Substrate specificity of the Trypanosoma cruzi trans-sialidase.

Trypanosoma cruzi trypomastigotes acquire sialic acid (SA) from host glycoconjugates by means of a plasma membrane-associated trans-sialidase (TS). Here we study the substrate specificity of TS, which differs from all known sialyltransferases in that it does not require cytidine monophosphate (CMP)-SA as donor. The T. cruzi TS reversibly transfers SA to saccharides with terminal beta-Gal (but not alpha-Gal) residues. Donors are saccharides with SA linked to terminal beta-Gal residues by (alpha 2-3), but not (alpha 2-6) bonds. The type of beta-linkage of the terminal Gal residue is of minor importance (beta 1-4 and beta 1-6 are slightly better than beta 1-3), whereas chain length and the structure of additional vicinal sugar residues are not relevant. SA on the surface of living trypomastigotes of T. cruzi is transferred back and forth between the parasite surface and acceptor molecules with terminal beta-Gal, either in solution or on the surface of neighbouring mammalian cells. Addition of fucose residue on or close to the terminal galactose impairs TS activity. As a consequence, the enzyme acts poorly on the E-selectin ligand sialyl-Lewisx and its precursor Lewisx, and in vitro adhesion of TS-treated neutrophils to L-cells expressing L-selectin is not affected. Modifications in the structure of the (alpha 2-3)-linked N-acetyl-neuraminic acid (Neu5Ac) (deoxy or methoxy) of the donor molecules do not impair transfer if the changes are at C9, whereas changes at C4, C7 and C8 impair the ability to donate the modified SA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid composition of Trypanosoma cruzi.

1. Lipid composition of Trypanosoma cruzi epimastigote form in culture consist of 35% of phospholipids and 65% of neutral lipids. 2. Among the phospholipids, phosphatidylcholine is the more abundant (44%), followed by phosphatidylethanolamine (28%), phosphatidylinositol (12%), sphingomyelin (4%), and smaller amounts of cardiolipin, phosphatidic acid, lysolecithin, phosphatidylserine (traces), and an unidentified phospholipid (3%). 3. Pulse labeling with 32P showed highest specific incorporation in phosphatidylethanolamine, followed by phosphatidylinositol and phosphatidylcholine, suggesting a more active role for phosphatidylethanolamine in these organisms.     

Heterogeneity of the kinetoplast DNA molecules of Trypanosoma cruzi.

Kinetoplast DNA (kDNA) of the culture form of Trypanosoma cruzi is cleaved by restriction endonucleases (HpaII, HindII, EcoRI, and HaeIII). The analysis of the cleavage patterns proves that the minicircles (free circulargenome units) are heterogeneous in base sequences. The same results are obtained with the complex kDNA network which is composed of the association of minicircles and linear molecules. Kinetic studies of the renaturation of kDNA previously cleaved by HpaII into fragments of the genome unit size show at least two populations of molecules. About 75% of these molecules correspond to the fast renaturing population having the molecular complexity of the minicircles. The molecules of the slow renaturing population have a much higher molecular complexity than the minicircles and do not seem to be related to the majority of the long linear molecules.     

Trypanosoma cruzi: Mutations in the 3' untranslated region of calmodulin gene are specific for lineages T. cruzi I, T. cruzi II, and the Zymodeme III isolates

Populations of Trypanosoma cruzi can be clustered in two main phylogenetic lineages, T. cruzi I and T. cruzi II and a third group denominated Zymodeme III (ZIII) has been described. Using 23 isolates representing the two major T. cruzi groups and the Zymodeme III, the 3' untranslated region (3'UTR) of the calmodulin gene was analyzed. Several mutations located on a 330 bp segment of this 3'UTR were observed, among which three important insertion/deletion events, namely (1) a dinucleotide AG present only in ZIII isolates; (2) a 13 bases purine block missing only in ZIII; and (3) a five base GT block in T. cruzi II. Minimum free energy dot plots show that T. cruzi I and T. cruzi ZIII exhibit similar patterns of optimal and sub-optimal folding of this segment. These mutations in 3'UTR of calmodulin raise the possibility that T. cruzi I and ZIII group are sharing common functional routes.     

