**4. Conclusion**

168 Gel Electrophoresis – Advanced Techniques

Gomes et al (2009) investigated the production of peptidases, especially cruzipain, as well as the protein surface distribution in four newly sylvatic isolates of *T. cruzi* belonging to the Z3

The differences in peptidase expression between TCI and TCII phylogenetic groups have recently been investigated. Since *T. cruzi* isolates from sylvatic triatomines were included in the third phylogenetic group, named Z3, our investigation contributes to investigate the expression of surface polypeptides and the major cysteine peptidase from the Z3 parasite population, thereby furthering understanding on the genetic variability in the pathogenesis of Chagas disease. In this context, we carried out an identification of the protein profile and peptidase from epimastigotes (replicative forms of this parasite) of sylvatic isolates of *T. cruzi* (classified as Z3) from triatomines captured in Santa Maria Madalena (SMM) in the State of Rio de Janeiro. The separation of soluble whole proteins revealed a different protein profile, with approximately 35 polypeptides presenting apparent molecular masses from 118 to 25 kDa in all the samples. The proteolytic activity was determined by zymograms analysis of all the samples, using SDS-polyacrylamide gel electrophoresis containing gelatin as substrate. Our main results demonstrate a major band of 45 kDa sensible to E-64, a powerful cysteine peptidase inhibitor, in all the samples. In order to confirm this data, western blotting was performed using the anti-cruzipain polyclonal antibody. These findings showed a strong polypeptide band with an apparent molecular mass between 40 and 50 kDa in all the sylvatic isolates: SMM10; SMM53; SMM88 and SMM98 respectively

Fig. 5. A – Gelatin-SDS-PAGE showing the proteolytic activity profiles of *T. cruzi* sylvatic isolates. Parasites (SMM10, SMM53, SMM88, SMM98, and Dm28c) grown for 7 days were harvested and lysed by SDS. The gel was incubated in 50 mM sodium phosphate buffer, pH 5.5, supplemented with 2 mM DTT for 40 h at 37°C; B- Western blotting showing the reactivity of cellular polypeptides of *T. cruzi* sylvatic isolates with the anti-cruzipain polyclonal antibody. Numbers on the left indicate the relative molecular mass markers,

These results show the presence of a main cysteine peptidase, cruzipain, in the sylvatic isolates of *T. cruzi* from Santa Maria Madalena, in the State of Rio de Janeiro (Gomes et al., 2009). We also observed another gelatinolyti activity of 66 kDa that was recognized by the anti-cruzipain antibody, probably a cruzipain isoform; since cruzipain is a high mannose-

**3.4 Proteolytic profile of** *T. cruzi* **isolates from Rio de Janeiro** 

genotype.

and also Dm28c (Figure 5).

expressed in kilodaltons.

*Trypanosoma cruzi* shows considerable heterogeneity among populations isolated from sylvatic and domestic cycles. Despite of knowledge concerning the genome of these flagellated organisms and their main protein families, very little is known about these parasites isolated from triatomine bugs captured from field, as well as *T. cruzi* extracted from sylvatic mammals. In this context, we do hereby highlight the importance of molecular studies on *T. cruzi* sylvatic isolates collected by blood culture from vertebrate hosts and/or from triatomine vectors, *Triatoma vitticeps*, in Triunfo location, 2nd district of Santa Maria Madalena city, Northern region of Rio de Janeiro State, Brazil. The results of our investigations with *T. cruzi* samples isolated from sylvatic triatomine insects revealed that these parasites belong to a phylogenetic group called ZIII, and proteolytic analyzes evidenced the presence of a key peptidase cysteine, cruzipain, in all samples of sylvatic *T. cruzi* isolates from Santa Maria Madalena - Rio de Janeiro (Brazil), which was confirmed by anti-cruzipain antibody recognition. Taken together, our results can corroborate in understanding the role of proteolytic enzymes in determining the virulence of these microorganisms, as well as genetic variability of Z3 population in Chagas disease pathogenesis.
