brucei within its insect vector.Since a conventional gene knockout approach was precluded, wedeveloped a dominant-negative strategy, in which fusion proteinsconsisting of a bacterial sialidase and trypanosome proteinswere expressed in T.brucei and T.cruzi. The strong recombinantsialidase activity shifted the reaction equilibrium from sialicacid transfer to hydrolysis, in this way creating a sialic-acid-negativephenotype. Taking advantage of a recently introduced inducibleexpression system, we were able to control the expression ofsialidase fusion proteins in T.brucez. Reversion of the sialic-acid-negativestate to wild-type sialylation was accomplished by selectiveinhibition of the foreign sialidase, leaving the parasite trans-sialidaseunaffected. Both desialylation and resialylation of trypanosomeswas rapidly achieved. Our results show that neither T.bruceinor T.cruzi require sialic acids for survival in vitro, rulingout the involvement of sialylation in cell surface integrity.The versatile system introduced here will allow a detailed invivo study of the role of trans-sialylation during the trypanosomeinfection cycle. Furthermore, cell-surface sialic acids areimplicated in a multitude of (patho-) biochemical processesin other organisms. The quantitative and qualitative manipulationof cell surface sialic acids, by expressing of counteractingenzymes, constitutes a novel approach with potentially broadapplications in glycobiology. sialidase trans-sialidase sialic acids PARP procyclin dominant-negative phenotype     

Serine hydroxymethyltransferase activity in Trypanosoma cruzi, Trypanosoma rangeli and American Leishmania spp

Serine hydroxymethyltransferase (SHMT) was studied in several American trypanosomatids, Trypanosoma cruzi epimastigotes displaying, in contrast with T. rangeli, high enzymatic activity. Several Leishmania spp. members, including L. braziliensis, L. mexicana and L. garnhami promastigotes, under identical assay conditions, showed low enzymatic activity. The T. cruzi and leishmanial enzymes presented several different kinetic properties, and thus apparent Km for THF was 0.30 mM for the trypanosomal SHMT vs 0.60 mM for the leishmanial enzyme, while the apparent Km for serine was 0.40 mM for trypanosomal SHMT vs 0.15 mM for leishmanial enzyme. There were significant variations in the specific activity of SHMT between the several different trypanosomatids strains studied, but the meaning of these results is not clear because they showed no correlation either with taxonomy or infectivity.     

Phylogenetic position of the giant anuran trypanosomes Trypanosoma chattoni,Trypanosoma fallisi,Trypanosoma mega,Trypanosoma neveulemairei,and Trypanosoma ranarum inferred from 18S rRNA gene sequences

Phylogenetic relationships within the kinetoplastid flagellates were inferred from comparisons of small-subunit ribosomal RNA gene sequences. These included 5 new gene sequences, Trypanosoma fallisi (2,239 bp), Trypanosoma chattoni (2,180 bp), Trypanosoma mega (2,211 bp), Trypanosoma neveulemairei (2,197 bp), and Trypanosoma ranarum (2,203 bp). Trees produced using maximum-parsimony and distance-matrix methods (least-squares, neighbor-joining, and maximum-likelihood), supported by strong bootstrap and quartet-puzzle analyses, indicated that the trypanosomes are a monophyletic group that divides into 2 major lineages, the salivarian trypanosomes and the nonsalivarian trypanosomes. The nonsalivarian trypanosomes further divide into 2 lineages, 1 containing trypanosomes of birds, mammals, and reptiles and the other containing trypanosomes of fish, reptiles, and anurans. Among the giant trypanosomes, T. chattoni is clearly shown to be distantly related to all the other anuran trypanosome species. Trypanosoma mega is closely associated with T. fallisi and T. ranarum, whereas T. neveulemairei and Trypanosoma rotatorium are sister taxa. The branching order of the anuran trypanosomes suggests that some toad trypanosomes may have evolved by host switching from frogs to toads.     

Action of Trypanosoma rangeli in infections with virulent Trypanosoma cruzi populations

In experimental murine infections with Trypanosoma rangeli it has been observed development immune response to Trypanosoma cruzi. The aim of the present work was to analyze the result of antigenic stimuli and the protective effect with T. rangeli in T. cruzi infections. Mice groups immunized with metacyclic trypomastigotes of T. rangeli (Choach -2V strain), derived from haemolymph and salivary gland and reinfected with T. cruzi virulent populations (Tulahuen strain, SA strain and Dm28c clone) from infected in vitro cells, showed decrease severity of disease outcomes, low parasitemia levels and 100% survival of all mice immunized, in comparison with groups infected only with T. cruzi populations, which demonstrated tissue affection, high parasitemia levels and the death of all animals. The above mentioned data contribute to understand the biological behaviour of T. cruzi and T. rangeli and their interaction with vertebrate host.     

