**3.2 Molecular profile of** *T. cruzi* **isolates from Rio de Janeiro**

The results obtained by means of molecular analysis revealed that the isolates have similar profiles, except for sample SMM1 (area F). Samples SMM10, SMM53, SMM88, SMM98 (area A), SMM36 and SMM82 (area B) revealed the presence of 150 bp, indicating that they belong to the zymodeme III group (Z3; Figure 4). Likewise, sample SMM1 from area F showed similarity to Z3 (150 bp), but also presented another band that may be related to the TcII profile (250 bp) and was very similar to the reference strain CL Brener (Figure 4). The phylogenetic position of Z3 has been much debated. According to some authors, the numerical taxonomy based on 24 isoenzymatic Z3 profiles is more closely associated with Z1 (TcII) than with Z2 (TcI) (Ready & Miles, 1980). However, other works place Z3 in an intermediate position between Z1 and Z2 (Stothard et al., 1998). Our study revealed one isolate (SMM1) with a hybrid profile associated with Z3 and TcII. This result may corroborate the hypothesis that this isolate is the product of a

Molecular and Proteolytic Profiles of

(dos Santos, 2011).

*Trypanosoma cruzi* Sylvatic Isolates from Rio de Janeiro-Brazil 167

Proteases are essential for all life forms. They are involved in a multitude of physiological reactions, ranging from simple digestion of proteins for nutritional purposes, to highlyregulated metabolic cascades (e.g. proliferation and growth, differentiation, signaling and death pathways), and are essential for homeostatic control in both prokaryote and eukaryote cells (Rao et al., 1998). Proteases are also essential molecules in viruses, bacteria, fungi and protozoa, for their colonization, invasion, dissemination and evasion of host immune responses, mediating and sustaining the infectious disease process. Collectively, proteases participate in different steps of the multifaceted interaction events between microorganism and host structures, being considered as virulent attributes. Consequently, the biochemical characterization of these proteolytic enzymes is of interest not only for understanding proteases in general, but also for understanding their roles in microbial infections, and thus, their use as targets for rational chemotherapy of microbial diseases (Santos, 2010)

Proteases are subdivided into two major groups, depending on their site of action: exopeptidases and endopeptidases. Exopeptidases cleave the peptide bond proximal to the amino (NH2) or carboxyl (COOH) termini of the proteinaceous substrate, whereas endopeptidases cleave peptide bonds within a polypeptide chain. Based on their site of action at the NH2 terminal, the exopeptidases are classified as aminopeptidases, dipeptidyl peptidases or tripeptidyl peptidases that act at a free NH2 terminus of the polypeptide chain and liberate a single amino acid residue, a dipeptide or a tripeptide, respectively. Carboxypeptidases or peptidyl peptidases act at the COOH terminal of the polypeptide chain and liberate a single amino acid or a dipeptide (which can be hydrolyzed by the action of a dipeptidase). Carboxypeptidases can be further divided into three major groups: serine, metallo and cysteine carboxypeptidases, based on the functional group present at the active site of the enzymes. Similarly, endopeptidases are classified according to essential catalytic residues at their active sites in: serine, metallo, glutamic, threonine, cysteine and aspartic endopeptidases. Conversely, there are a few miscellaneous proteases that do not precisely fit

Cysteine peptidases from parasitic protozoa have been characterized as factors of virulence and pathogenicity in several human and veterinary diseases. *T. cruzi* contains a major cysteine peptidase named cruzipain (also known as cruzain or GP57/51), which is present in different developmental forms of the parasite, although at variable levels (Dos Reis et al., 2006). Cruzipain is a papain-like peptidase that shares biochemical characteristics with both cathepsin L and cathepsin B (Cazzulo et al., 1990b). Cysteine peptidases have already been detected in many species of Trypanosomatidae, and are regarded as essential for the survival of several parasitic protozoa. The enzyme has been shown to be lysossomal, and is located in an epimastigote-specific pre-lysossomal organelle called the 'reservossome', which contains proteins that are digested during differentiation to metacyclic trypomastigotes (Soares et al., 1992). Some authors have suggested a second location of enzyme isoforms in the plasma membrane, associated with a glycosylphosphatidylinositol (GPI) anchor (Elias et al., 2008). These isoforms were present in epimastigotes, amastigotes and trypomastigotes, and reacted with polyclonal anti-cruzipain sera, thereby becoming an immunodominant antigen that is recognized by the sera of human patients with chronic Chagas disease (Martínez et al., 1991). Recently, the peptidase expression analysis of fresh field sylvatic isolated strains of *T. cruzi* showed a heterogeneous profile of cysteine proteolytic activities in the main phylogenetic groups TCI and TCII (Fampa et al., 2008).