Characterization of the myo-inositol transport system in Trypanosoma cruzi.

myo-inositol is a growth factor for mammalian cells as well as for the pathogenic protozoa Trypanosoma cruzi. Most of the cell surface molecules in this organism rely on myo-inositol as the biosynthetic precursor for phosphoinositides and glycosylated phosphatidylinositols. The aim of this work was to investigate the process of myo-inositol translocation across the parasite cell membrane. myo-Inositol uptake was concentration-dependent in the concentration range 0.1-10 microM with maximal transport obtained at 8 microM. Using sodium-free buffers, where Na+ was replaced by choline or K+, myo-inositol uptake was inhibited by 50%. Furosemide, an inhibitor of the ouabain-insensitive Na+-ATPase, inhibited the Na+-dependent and Na+-independent myo-inositol uptake by 68 and 33%, respectively. In contrast, ouabain, an (Na++/K+) ATPase inhibitor, did not affect transport. Part of the myo-inositol uptake is mediated by active transport as it was inhibited when energy metabolism inhibitors such as carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (34%), 2,4-dinitrophenol (50%), KCN (71%) and NaN3 (69%) were added to the medium, or the temperature of the medium was lowered to 4 degrees C. The addition of glucose (5-50 mM) or mannose (10 mM) did not change the myo-inositol uptake, whereas the addition of 10 mM nonlabeled myo-inositol totally inhibited this transport, indicating that the transporter is specific for myo-inositol. Phloretin (0.3 mM) and phoridzin (5 mM), but not cytochalasin B, were efficient inhibitors of myo-inositol uptake. A portion of the accumulated myo-inositol is converted to inositol phosphates and phosphoinositides. These data show that myo-inositol transport in T. cruzi epimastigotes is mediated by at least two specific transporters - one Na+-dependent and the other Na+-independent.     

Epoxide hydrase in Trypanosoma cruzi epimastigotes.

1. Microsomal fractions from Trypanosoma cruzi epimastigotes catalyze the hydration of styrene oxide to styrene glycol. The activity is linear up to 45 min of incubation, is proportional to microsomal protein concentration within certain range, and has an optimum pH of 8.5. 2. Double-reciprocal plots indicate a Km value of 5.3 . 10(-4) M for styrene oxide and a V of 29.6 pmol of styrene glycol formed/min per mg protein at 37 degrees C. 4-Chlorophenyl-2,3-epoxypropyl either (Ki = 2.08 . 10(-4) M) and juvenile hormone I (Ki = 2.7 . 10(-4) M) are competitive inhibitors; whereas, 1-chloro-2,3-epoxypropane is a non-competitive inhibitor. The enzyme is induced about three-fold by 5 mM phenobarbital in the growth medium. 3. The epoxide hydrase is not activated by detergents but rather inhibited by concentrations of Tween-80 and Lubrol as low as 0.025%. 4. Experiments with intact cells indicate that about 3% of [8-14C]styrene oxide penetrates after 90 min of incubation; whereas, over 30% of juvenile hormone I is found intracellularly after the same incubation period. Intracellular styrene oxide is hydrated to styrene glycol to a significant extent and the in vivo hydration is increased by pretreatment with phenobarbital and inhibited upon the addition of 4-chlorophenyl-2,3-epoxypropyl ether. Only a small amount of the intracellular juvenile hormone I is recovered as the corresponding diol ester.     

Tissue tropism of different Trypanosoma cruzi strains.

A systematic study of the distribution of intracellular parasites in the organs and tissues was performed in groups of mice inoculated with 4 different Trypanosoma cruzi strains. An extremely high parasitism of spleen, liver, and bone marrow was observed in mice inoculated with Y and Berenice strains; with CL strain, however, parasites were almost absent in those organs. Bloodstream forms apparently present differences which facilitate or prevent their uptake by macrophages from the mononuclear phagocytic system. Parasitism of the smooth muscle from hollow organs was significantly higher with ABC and Berenice strains than with Y and CL. The importance of the distribution of intracellular stages in the pathogenesis of the disease is discussed.     

