Molecular and biologic characterization of Leishmania parasites implicated in an epidemic outbreak in northwestern Argentina.

Leishmania (Viannia) braziliensis and its variants were implicated in the epidemic outbreak of mucocutaneous leishmaniasis that occurred in Salta, northwestern Argentina, in 1985. A total of 24 suspected, untreated cases were evaluated clinically and parasitologically. Four of five stable isolates were consistent with the reference strain of L. (V.) braziliensis as determined by monoclonal antibodies and indirect immunofluorescence or radioimmunobinding assays. Zymodeme analysis in agarose gels showed a close relationship with L. (V.) guyanensis and L. (V.) panamensis. All zymograms obtained with polyacrylamide gels belonged to the subgenus Viannia; the patterns were different from, but very closely related to, the reference strains of L. (V.) braziliensis as determined by dendrogram analysis. Hamsters infected with two isolates showed a pattern consistent with L. (V.) braziliensis. The pattern of development in the gut of Lutzomyia longipalpis was consistent with members of Viannia.

Identification of Trypanosoma cruzi zymodemes by kinetoplast DNA probes.

Analysis of zymograms of extracts of Trypanosoma cruzi isolated from different hosts in Argentina allowed characterization of 12 zymodemes or "isozymic strains," only six of which were found in human patients. Two of these six zymodemes (Z1 and Z12) were widely distributed and found in more than 80% of human patients. These two "major natural clones" differed significantly in pathogenic activity. Because the groupings obtained by studying enzymes and kinetoplast DNA (kDNA) were similar, it is possible to identify the zymodeme by analyzing kDNA. A 290-bp fragment was amplified by PCR using primers for the sequences flanking the hypervariable regions of kDNA minicircles. Labeled probes for this fragment, prepared from Z1 and Z12 reference stocks, hybridized specifically with PCR-amplified kDNA from parasite stocks, allowing identification of zymodemes.

Genetic variation among Trypanosoma cruzi populations.

The analysis of enzyme electrophoretic patterns in 141 extracts of Trypanosoma cruzi isolated from humans and domesticated and wild animals from the endemic zone of Chagas' disease in Argentina allowed their characterization into 12 different "isozymic strains" or zymodemes (Z1 to Z12). In humans, the parasites most frequently found belong to Z1 and Z12. These zymodemes differ significantly in pathogenic activity, Z1 having fewer deleterious effects than Z12. Because of the good correlation existing between zymodemes and the groups of parasites characterized by the study of kinetoplast DNA (kDNA), it is possible to identify zymodemes by analyzing kDNA. A 270-base-pair probe of the highly variable region of minicircles (HVRm) of kDNA, purified from Z1 and Z12 reference stocks, hybridized specifically with PCR-amplified HVRm from T. cruzi isolates, allowing identification of the "major natural clones." It is suggested that this technique of zymodeme identification could be useful for the prognosis of the probable evolution of the disease.

Characterization of Trypanosoma cruzi populations by zymodemes: correlation with clinical picture.

Trypanosoma cruzi isolated from 55 chronic chagasic patients were grouped into isozymic strains on the basis of electrophoretic patterns for a set of six enzymes. The total sample showed a distribution of asymptomatic (63.6%) and clinically ill (36.4%) patients similar to that generally reported for Chagas' disease. Six of the 12 zymodemes known to exist in Argentina have been isolated from humans. Only two (Z1 and Z12) are frequent and widely distributed in the endemic area. These two zymodemes differ significantly in their pathogenicity. The proportion of asymptomatic patients was higher with the Z1 zymodeme (81.1%) than with the Z12 zymodeme (27.3%). The incidence of heart alterations was lower in Z1 than in Z12 zymodeme patients (18.9% versus 72.7%). Clinically evident acute disease was seen in 36.3% of cases with zymodeme Z12 and in 8.1% of cases with zymodeme Z1. The differences between the two prevalent zymodemes in Argentina are statistically significant. These observations indicate that the Z1 T. cruzi is a more benign strain than Z12. Patients infected with Z1 would be more likely to be asymptomatic for a longer time than those infected with Z12. The risk of cardiac lesion would be greater for patients harboring Z12 T. cruzi than for those with Z1. The results suggest that strain identification could be a useful prognostic tool.