into the standard classification (dos Santos, 2010, 2011).

mixture of parasite populations, since the vector in wild environments may feed on several vertebrate hosts. This complexity was demonstrated in the State of Rio de Janeiro by Fernandes et al. (1999), who showed a preferential association of the two lineages of *T. cruzi* with different hosts. They suggest that the vector *T. vitticeps* is involved in the transmission cycle among mammals infected by lineage 2 in the municipality of Teresópolis, and in the transmission cycle of primates in municipality of Silva Jardim. The hybrid profile found in these samples may indicate a possibility that the vector *T. vitticeps* does not only participate in the wild cycle of the disease.

The main purpose of typing of isolates of *T. cruzi* is to identify strains with different epidemiological and/or clinical characteristics of Chagas disease. Our results corroborate other descriptions in the literature, and contribute to the knowledge and records of the profile of some additional wild isolates of *T. cruzi* in regions not yet affected by the disease. Added to the complexity observed between the isolates is the finding that the Z3 profile is divided into two groups, called Z3a and Z3b (Mendonça et al., 2002). Our laboratory is interested in investigating whether such a dichotomy occurs among the Z3 isolates obtained from *T. vitticeps* in this area of study.

Fig. 4. PCR Multiplex – Mini-exon. The gel of agarose for electrophoresis was amplified using isolates of *Trypanosoma cruzi* of reference that possess approach bands of TCI, compared to TCII, Z3 and *Trypanosoma rangeli* and with *T. cruzi* sylvatics isolates from Rio de Janeiro. The isolates was performed using 25 ng of genomic DNA extracted using the phenol–chloroform method. Five primers were used: for Tc1 (5′-TTG CTC GCA CAC TCG GCT GCAT-3′), for Tc2 (5′-ACA CTT TCT GTG GCG CTG ATC G-3′), for Z3 (CCG CGW ACA ACC CCT MAT AAA AAT G-3′), for Tr (CCT ATT GTG ATC CCC ATC CCC ATC TTC G-3′), and for the mini-exon (5′ TAC CAA TAT AGT ACAGAA ACT G-3′). Lane 1. Molecular weight marker (100bp DNA ladder), 2. SMM98, 3. SMM36, 4. SMM82, 5. *T. rangeli*, 6. CL Brener, 7. DM28c, 8. JJ, 9. Molecular weight marker (100bp DNA ladder), 10.SMM1, 11. SMM10, 12. SMM53, 13. SMM88, 14. *T. rangeli*, 15. CL Brener, 16. DM28c, 17. JJ, 18. Molecular weight marker (100bp DNA ladder), 19. negative control (no DNA added). bp = base pairs.

#### **3.3 Proteolytic enzymes**

Despite the existing knowledge of this flagellate genome and its main families of proteins, little is known about these parasites isolated from triatomines captured in the field, as well *T. cruzi* in mammals of wild origin. Proteolytic enzymes are reported to play an important role in determining the virulence of these microorganisms.

mixture of parasite populations, since the vector in wild environments may feed on several vertebrate hosts. This complexity was demonstrated in the State of Rio de Janeiro by Fernandes et al. (1999), who showed a preferential association of the two lineages of *T. cruzi* with different hosts. They suggest that the vector *T. vitticeps* is involved in the transmission cycle among mammals infected by lineage 2 in the municipality of Teresópolis, and in the transmission cycle of primates in municipality of Silva Jardim. The hybrid profile found in these samples may indicate a possibility that the vector *T. vitticeps* does not only participate in