Ceramide and inositol content of the lipopeptidophosphoglycan from Trypanosoma cruzi.

Inositol (2.5%), 17-methyl-sphinganine (4.8%) and sphinganine (1.5%) have been identified as constituents of the lipopeptidophospholycan isolated from whole cells of epimastigote forms of $\text{Trypanosoma cruzi}$. The branched chain base was characterized by combined gas chromatography — mass spectrometry.     

Adenosine triphosphatase activities in Trypanosoma cruzi.

1. Subcellular fractions obtained from epimastigotes of Trypanosoma cruzi, disrupted by three different procedures, contained in addition to the already known Mg2+-activated adenosine triphosphatase (ATPase; E.C.3.6.1.4), a Ca2+-ATPase activity. 2. The Ca2+-ATPase (a) was activated by low concentrations of CaCl2 (apparent Ka, 80 microM); (b) had a Km for ATP of 0.6 mM (at 1 mM CaCl2, pH 8.0); (c) presented a broad pH curve (optimum 7.1-8.6); and (d) was insensitive to oligomycin concentrations which inhibited the Mg2+-ATPase present in the same preparations. 3. All attempts to find a (Na+-K+)-activated, ouabain-inhibited, ATPase have been unsuccessful, in spite of the fact that living epimastigoes of T. cruzi are able to concentrate K+ and exclude Na+ from the medium.     

The karyotype and ploidy of Trypanosoma cruzi.

Little is known of the number or organization of chromosomes in Trypanosoma cruzi, the protozoan parasite responsible for Chagas' disease in man in the New World. Straightforward cytogenetic analysis is precluded because trypanosome chromosomes fail to condense during the cell cycle. We have size-fractionated the chromosome-sized DNA molecules of representative T. cruzi strains by pulsed field gradient (PFG) gel electrophoresis and located several housekeeping genes by Southern blotting using cDNA probes from the related trypanosome T. brucei. We show that DNA molecules from homologous chromosomes of T. cruzi migrate differently in the PFG system and infer that T. cruzi epimastigotes are at minimum diploid. In contrast to T. brucei, mini-chromosomes are absent in T. cruzi. All the housekeeping genes studied hybridize to DNA molecules which can be resolved in the PFG system, suggesting that T. cruzi may have no chromosomes larger than a few megabase pairs.     

HMG-like chromosomal proteins in Trypanosoma cruzi.

HMG-like chromosomal proteins from Trypanosoma cruzi were studied. Four HMG-like proteins, designated HMG A, HMG-B, HMG-C, and HMG-E, were isolated and found to have molecular weights of 35.5 kd, 27.5 kd, 21.8 kd and 10.4 kd, respectively. Immunological relatedness was demonstrated between the mammalian HMG 1,2 and the HMG-A and HMG-B from T. cruzi. The relative amounts of HMG-C and HMG-E proteins vary in T. cruzi depending to the proliferative stage of the cells. HMG-E protein is increased in proliferating cells when compared to its level in non-proliferating cells. HMG-C is increased in the non-proliferating cells. Probably, the shifts observed in the relative amounts of HMG-like proteins are related to the proliferating cells of this flagellate. The results are consistent with those described for other lower eukaryotes where the HMG-like proteins isolated are similar but not identical to HMG proteins from vertebrates.     

NAD-linked glutamate dehydrogenase in Trypanosoma cruzi.

1. Epimastigotes of Trypanosoma cruzi, Tulahuén strain, contained a NAD-linked glutamate dehydrogenase (EC 1.4.1.3), in addition to the already known NADP-linked enzyme enzyme (EC 1.4.1.4). 2. The partially purified NAD-linked enzyme had a higher molecular weight and was much more labile than the NADP-linked enzyme, and was inhibited by purine nucleotides. 3. These results further emphasize the difference in glutamate metabolism between the parasite and its mammalian host.