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Four iron superoxide dismutase (SODI, SODII, SODIII, and SODIV) activities with pI values of 6.9, 6.8, 5.25, and 3.8, respectively, were isolated from epimastigote forms of the Maracay strain of Trypanosoma cruzi cultivated at 28 degrees C in Grace's medium supplemented with 10% heat-inactivated fetal bovine serum. The activity of SODe (pI 3.8), which coincides with that of SODIV, was detected in Grace's medium without serum in which T. cruzi epimastigotes were cultivated for 24 hours at 28 degrees C. SODe, which was excreted into the medium by the parasite, was immunogenic and antibodies to SODe were detected in serum to a dilution of 1:2,500 by Western blot. The role of SODe is related to the establishment of the parasite within the host, and its high immunogenicity and specificity make it a useful molecular marker in diagnosing infection with this parasite. To validate a Western blot result using SODe as a antigen fraction, 1,029 sera of individuals from 11 municipalities in the state of Queretaro, Mexico were analyzed. Sampling was done randomly and results were compared with those for the same sera with three conventional serologic methods: an enzyme-linked immunosorbent assay (ELISA), indirect hemagglutination (IHA), and an indirect immunofluorescence assay (IFA) to detect antibodies to T. cruzi SODe. Samples that were positive by these three techniques were also positive by the Western blot method. The seroprevalence values for SODe (8.16% by ELISA and Western blot) in Queretaro were considerably higher than those reported in regions of Mexico considered to be endemic for Chagas disease. These results support the use of SODe in the serodiagnosis of Chagas disease.

We have isolated and biochemically characterized two iron superoxide dismutases activities (SODI and SODII) from a plant trypanosomatid isolated from Euphorbia characias. The isoenzyme FeSODII has immunogenic capacity, and the positivity of the anti-SODII serum persists to a dilution of 1/40,000, by Western blot. In addition, Western blot has been used to test the positivity of the anti-SODII serum against antigen fractions (SOD) from 17 isolates belonging to the family Trypanosomatidae and for which we had previously determined the isoenzymatic profile. The reaction proved positive only with those plant isolates considered to belong to the genus Phytomonas, whereas there was no reaction of the anti-SODII serum, against the antigen fractions from the species Trypanosoma cruzi, Leishmania donovani, Herpetomonas samuelpessoai, Herpetomonas davidi, Crithidia luciliae and Leptomonas collosoma. FeSODII is located mainly over the entire surface of the parasite, as well as in the nucleus, glycosomes and membranes. The above makes FeSODII promising as a molecular tool for diagnosis and identification, and as a potential chemotherapeutic target for designing drugs aimed at controlling not only of the diseases caused by Phytomonas species, but also for the great metabolic similarity to other trypanosomatids of animals and humans, it may be possible for these results to be extrapolated. Moreover, the sequencing of the amino-terminal end of the FeSODII enables the design of primers that in the near future will make it possible to sequence the gene of this isoenzyme.

No abstract

Two superoxide dismutases (SODI and SODII) have been purified by differential centrifugation, fractionation with ammonium sulphate followed by chromatographic separation (ionic exchange and affinity), from a plant trypanosomatid isolated from Euphorbia characias, and then characterized for several biochemical properties. Both enzymes were insensitive to cyanide but sensitive to hydrogen peroxide, properties characteristic of iron-containing superoxide dismutase. SODI had a molecular mass of approximately 66 kDa, whereas the molecular mass of SODII was approximately 22 kDa, both enzymes showing single bands. The isoelectric points of SODI and SODII were 6.8 and 3.6, respectively. The enzymatic stability persisted at least for 6 months when the sample was lyophilized and preserved at -80 degrees C. Digitonin titration and subcellular fractionation showed that both enzymes were in the cytoplasmic fraction, although part of SODII isoenzyme was also associated with glycosomes. We assayed these activities (SOD) in 18 trypanosomatid isolates on isoelectric focusing gels, and have demonstrated that the SOD is a biochemical marker sufficient to identify a trypanosomatid isolated from a plant as belonging to the genus Phytomonas and to distinguish between a true Phytomonas and other trypanosomatids that are capable of causing transient infections in plants.

The use of natural products for the treatment of protozoal infections (Leishmania and Trypanosoma spp.) is well known and has been documented since ancient times. We have already established an in vitro culture system using mammalian host cells (Vero) infected with Trypanosoma cruzi in which the time course of parasite growth is determined quantitatively. This system was used to screen anti-T. cruzi agents using two experimental models: simultaneous cell infection and compound addition or preincubation of the parasite with the test compound prior to cell infection. Among 64 diterpenoid alkaloids tested, including C₁₉ and C₂₀ skeletons, five C₂₀ compounds were active on T. cruzi epimastigotes: azitine, isoazitine and 15,22-O-diacetyl-19-oxodihydroatisine had moderate effects on the parasite, while atisinium chloride and 13-oxocardiopetamine were potent T. cruzi epimastigote growth inhibitors with activity levels similar to that of benznidazole, used as the reference drug. Additionally, these compounds decreased the ability of metacyclic forms to invade mammalian cells, their intracellular replications and their transformation into trypomastigotes, with no toxicity to the host cell. These results suggest that these alkaloids are structural leads of clinically active compounds against T. cruzi and probably other members of the Trypanosomatidae.

An excreted iron superoxide dismutase (FeSODe)

We have characterized diverse strains or species of Leishmania isolated in humans that are currently circulating throughout Peru, by means of isoenzymatic characterization, kDNA analysis by restriction enzymes, and multiplex PCR assay. The cluster analysis gave five groups. Cluster 1 includes L. (L.) donovani together with the isolates LP4 and LP7, forming the donovani complex. Thus, this complex corresponds to the New World visceral form, L. (L.) chagasi. Cluster 2 is formed by the isolates LP1-LP3, LP6, LP10, LP9, and LP11, phylogenetically intermediate between Cluster 1 and Cluster 3, or they can be treated as hybrids. Cluster 3 is divided into two subgroups: one formed by L. (V.) peruviana, together with the isolates LP14 and LP5, and the second one formed by L. (V.) brazilensis and the isolate LP8. These two subgroups form part of the brazilensis complex. The three strains of L. (L.) infantum [L. (L.) infantum I and II and la LSI] make up Cluster 4. In Cluster 5, we include the three Mexican strains (LM1-LM3) forming one subgroup while we would place L. (L.) amazonensis in another subgroup. These two subgroups would comprise the complex mexicana.