**1.2 Serine protease inhibitors: ecotins and** *Trypanosomatida* **ISPs**

Ecotins are serine protease inhibitors initially described in *E. coli* bacteria and named for their capacity to inhibit the digestive enzyme trypsin—*E. coli* trypsin inhibitor [8]. The *E. coli* ecotin has a molecular weight of 18 kDa and is expressed in the cellular periplasm with a homodimeric active form. It inhibits serine proteases of family S1A, including trypsin, chymotrypsin, neutrophil elastase, and cathepsin G [24, 25]. Ecotin activity protect cells against exogenous serine peptidases involved in various biological processes, including coagulation and fibrinolysis; this capacity for inhibiting a considerable number of different proteins differentiates ecotin from most other serine protease inhibitors, which generally are highly specific [26–29].

Trypanosomatids are the only eukaryotes with genes coding for ecotin analogs, described for the first time in 2005 by Ivens et al. in *L. major*, with three variants that were named ISP1, ISP2, and ISP3 [9, 30]. In *L. major*, the ISP1 and ISP2 ecotins have 16.5 and 17.5 kDa, respectively, and while structurally similar to the *E. coli* ecotin, their amino acid sequence identity is only 36% [31], and they have different patterns of expression and inhibitory activity in the various stages of the parasite life cycle [30]. In *L. major,* the ISP1 variant is expressed in larger quantities in the life cycle forms living in the insect host, and knockout studies with this gene suggest that it has endogenous functions, mainly in the flagellar formation process [31]. Also in *L. major*, ISP2 expression occurs in all life cycle stages and there are evidences that this enzyme participates in the parasite macrophage infection process in hosts, by inhibiting serine proteases such as neutrophil elastase in vertebrates. There is evidence that *Leishmania* parasites with knocked down ISP2 suffer more intense phagocytosis by host macrophages [30, 32, 33]. *E. coli* and other bacteria that have periplasmic ecotin use it to evade hosts' immune systems, and *L. major* employs its ISP2 inhibitor in a similar fashion [26, 34]. The lack of genes coding for ecotin target enzymes (the S1A family of serine proteases) in both *E. coli* and *L. major* is a strong indicator of the probable role of ecotins in these species' interactions with vertebrate hosts [30].

An ISP2 homolog has been found in *T. cruzi* with a high degree of sequence similarity to the *L. major* gene [35]. BLAST searches in the NCBI GenBank database reveal that other members of the genus *Trypanosoma* also possess ISP2 homologs, as well as close relatives in the order Trypanosomatida such as *Leptomonas* spp., most papers published on the subject have focused on *Leishmania* ISPs. It is probable that, due to both its conservation in various species of trypanosomatids and its flexible functional properties, ecotin homologs have offered some fitness gain to trypanosomatids with vertebrate hosts. Also, the conservation of ISPs in various species indicates an origin in the common ancestor of Trypanosomatida. The similarity between trypanosomatid ISPs and bacterial ecotins makes us raise the hypothesis of a lateral gene transfer between *E. coli-*like bacteria and the common ancestor of the various Trypanosomatida genera as the origin of ISPs [30, 36]. Recent research suggest that this kind of lateral gene transfer has been essential in this group's evolutionary history [22]. The bacterial endosymbionts in Kinetoplastida are in class Betaproteobacteria [37]. This group contains vertebrate infecting species that not only possess ecotinencoding genes, but that depends on those ecotins being expressed to maintain their virulence [34], which may be another hint of ancestral lateral gene transfers between Betaproteobacteria and Trypanosomatida.

*Leishmania* and *Trypanosoma* parasites are responsible for a number of severely neglected tropical diseases, as officially listed by the World Health Organization [38]. Multiple sources indicate that ecotin and its homologs are connected to these parasites' infective capacity, but research in this specific subject is still timid, especially in the *Trypanosoma* genus. Neglected tropical diseases like Chagas' disease (caused *by T. cruzi*) and African sleeping sickness (caused by *T. brucei*) are neglected for socio-historical reasons, as these afflictive diseases rarely, if ever, occur in developed countries.

The evolution of the genes encoding for trypanosomatid ISPs can shed light not only on the group's evolutionary history but also on the overall importance of this enzyme for future researchers. In the next few pages, we show evidence for a common ancestry of ISPs in extant trypanosomatids using both phylogenetic inferences and a novel method for gene loci analysis. Also, we demonstrate the antigenicity of *T. cruzi* ISP2 in the murine model of Chagas' disease and the presence of a higher concentration of antibodies against TcISP2 in the chronic phase of infection.

*Exploring the Evolutionary Origin and Biological Role of the* Trypanosoma cruzi *Ecotin-Like… DOI: http://dx.doi.org/10.5772/intechopen.109929*