The main purpose of typing of isolates of *T. cruzi* is to identify strains with different epidemiological and/or clinical characteristics of Chagas disease. Our results corroborate other descriptions in the literature, and contribute to the knowledge and records of the profile of some additional wild isolates of *T. cruzi* in regions not yet affected by the disease. Added to the complexity observed between the isolates is the finding that the Z3 profile is divided into two groups, called Z3a and Z3b (Mendonça et al., 2002). Our laboratory is interested in investigating whether such a dichotomy occurs among the Z3 isolates obtained

Fig. 4. PCR Multiplex – Mini-exon. The gel of agarose for electrophoresis was amplified using isolates of *Trypanosoma cruzi* of reference that possess approach bands of TCI, compared to TCII, Z3 and *Trypanosoma rangeli* and with *T. cruzi* sylvatics isolates from Rio de Janeiro. The isolates was performed using 25 ng of genomic DNA extracted using the phenol–chloroform method. Five primers were used: for Tc1 (5′-TTG CTC GCA CAC TCG GCT GCAT-3′), for Tc2 (5′-ACA CTT TCT GTG GCG CTG ATC G-3′), for Z3 (CCG CGW ACA ACC CCT MAT AAA AAT G-3′), for Tr (CCT ATT GTG ATC CCC ATC CCC ATC TTC G-3′), and for the mini-exon (5′ TAC CAA TAT AGT ACAGAA ACT G-3′). Lane 1. Molecular weight marker (100bp DNA

ladder), 2. SMM98, 3. SMM36, 4. SMM82, 5. *T. rangeli*, 6. CL Brener, 7. DM28c, 8. JJ, 9. Molecular weight marker (100bp DNA ladder), 10.SMM1, 11. SMM10, 12. SMM53, 13. SMM88, 14. *T. rangeli*, 15. CL Brener, 16. DM28c, 17. JJ, 18. Molecular weight marker (100bp

Despite the existing knowledge of this flagellate genome and its main families of proteins, little is known about these parasites isolated from triatomines captured in the field, as well *T. cruzi* in mammals of wild origin. Proteolytic enzymes are reported to play an important

DNA ladder), 19. negative control (no DNA added). bp = base pairs.

role in determining the virulence of these microorganisms.

the wild cycle of the disease.

from *T. vitticeps* in this area of study.

**3.3 Proteolytic enzymes** 

Proteases are essential for all life forms. They are involved in a multitude of physiological reactions, ranging from simple digestion of proteins for nutritional purposes, to highlyregulated metabolic cascades (e.g. proliferation and growth, differentiation, signaling and death pathways), and are essential for homeostatic control in both prokaryote and eukaryote cells (Rao et al., 1998). Proteases are also essential molecules in viruses, bacteria, fungi and protozoa, for their colonization, invasion, dissemination and evasion of host immune responses, mediating and sustaining the infectious disease process. Collectively, proteases participate in different steps of the multifaceted interaction events between microorganism and host structures, being considered as virulent attributes. Consequently, the biochemical characterization of these proteolytic enzymes is of interest not only for understanding proteases in general, but also for understanding their roles in microbial infections, and thus, their use as targets for rational chemotherapy of microbial diseases (Santos, 2010) (dos Santos, 2011).

