A biochemical comparison of glucosephosphate isomerase isozymes from Trypanosoma cruzi

The glucosephosphate isomerase (D-glucose-6-phosphate Ketol-isomerase, EC 5.3.1.9) isozymes of Trypanosoma cruzi were characterized with respect to their native and subunit molecular size, isoelectric point and in vitro thermostability. The molecular weight data are consistent with a dimeric enzyme structure. The apparent native and subunit size homogeneity and differences in pI values imply that the electrophoretic mobility differences of isozymes in native gels are determined by their molecular charge. Minor differences in peptide maps indicate the existence of some heterogeneity in the primary structure of the isozymes. The stability of triple-banded glucosephosphate isomerase electrophoretic profiles was confirmed, supporting the view that these phenotypes represent non-interconvertible enzyme species.     

Comparative studies of Trypanosoma vespertilionis Battaglia and Trypanosoma dionisii Bettencourt & França

SYNOPSIS. In diphasic blood agar media Trypanosoma vespertilionis developed spheroid clusters as compared to rather long, sausage-shaped (sometimes branched) clusters formed by Trypanosoma dionisii. The former species attained a greater population density (∼6 × 10⁷ organisms/ml) than the latter (∼ 2 × 10⁷ organisms/ml). Greater numbers of epimastigotes, some in active binary divisions, were observed during the logarithmic phase of growth, and morphologic changes occurred during cultivation which correlated with increased acidity and a depletion of glucose. Maximum numbers of trypomastigote forms were found during the stationary and early death phases. Most of the forms observed after 20 days were sphaeromastigotes. Glucose concentrations decreased to 0 M in T. vespertilionis and to 4.4 × 10⁻⁵ M in T. dionisii cultures during the stationary and death phases. By the 12th day of incubation cultures of T. vespertilionis were more acid (pH 5.5) than those of T. dionisii (pH 6.5). No antigenic changes during cultivation of each of the parasites were detected by immunodiffusion. Trypanosoma vespertilionis and T. dionisii contained common and specific antigens. At least 2–3 common antigens were detected in extracts reacted against heterologous antisera. Specific antigens were observed as nonidentical lines formed by extracts reacted against homologous and heterologous antisera and with antisera absorbed with heterologous antigens. At least 2 specific antigens were evident in extracts of T. vespertilionis and 1 in extracts of T. dionisii.     

Trypanosoma cruzi histones. Further characterization and comparison with higher eukaryotes

Histones extracted from T. cruzi chromatin were analyzed in three electrophoretic systems. Our results show that a basic protein with some properties similar to those of histone H1 from higher eukaryotes is present in T. cruzi. However this protein presents different electrophoretic mobilities than H1 histone from higher eukaryotes in all three electrophoretic systems tested. Considering the marked differences observed in the electrophoretic mobilities of T. cruzi histones as compared with those from higher eukaryotes, it is proposed that histones are conservative proteins primarily with regard to their function.     

Morphological comparison of axenic amastigogenesis of trypomastigotes and metacyclic forms of Trypanosoma cruzi

Amastigogenesis occurs first when metacyclic trypomastigotes from triatomine urine differentiate into amastigotes inside mammalian host cells and a secondary process when tissue-derived trypomastigotes invade new cells and differentiate newly to amastigotes. Using scanning electron microscopy, we compared the morphological patterns manifested by trypomastigotes and metacyclic forms of Trypanosoma cruzi during their axenic-transformation to amastigotes in acidic medium at 37 C. We show here that in culture MEMTAU medium, secondary and primary axenic amastigogenesis display different morphologies. As already described, we also observed a high differentiation rate of trypomastigotes into amastigotes. Conversely, the transformation rate of in vitro-induced-metacyclic trypomastigotes to amastigotes was significantly slower and displayed distinct patterns of transformation that seem environment-dependent. Morphological comparisons of extracelullar and intracellular amastigotes showed marked similarities, albeit some differences were also detected. SDS-PAGE analyses of protein and glycoprotein from primary and axenic extracelullar amastigotes showed similarities in glycopeptide profiles, but variations between their proteins demonstrated differences in their respective macromolecular constitutions. The data indicate that primary and axenic secondary amastigogenesis of T. cruzi may be the result of different developmental processes and suggest that the respective intracellular mechanisms driving amastigogenesis may not be the same.     