Cultural and physiological observations on Trypanosoma rhodesiense and Trypanosoma gambiense. 1949

1. A diphasic medium of simple preparation is described for the indefinite cultivation of T. rhodesiense and T. gambiense. 2. The chief advantage of the medium is that it contains rabbit blood and thus obviates the necessity of using human blood. 3. The flagellates develop only to the proventricular stage; hence the cultures are noninfective. 4. The proventricular forms of both T. rhodesiense and T. gambiense consume sugar with the concomitant formation of acid. They are aerobic fermenters. 5. Very little, if any, ammonia is produced by the living parasites.     

Sialoglycolipids in Trypanosoma cruzi

Mild oxidation of epimastigote forms of T.cruzi followed by sodium borotritide reduction incorporates radioactivity into glycolipid fractions. Column chromatography on silica gel of the chloroform:methanol (2:1) extract separated two main peaks of radioactivity. Treatment with neuraminidase released 30% and 18% of the radioactivity, respectively. Paper chromatography showed peaks of radioactivity with relative migration to NANA7 of 1.33 in fraction A and 1.33 and 1.51 in fraction B. When unlabeled cells were submitted to a Folch extraction, thin layer chromatography of the upper phase showed at least two components detected with the resorcinol-copper reagent. Enzymatic and mild acid hydrolysis released a sialic acid with a migration relative to NANA of 1.22. These results suggest that a substituted sialic acid is present in glycolipids of the epimastigote form of T.cruzi.     

Trypanosoma evansi in Asia

Trypanosoma evansi has the widest geographical range of all the pathogenic trypanosome species and infects domesticated livestock in many countries of South America, Africa and Asia. In spite of this wide distribution, T. evansi has been less intensively investigated than the African tsetse-transmitted trypanosomes and there is correspondingly less information available on the incidence and economic importance of the disease that it causes. Many of the new techniques in immunology and molecular biology, which have provided much fundamental information on the tsetse-transmitted trypanosomes, have yet to be applied to T. evansi. Interest in T. evansi is increasing and a Working Group has now been established to coordinate and promote future research (Box 1). T. evansi is an important aetiological agent of disease in the livestock of Asia; this article evaluates both the historical perspective and our current knowledge of the epidemiology and pathology of T. evansi in this region.     

Kinetics of S-adenosylmethionine cellular transport and protein methylation in Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

African trypanosomes of the Trypanosoma brucei group are agents of disease in man and animals. They present unique biochemical characteristics such as the need for preformed purines and have extensive salvage mechanisms for nucleoside recovery. In this regard we have shown that trypanosomes have a dedicated transporter for S-adenosylmethionine (AdoMet), a key metabolite in transmethylation reactions and polyamine synthesis. In this study we compared the apparent kinetics of AdoMet transport, cytosolic AdoMet pool formation, and utilization of AdoMet in protein methylation reactions using two isolates: Trypanosoma brucei brucei, a veterinary parasite, and Trypanosoma brucei rhodesiense, a human pathogen that is highly refractory and has greatly reduced susceptibility to standard trypanocidal agents active against T. b. brucei. The apparent Km values for [methyl-3H]AdoMet transport, derived by Hanes-Woolf analysis, for T. b. brucei was 4.2 and 10 mM for T. b. rhodesiense, and the Vmax values were 124 and 400 micromol/liter/min, respectively. Both strains formed substantial cytosolic pools of AdoMet, 1600 nmol/10(9) T. b. brucei and 3500 nmol/10(9) T. b. rhodesiense after 10 min incubation with 25 mM exogenous AdoMet. Data obtained from washed trichloroacetic acid precipitates of cells incubated with [methyl-3H]AdoMet indicated that the rate of protein methylation in T. b. brucei was fourfold greater than in T. b. rhodesiense. These results demonstrate that the unique rapid uptake and utilization of AdoMet by African trypanosomes is an important consideration in the design and development of new agents of potential use in chemotherapy.