Major cysteine protease (cruzipain) in Z3 sylvatic isolates of Trypanosoma cruzi from Rio de Janeiro, Brazil.

Trypanosoma cruzi, the etiologic agent of Chagas' disease, is represented by a set of parasites which circulate between man, vectors, domestic and wild animals. Recently, our group isolated from Triatoma vitticeps strains of T. cruzi that were characterized as belonging to the Z3 phylogenetic lineage. Since very little is known about the biological and/or biochemical markers of sylvatic Z3 isolates, we have studied the protein and protease profiles of distinct Z3 isolates designated as SMM10, SMM53, SMM88, and SMM98. By means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both quantitative and qualitative differences were observed in the protein profiles of these strains. All strains produced an acidic cysteine protease of 45 kDa, resembling cruzipain activity. The strain SMM10 synthesized an additional 55 kDa metalloprotease. Using Western blotting and anti-cruzipain antibody to detect cruzipain-like molecules, a 40-kDa reactive molecule was identified in all strains; in the strain SMM10, an 80-kDa protein was also reacted. Studies about cruzipain isoforms from sylvatic parasites could be valuable tools in the comprehension of the genetic variability in the pathogenesis of Chagas' disease.

Suppression of the prophenoloxidase system in Rhodnius prolixus orally infected with Trypanosoma rangeli.

Investigations were carried out to compare aspects of the prophenoloxidase (proPO)-activating pathway in Rhodnius prolixus hemolymph in response to oral infection and inoculation of the insects with two developmental forms of Trypanosoma rangeli epimastigotes strain H14. In vivo experiments demonstrated that in control insects fed on uninfected blood, inoculation challenge with short epimastigotes resulted in high phenoloxidase (PO) activity. In contrast, previous feeding on blood containing either short or long epimastigotes was able to suppress the proPO activation induced by thoracic inoculation of the short forms. In vitro assays in the presence of short epimastigotes demonstrated that control hemolymph or hemolymph provided by insects previously fed on blood containing epimastigotes incubated with fat body homogenates from control insects significantly increased the PO activity. However, fat body homogenates from insects previously fed on blood containing epimastigotes, incubated with hemolymph taken from insects fed on control blood or blood infected with epimastigotes, drastically reduced the proPO activation. The proteolytic activity in the fat body homogenates of control insects was significantly higher than in those obtained from fat body extracts of insects previously fed on blood containing epimastigotes. These findings indicate that the reduction of the proteolytic activities in the fat body from insects fed on infected blood no longer allows a significant response of the proPO system against parasite challenge. It also provides a better understanding of T. rangeli infection in the vector and offer novel insights into basic immune processes in their invertebrate hosts.

Effects of gamma irradiation on the development of Trypanosoma rangeli in the vector Rhodnius prolixus.

Studies on the effects of gamma radiation on the infectivity of Trypanosoma rangeli (strain H14) for the vector Rhodnius prolixus revealed that (i) the LD(50) (lethal dose for 50% of bugs) for uninfected insects was 4147 rads; (ii) irradiated insects with a dose of 1200 rads subsequently infected with the flagellates exhibited a mortality of 45%, while uninfected irradiated insects showed a mortality of 5%, and infected nonirradiated insects exhibited 10% mortality; (iii) flagellates were present in the hemolymph of irradiated insects 7 days postinfection (p.i.), while in nonirradiated insects the parasites appeared in the hemocoel 18 days p.i.; (iv) T. rangeli infection decreased the number of hemocytes significantly and induced the formation of nodules in the hemolymph of both irradiated and nonirradiated insects; and (v) gamma irradiation affected the ultrastructural organization of the epithelial cells of the small intestine, principally the perimicrovillar membranes and microvilli. In this paper, we discuss the significance of the intestinal microenvironment of R. prolixus with regard to its interaction with T. rangeli.

Metalloproteases in Trypanosoma rangeli-infected Rhodnius prolixus

Protease activities in the haemolymph and fat body in a bloodsucking insect, Rhodnius prolixus, infected with Trypanosoma rangeli, were investigated. After SDS-polyacrylamide gel electrophoresis containing gelatin as substrate, analysis of zymograms performed on samples of different tissues of controls and insects inoculated or orally infected with short or long epimastigotes of T. rangeli, demonstrated distinct patterns of protease activities: (i) proteases were detected in the haemolymph of insects which were fed on, or inoculated with, short epimastigotes of T. rangeli (39 kDa and 33 kDa, respectively), but they were not observed in the fat body taken from these insects; (ii) protease was also presented in the fat bodies derived from naive insects or controls inoculated with sterile phosphate-saline buffer (49 kDa), but it was not detected in the haemolymph of these insects; (iii) no protease activity was observed in both haemolymph and fat bodies taken from insects inoculated with, or fed on, long epimastigotes of T. rangeli. Furthermore, in short epimastigotes of T. rangeli extracts, three bands of the protease activities with apparent molecular weights of 297, 198 and 95 kDa were detected while long epimastigotes preparation presented only two bands of protease activities with molecular weights of 297 and 198 kDa. The proteases from the insect infected with T. rangeli and controls belong to the class of either metalloproteases or metal-activated enzymes since they are inhibited by 1,10-phenanthroline. The significance of these proteases in the insects infected with short epimastigotes of T. rangeli is discussed in relation to the success of the establishment of infection of these parasites in its vector, R. prolixus