The karyotype of Trypanosoma cruzi Dm 28c: comparison with other T. cruzi strains and trypanosomatids

Chromosome-sized DNA molecules from Trypanosoma cruzi clone Dm 28c were analyzed and compared with other T. cruzi strains and monogenetic trypanosomatids by orthogonal field alteration gel electrophoresis. The results showed that T. cruzi Dm 28c displays at least 18 chromosomes ranging from 550 to more than 1500 kb and that in general the trypanosomatids have smaller chromosomes distributed in the size range from 300 to 1500 kb. With the exception of T. cruzi strain G49, there is no evidence of minichromosomes, suggesting they are not widely distributed among different isolates of the parasite. The hybridization of T. cruzi chromosomal Southern blots with probes for T. cruzi-specific genes showed that their location can change from one strain to another, supporting the idea of the plasticity of the parasite genome. Furthermore, the chromosome pattern is strictly conserved during the transformation of T. cruzi Dm 28c epimastigotes to metacyclic trypomastigotes, suggesting that extensive chromosomal rearrangements do not occur during at least part of the life cycle of the parasite.     

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.     

Intermediary metabolism of Trypanosoma cruzi

In this article, Julio Urbino discusses the characteristics o f the intermediary metabolism of Trypanosoma cruzi (the causative agent of Chagas disease), which are responsible for the unusual capacity of this parasite to use carbohydrates or amino acids as carbon and energy sources without drastic changes in its catabolic enzyme levels(1-3). Many, but not all, o f the metabolic capabilities of this organism are shared with Leishmania and the procyclic form o f the African trypanosomes, and the reviewer presents a metabolic model which is also consistent with the information available on these other parasites(2,4).     

The gamma-glutamyltranspeptidase of Trypanosoma cruzi

Trypanosoma cruzi epimastigotes show gamma-glutamyltranspeptidase activity which has characteristics significantly different than the mammalian enzyme. The protozoan enzyme is localized in the cytosolic fraction, it has a Km of 1.6 mM and a Vmax of 17.4 nmol/min/mg protein with L-gamma-glutamyl-p-nitroanilide as gamma-glutamyl donor, and an optimun pH range from 7.5 to 8.0. The best amino acid acceptors were L-histidine, L-asparagine, L-aspartate, L-glutamate and L-proline, but L-glutamine was a very poor acceptor. The enzyme was very sensitive to inhibition by 6-diazo-5-oxo-L-norleucine (k2 = 4.0 X 10(5)/M per min) and O-diazo-acetyl-L-serine (k2 = 1.1 X 10(4)/M per min). Phenobarbital (k2 = 8.38/M per min) and L-serine borate (Ki = 34 mM) were poor inhibitors. The activity of the enzyme was not correlated with the logarithmic phase of growth of the parasites and steadily decreases with the age of the cultures.     

Cell signalling and Trypanosoma cruzi invasion

Mammalian cell invasion by the protozoan pathogen Trypanosoma cruzi is critical to its survival in the host. To promote its entry into a wide variety of non-professional phagocytic cells, infective trypomastigotes exploit an arsenal of heterogenous surface glycoproteins, secreted proteases and signalling agonists to actively manipulate multiple host cell signalling pathways. Signals initiated in the parasite upon contact with mammalian cells also function as critical regulators of the invasion process. Whereas the full spectrum of cellular responses modulated by T. cruzi is not yet known, mounting evidence suggests that these pathways impinge on a number of cellular processes, in particular the ubiquitous wound-repair mechanism exploited for lysosome-mediated parasite entry. Furthermore, differential engagement of host cell signalling pathways in a cell type-specific manner and modulation of host cell gene expression by T. cruzi are becoming recognized as essential determinants of infectivity and intracellular survival by this pathogen.     

Trypanosoma cruzi trans-sialidase and cell invasion

Trypanosoma cruzi does not synthesize sialic acid but does contain a trans-sialidase, an enzyme capable of transferring sialic acid between host glycoconjugates and the parasite. Sialic acids are negatively charged carbohydrates attached to the terminal non-reducing end of glycoproteins and glycolipids, and their presence can dramatically influence many cell-surface recognition processes. Since sialic acids have been implicated in several ligand-receptor interactions, including the interaction of pathogenic viruses, bacteria and protozoans with their hosts, the expression of trans-sialidase and the acquisition of sialic acid by T. cruzi may be relevant to the interaction of the parasite with the host, and consequently may influence the pathobiology of Chagas disease. In this review, Sergio Schenkman and Daniel Eichinger discuss recent data about the structure and function of T. cruzi trans-sialidase.