Genetic structure of Trypanosoma cruzi populations from Argentina estimated from enzyme polymorphism.

Isolates of Trypanosoma cruzi from human patients, domestic and sylvatic animals and vector insects were obtained in different areas of Argentina. Electrophoretic patterns of enzymes from extracts of 95 isolates were analysed. On the basis of zymograms providing information on 10 loci, 12 zymodemes are described according to their genotypes. Data presented show fixed heterozygosity, absence of segregation of genotypes, significant departures from Hardy-Weinberg equilibrium, and over-represented genotypes. This evidence supports the hypothesis that sexual reproduction is very restricted or absent in this parasite. The proportion of polymorphic loci is 80%. The expected mean heterozygosity per locus (He) is 0.43, while the observed value (Ho) is 0.24. Differences between these values may be explained by accepting a basically clonal structure for T. cruzi. The data matrix of 12 zymodemes using 28 characters was analysed using a Wagner parsimony algorithm. Two equally most parsimonious unrooted trees were generated; both have 39 steps. The results show clusters clearly separated according to the geographical origin of the stocks. There are some indications of some correlations between genetic composition of the parasite and the clinical picture of the infection in human patients.

Characterization of Trypanosoma cruzi from Argentina by electrophoretic zymograms.

Polyacrylamide gel electrophoretic patterns for six enzymes in 73 isolates and 38 clones of Trypanosoma cruzi from different areas of Argentina were classified into 12 zymodemes. The isolates were obtained from human patients with acute, chronic or congenital Chagas' disease, vector insects, domestic and sylvatic animals. Two out of 8 isolates cloned were shown to be heterogeneous. Zymodemes 1 and 12 exhibit widespread geographic distribution; isolates belonging to both zymodemes account for 55% of the total analyzed. The other zymodemes are not widely geographically dispersed. Although there is a clear predominance of zymodeme 1 among asymptomatic patients, the data do not show a clear relationship between particular zymodemes and the clinical picture. The results suggest that the sylvatic and domestic transmission cycles overlap. This remarkable heterogeneity of T. cruzi in Argentina supports the possible multiclonal origin of these parasite populations.

Subcellular localization of branched-chain amino acid aminotransferase and lactate dehydrogenase C4 in rat and mouse spermatozoa.

Spermatozoa isolated from rat and mouse epididymes show a relatively high branched-chain amino acid aminotransferase (leucine aminotransferase, EC 2.6.1.6) activity. There is a significant reduction of leucine aminotransferase and of the isoenzyme C4 of lactate dehydrogenase (EC 1.1.1.27) in the gametes during their epididymal transit. Studies of patterns of liberation of the leucine aminotransferase and of the lactate dehydrogenase C4 from intact spermatozoa, treated with increasing concentrations of digitonin, indicate that both enzymes have the same dual subcellular location, i.e. in the cytosol and in the mitochondria.

Trypanosoma cruzi isolates from Argentina and Chile grouped with the aid of DNA probes.

Fifty-two isolates and several clones from Trypanosoma cruzi, the agent of Chagas' disease, were analyzed using cloned minicircles or total kinetoplast DNA as probes. Isolates were obtained from triatomines, guinea pigs and infected humans in the Central and Northern regions of Argentina and the North of Chile. 35% of all the randomly selected isolates could be identified with one cloned minicircle probe. This widely distributed T. cruzi group was detected on both sides of the Andes mountain range (Argentina and Chile) in Triatoma infestans as well as in human infections. Most of the other isolates could be grouped with four kinetoplast DNAs as probes, but their geographical distribution seems to be restricted as compared with the one mentioned above. These results confirm the heterogeneity of T. cruzi subspecies in nature and the usefulness of DNA probes to group them.

Characterization by electrophoretic zymograms of 19 Trypanosoma cruzi clones derived from two chronic chagasic patients.

1. Electrophoretic patterns of aspartate aminotransferase, glucose-6-phosphate dehydrogenase, phosphoglucomutase, glucose-phosphate isomerase, malic enzyme and alcohol dehydrogenase have been analyzed in extracts from Trypanosoma cruzi, Tulahuén strain, 19 clones derived from isolates obtained from two chronic chagasic patients from Argentina and from Brazilian stocks Silvio X10/1 (zymodeme 1), Esmeraldo/1 (zymodeme 2), and CAN-III/1 (zymodeme 3). 2. The clones isolated from one of the patients were genetically heterogeneous. 3. Phosphoglucomutase and glucose phosphate isomerase patterns for the clones analyzed clearly differ from those of the Brazilian stocks. 4. Grouping of clones on the basis of isozyme patterns showed some correlation with that based on total DNA per organism. 5. Under the experimental conditions used, the polyacrylamide gel electrophoresis micromethod employed was advantageous over starch gel electrophoresis.

