Sulfated moieties from the native C-T domain of cruzipain enhance the immunopathogenesis of experimental chronic Chagas disease

SOPRANO, LL; ACOSTA, DM; FERRERO, MR; GARCIA, GA; ESTEVA, MI; COUTO, AS; DUSCHAK, VG

Ascochinga, Cordoba

Congreso; XXIV Reuni¨®n Anual de la Sociedad Argentina de Protozoolog¨ªa; 2010

Sociedad Argentina de Protozoologia

Trypanosoma cruzi contains a major cysteine proteinase, cruzipain (Cz). This lysosomal enzyme bears an unusual C-terminal domain (C-T) that contains a number of post-translational modifications and is responsible for most antibodies in natural and experimental infections. Progress in the structural characterization by UV-MALDI-TOF MS analysis, revealed the presence of sulfated high-mannose type oligosaccharides in the C-T, which are essential for cruzipain recognition by IgG antibodies from the serum of chagasic patients. In order to evaluate the immune responses to sulfated moieties on Cz, BALB/c mice were immunized with purified Cz and C-T prior and after desulfation treatment. Humoral immune response to sulfates on Cz or C-T was mainly IgG2b. Antibody responses to natural sulfated Cz were strongly biased toward IgG1 with lower levels of IgG2a after immunization, while immunization with sulfate-depleted cruzipain produced dominant IgG2a antibody responses. When immune cells from C-T immunized mice stimulated with Cz or C-T were analyzed by ELISA assays showed high levels of IL-4. By contrast, levels of INF-¦Ã increased in supernatants of cells from mice immunized with sulfate-depleted antigens. Moreover, immunization with C-T elicited ultrastructural abnormalities in heart and skeletal muscle tissues. Surprisingly, pathological alterations were not observed in tissues from mice immunized with sulfate-depleted-C-T. In addition, a lower labeling was observed by immunogold-electron microscopy using myosin-adsorbed sera specific for Cz and C-T compared to C-T immunized mice in both tissues. BALB/c mice immunized with C-T prior and after desulfation treatment, were further challenged with a sublethal doses of trypomastigotes. In addition to the high mortality found in mice group previously immunized with C-T, pathological abnormalities were observed in skeletal muscle tissue from mice of this group that survived post infection. Western blot analysis of cardiac and muscle tissue homogenates from infected mice previously immunized with C-T, confronted with rabbit sera specific for Cz, showed significant differences by comparison with those previously immunized with sulfate-depleted C-T.  Our results highlight sulfated oligosaccharides as main components of the C-T domain from Cz tsuggesting that sulfated  moieties from C-T play an enhancer role in the immunopathogenesis of chronic Chagas disease. Supported by CONICET-ANPCYT-UBA.INP-ANLIS-Malbr¨¢n