Proteases are subdivided into two major groups, depending on their site of action: exopeptidases and endopeptidases. Exopeptidases cleave the peptide bond proximal to the amino (NH2) or carboxyl (COOH) termini of the proteinaceous substrate, whereas endopeptidases cleave peptide bonds within a polypeptide chain. Based on their site of action at the NH2 terminal, the exopeptidases are classified as aminopeptidases, dipeptidyl peptidases or tripeptidyl peptidases that act at a free NH2 terminus of the polypeptide chain and liberate a single amino acid residue, a dipeptide or a tripeptide, respectively. Carboxypeptidases or peptidyl peptidases act at the COOH terminal of the polypeptide chain and liberate a single amino acid or a dipeptide (which can be hydrolyzed by the action of a dipeptidase). Carboxypeptidases can be further divided into three major groups: serine, metallo and cysteine carboxypeptidases, based on the functional group present at the active site of the enzymes. Similarly, endopeptidases are classified according to essential catalytic residues at their active sites in: serine, metallo, glutamic, threonine, cysteine and aspartic endopeptidases. Conversely, there are a few miscellaneous proteases that do not precisely fit into the standard classification (dos Santos, 2010, 2011).

Cysteine peptidases from parasitic protozoa have been characterized as factors of virulence and pathogenicity in several human and veterinary diseases. *T. cruzi* contains a major cysteine peptidase named cruzipain (also known as cruzain or GP57/51), which is present in different developmental forms of the parasite, although at variable levels (Dos Reis et al., 2006). Cruzipain is a papain-like peptidase that shares biochemical characteristics with both cathepsin L and cathepsin B (Cazzulo et al., 1990b). Cysteine peptidases have already been detected in many species of Trypanosomatidae, and are regarded as essential for the survival of several parasitic protozoa. The enzyme has been shown to be lysossomal, and is located in an epimastigote-specific pre-lysossomal organelle called the 'reservossome', which contains proteins that are digested during differentiation to metacyclic trypomastigotes (Soares et al., 1992). Some authors have suggested a second location of enzyme isoforms in the plasma membrane, associated with a glycosylphosphatidylinositol (GPI) anchor (Elias et al., 2008). These isoforms were present in epimastigotes, amastigotes and trypomastigotes, and reacted with polyclonal anti-cruzipain sera, thereby becoming an immunodominant antigen that is recognized by the sera of human patients with chronic Chagas disease (Martínez et al., 1991). Recently, the peptidase expression analysis of fresh field sylvatic isolated strains of *T. cruzi* showed a heterogeneous profile of cysteine proteolytic activities in the main phylogenetic groups TCI and TCII (Fampa et al., 2008).

Molecular and Proteolytic Profiles of

*T. cruzi*.

**4. Conclusion** 

pathogenesis.

FIOCRUZ.

**6. References** 

306, ISSN 1020-4989.

**5. Acknowledgment** 

*Trypanosoma cruzi* Sylvatic Isolates from Rio de Janeiro-Brazil 169

type glycoprotein containing about 10% carbohydrate, its molecular mass can be estimated from the sequence, considering two high-mannose oligosaccharide chains, as about 40 kDa. However, this enzyme can present anomalous behavior in SDS-PAGE, yielding apparent molecular mass values of 35 to 60 kDa depending on the experimental conditions. The cysteine peptidases from parasites, including *T. cruzi*, have proven to be valuable targets for chemotherapy. Due to the biological importance of cruzipain in the life cycle of *T. cruzi*, many studies have sought to build specific inhibitors against the active core of this enzyme, in order to obtain a new drug capable of providing protection against human infection by

*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

The authors would like to thank all the members of Setor de Entomologia Forense from Laboratório de Transmissores de Leishmanioses at Instituto Oswaldo Cruz- FIOCRUZ for the encouragement and help, especially to Prof. Catarina Macedo Lopes, who helped and made some figures of this chapter. The financial support CAPES, CNPq, FAPERJ and

Aragão, M.B. & Souza, S.A.(1967). Triatoma infestans colonizado em domicílios da baixada

Ávila-Pires, F.D. (1976). Ecology of small mammals in relation to sylvan and domestic

*Medicina Tropical*, Vol.5, No.1, (August 1971), ISSN 0037-8682.

fluminense, Estado do Rio de Janeiro, Brasil. *Revista da Sociedade Brasileira de* 

transmission cycles. In new approaches in American tripanosomiasis research. *Pan American Health organization Scientific Publication*, Vol. 318, (March 1976), pp.301-

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 genotype.
