**3.1 Phylogenetic inferences and TcISP2 origin and role in Chagas' disease**

Maximum likelihood phylogenetic inference resulted in the tree represented in **Figure 3**, with *E. coli* ecotin as the outgroup and color-coded ISPs 1, 2, and 3. The tree topology strongly indicates that the ISP ecotin homologs have differentiated from each other a long time ago, and it is probable that at least ISP1 and ISP2 have been with these organism's genome at least since the split between the *Leishmania* and *Trypanosoma* genera.

Using the loci image generation software described in detail in the author's master thesis [43] resulted in the images in **Figures 4** and **5**, showing the full chromosomes and a zoomed-in area of interest respectively. **Figure 4** is only useful for comparing *Leishmania* records as to the overall position in the chromosome: ecotin homologs occur only in chromosome 15 of these species, and the data for other species is either incomplete or badly annotated, resulting in huge contig sequences. **Figure 5** clears the image a little, but data for *T. grayi* and *Leptomonas pyrrhocoris* sequences is still fragmentary.

The images show up to five ecotin homologs in various *Leishmania* species. This duplication was not reported in previous papers. Sequence data analysis and visual genomic context inspection give strong support to the idea that ecotin homologs suffered various duplications and/or multiple events of lateral gene transference before the differentiation of modern Trypanosomatida genera. Closely examining **Figure 5**, it can be seen that *Leishmania* spp. mostly retained all five ISP copies, while trypanosomes lost at least a few of them. In these figures, the conserved hypothetical coding sequences (*CHP1-3*) and *katanin-like* labels are accessory labels: they serve to identify the complete ecotin loci and were helpful to identify possible genomic evolutionary events involving ISP1, ISP2, and ISP3.

We know ISPs came from bacteria by horizontal gene transfer in Kinetoplastida because they appear in no other eukaryotes. The unanswered question is "how." Looking at **Figures 4** and **5** and keeping the cladogram in **Figure 3** in mind, we can form a hypothesis for how trypanosomatids acquired ecotin homologs. The next paragraph is speculative, but given the evidence, it probably is not too far off-base.

## **Figure 3.**

*Maximum likelihood cladogram of ecotin homologs (ISPs), with ISP1, labeled in orange, ISP2 labeled in green, and ISP3 labeled in blue.*

The ancestor of all trypanosomatids either participated in multiple lateral gene transfers with ecotin-possessing bacteria or this event occurred only once and was followed by multiple gene duplications. If multiple gene transfers occurred, they probably happened no more than three times for ISPs 1, 2, and 3, and the additional ISP2 and ISP3 copies carried by *Leishmania* spp. are the result of a subsequent duplication. The positions of ISP1 and ISP2 in *T. brucei*, in the first and second ecotin loci respectively, with the ISP2 being probably homologous to *T. cruzi* ISP2, points to an early locus duplication, occurring before the two genera split. In this scenario, *Trypanosoma* *Exploring the Evolutionary Origin and Biological Role of the* Trypanosoma cruzi *Ecotin-Like… DOI: http://dx.doi.org/10.5772/intechopen.109929*

### **Figure 4.**

*Loci image generator result showing full GenBank records and all selected labels. Conserved hypothetical protein (CHP) 1 to 3, presenting highly similar amino acid sequences are represented by the same color. Seta direction indicates the orientation of the encoding gene according to genome annotation in GenBank.*

spp. subsequently lost copies of the gene. Their sequences show a much more compact genome when compared to *Leishmania* spp. in the images presented, leading to the suspicion that more deletions occurred in *Trypanosoma* species than in *Leishmania*, which would lend credence to the idea that *T. cruzi* and *T. brucei* lost some of their ISP copies. A possible sequence of events based on this limited dataset is this: the common Trypanosomatida ancestor had three ISP copies in the first locus (at around position 120 kbp in chromosome 15), get either via lateral transfers with bacteria or via a single lateral transfer followed by contiguous duplication. The ISP2 and ISP3 ancestors in this locus then suffered a simultaneous duplication event, creating the second locus at around 190 kbp. Subsequently, various species lost some of these copies.

The preservation of ISP2 in almost all species is an interesting fact and makes sense given the ample evidence of its importance against hosts' immune systems. Another interesting fact is that *T. brucei* parasites preserved the ISP1 variant in all cases, while *T. cruzi* lost the ISP1 gene. Since ISP1 seems to be involved in the development of motility and flagellar development in promastigotes inside the insect vector in *Leishmania* species [31], this could be a reason for its preservation in *T. brucei* and loss in *T. cruzi*. These species are members of section Salivaria and Stercoraria, respectively, with different life cycles and methods of transmission. While *T. cruzi* is transmitted by hemipterans, with infecting parasites deposited with their feces on the vertebrate host, *T. brucei* lives in the salivary gland of dipteran insects and is injected by their proboscis like the *Leishmania* species. It could be that the ISP1 ecotin variant

## **Figure 5.**

*Loci image generator result created with the same data as Figure 4 with a higher resolution and individual ISPs identified as ISP1, ISP2, and ISP3 with numbers; conserved hypothetical protein (CHP) 1 to 3, presenting highly similar amino acid sequences are represented by the same color; seta direction indicates the orientation of the encoding gene according to genome annotation in GenBank.*

gives some advantages to trypanosomatids with dipterans as their arthropod hosts. This association needs further investigation, resulting in data with potential public health applications.

These speculations are given to encourage further research. As tempting as it is to affirm their validity, our data set is very limited in scope and of very poor quality in some cases making bold affirmations. Automated genomic annotation can only go so far, and some of these sequences have errors, omissions, and other problems. Looking closely at the *L. mexicana* data in **Figure 5**, for example, it seems that the coding sequences between the first ISP occurrence and the CHP2 labeled gene should clearly be labeled as ISP2 and ISP3, but in the automated annotation, they appear as "*unknown proteins.*" Nevertheless, since the amount of available genomic data grows so fast, these speculations can be further developed as more data becomes available.

One thing this work clearly shows without a doubt is the ubiquity of large amounts of unreviewed genomic data online. The amount of retrievable information at very little monetary cost and using free-to-use bioinformatics tools is huge, and in this world of big data and exponentially falling sequencing costs, this fact will become more obvious as time passes. The next generation of budding biologists may well have to learn to program before they learn the names of all the plant and animal families.

*T. cruzi* presents only ISP2 in its chromosome 15, and its structural and biochemical characterization revealed high structural similarity with ecotin and strong inhibitory activity on human neutrophil elastase (NE) [44]. Ecotin appears to be a potent prokaryotic tool in evading the immune system. A classic example is that of the inhibition of NE: in humans, a serine peptidase produced in neutrophil granulocytes is one of the main immune defenses in combating pathogenic invaders. In

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

bacteria, one of the ways of action of NE is the cleavage of outer membrane protein A (OmpA, from English outer membrane protein A) in phagocytosed gram-negative bacteria, hindering their multiplication [47, 48]. This makes ecotin an important target for pharmacological research [49].