Subcellular localization of leucine aminotransferase and alpha-hydroxyacid dehydrogenase in Trypanosoma cruzi.

Homogenates of Trypanosoma cruzi epimastigotes (Tulahuén strain) show L-leucine aminotransferase activity (EC 2.6.1.6). Subcellular distribution of this enzyme and of alpha-hydroxyacid dehydrogenase, enzymes which share a common substrate/product (alpha-ketoisocaproate), has been studied by means of differential centrifugation, digitonin treatment of entire parasites, isopycnic centrifugation and determination of latency of enzymes in the large granule fraction. The results indicate that both enzymes have a dual localization, in the cytosol and in the mitochondrion, probably in the matrix. On the basis of this location, it is proposed that they operate in a shuttle system transferring reducing equivalents between the cytosol and the mitochondrion.

Inhibition by gossypol of oxidoreductases from Trypanosoma cruzi.

The effects of gossypol, a polyphenolic compound isolated from the cotton plant upon six oxidoreductases from cultured epimastigotes of Typanosoma cruzi were studied. Gossypol was a powerful inhibitor of the alpha-hydroxyacid and malate dehydrogenases, NAD-linked enzymes, and of glutamate dehydrogenase, malic enzyme and glucose-6-phosphate dehydrogenase, NADP-dependent enzymes. The drug did not have an effect on succinate dehydrogenase, a flavoprotein. The Ki values with respect to substrate were 0.73, 0.3 and 3.5 microM for alpha-hydroxyacid, malate and glutamate dehydrogenases, respectively, and 1.1, 0.19 and 7.8 microM with respect to the coenzyme. Inhibition was noncompetitive with respect to substrate and uncompetitive in relation to the coenzyme.

Effect of gossypol upon motility and ultrastructure of Trypanosoma cruzi.

Gossypol, a polyphenolic compound from the cotton plant, immobilizes and structurally alters cultured Trypanosoma cruzi epimastigotes. Ultrastructural changes observed in gossypol-treated parasites were first detected in the kinetoplast and mitochondrion. At 50 microM concentration, much disorganization was evident after 5 min of incubation. With 25 microM gossypol, the same effect occurred after 30 min. Most epimastigotes were rounded, containing various membranous structures that could not be related to known cell components.

Inhibitory action of gossypol on enzymes and growth of Trypanosoma cruzi.

Gossypol, a phenolic compound isolated from the cotton plant, is a powerful inhibitor of nicotinamide adenine dinucleotide-linked enzymes (alpha-hydroxyacid dehydrogenase and malate dehydrogenase) of Trypanosoma cruzi, the parasite that causes Chagas' disease. Parasites at the epimastigote stage that were incubated for 5 minutes with 100 micromolar gossypol were completely immobilized. Concentrations of gossypol as low as 0.01 micromolar markedly reduced the growth rate of T. cruzi in culture.

Branched-chain amino acid aminotransferase in mouse testicular tissue.

Branched-chain amino acid aminotransferase (L-leucine:2-oxoglutarate aminotransferase, EC 2.6.1.6) activity was determined in several tissues of the mouse. Testis homogenates presented a specific activity very close to that of heart extracts which were the most active. Enzyme activity was detectable in testes from 5-day-old mice and increased steadily during development to reach a maximum at the 20th day of life. The transaminase was present in the cytosol of testicular homogenates and also associated, probably in the matrix, with a special type of mitochondria present in spermatozoa and gametogenic cells. The enzyme from testis is active against the three branched-chain amino acids and catalyses the reaction in both directions. Highest activity and lowest Km were obtained with L-leucine. Activity with L-valine was the lowest. The enzyme from the mitochondrial fraction showed identical properties to that from the soluble phase. The possible participation of this aminotransferase in a shuttle system transferring reducing equivalents from cytoplasm to mitochondria is postulated.