Introduction

Having the potential of playing important roles in various diseases, extracellular vesicles (EVs) are one of the most primarily focused areas in today’s biology. Although introduced in late 80’s (Johnstone et al., 1987) as wasted vesicles and later as a significant role player in other complex functions like antigen presentation (Raposo et al., 1996), EVs are now confirmed as the mediators in intercellular exchange of genetic material in eukaryotes and multicellular organisms (Belting & Wittrup, 2008). By composition, they are mainly composed of lipid rafts (Théry et al., 1999; Wubbolts et al., 2003), membrane trafficking proteins (Hemler, 2003), transmembrane molecules, T cell-stimulating molecules, signaling molecules, and also small RNAs; mRNA and miRNA (Théry, Zitvogel & Amigorena, 2002; Valadi et al., 2007). These micros to nano sized vesicles have ability to circulate in intercellular milieu through almost every body fluid (Raposo & Stoorvogel, 2013).

EVs can be categorized according to the path/mechanism they choose to be released in the extracellular milieu. They can either be exosomes, which are 30–100 nm cup-shaped vesicles, released by exocytic fusion of multivesicular bodies (MVBs) and plasma membrane or ectosomes which are larger vesicles ranging around 100–1,000 nm in diameter and released directly from the plasma membrane by budding. Additionally, a third category may belong to apoptotic bodies, which are even larger than aforementioned categories having diameter of more than 1 μm and found to be originating from apoptotic cells (György et al., 2011).

Functionally, EVs can either be immune-stimulatory or immune-suppressive, depending on the cells they originate from. In previous studies, when it became clear that EVs were involved in transporting small RNAs (Valadi et al., 2007) that might be translated into proteins by recipient cells (Ratajczak et al., 2006; Valadi et al., 2007), the research regarding EVs exploded immensely. Other studies show that the contents of EVs, like extracellular RNAs (ExRNAs) are highly stable (Valadi et al., 2007; Skog et al., 2008) and represent both target and source environment (Choi et al., 2012; Sandvig & Llorente, 2012). Also, extracellular miRNAs (EXmiRNAs) are found to be capable of modulating gene expression in recipient cells (Mittelbrunn et al., 2011). Upon fusion of EVs with a target cell, alteration in the biology of the target tissue, through heterogeneous bioactive cargo is observed in many studies (Mittelbrunn et al., 2011; Montecalvo et al., 2012; Buck et al., 2014; Twu et al., 2013). Since proteins and RNAs carried by EVs are protected from hydrolysis or degradation in extracellular environment, they are considered to be a potential source of disease biomarkers (Keller et al., 2011).

Later, it was observed that parasites also release EVs (Silverman et al., 2010; Marcilla et al., 2012; Twu et al., 2013; Bernal et al., 2014); thus, hypothesis of involvement of these vesicles in host-pathogen interaction and immune response grew stronger. In fact, EVs extracted from Heligmosomoides polygyrus, a gastrointestinal nematode, have recently been shown to alter gene expression in host cells and suppress innate immune responses in mice (Buck et al., 2014).

These significant properties of EVs make them promising in therapeutic field of neglected diseases, Chagas disease included. Chagas disease, also called American Trypanosomiasis, is caused by the parasite T. cruzi. The triatomine bug (kissing bug), a bloodsucking insect that feeds on humans and animals, spreads this parasite through its feces. T. cruzi has distinct life stages which consist of three main developmental forms. The non-infective epimastigotes are found in the midgut of the bug, where they multiply by binary fission. Epimastigotes move to the hindgut and differentiate into metacyclic trypomastigotes that have capacity to infect mammalian cells. When the parasite enters the body, the trypomastigotes circulate in the blood, but do not divide. The trypomastigotes move to the cytoplasm and transform into amastigotes. The amastigotes, after many rounds of division, again transform back into trypomastigotes and enter the bloodstream, where they may invade cells in mammalian body or be transmitted to the insects during their meal of blood.

In a previous study, it`s been shown that T. cruzi releases at least two types of EVs; ectosomes and exosomes, generated by distinct pathways (Goncalves et al., 1991). In a study carried out in 2013, it was found that infective metacyclic forms release vesicles that carry virulence factors such as GP82 glycoproteins and mucins, while in contact with HeLa cells (Bayer-Santos et al., 2013). This suggests the possibility that EVs can be used as nano-carriers to deliver virulence and modulatory factors into the host cells. Furthermore, EVs have full potential to be used as delivery system for drugs, proteins, miRNAs/siRNAs, and other molecules (Fais et al., 2013). Jang & Gho (2014) showed that if EVs can be bio-engineered, then there is a great hope of target delivering of therapeutic agents which can be immensely helpful in revolutionizing vaccine development for Chagas disease. Since transcriptomic data enriches the information regarding small RNAs (miRNAs) which are key players in gene regulation (Ghildiyal & Zamore, 2009), studies regarding EVs transcriptomic data would help exploring different aspects of Chagas disease.

In this regard, another study was carried out in 2014 to analyze EVs extracted from T. cruzi (Bayer-Santos et al., 2014). In this study, EVs were extracted from epimastigotes and metacyclic trypomastigotes forms (clone Dm28c; two biological replicates) of T. cruzi. Total RNA extracted from both replicates was mixed before small RNA isolation, to obtain sufficient amount of small RNA. This procedure was performed for all sample-replicates. Briefly, the small RNA fraction of 16–40 nt was isolated from total RNA of metacyclic trypomastigote parental cells (mCell), epimastigote (eVes) and metacyclic-derived vesicles (mVes). Illumina GAIIx was used for sequencing cDNA library which was purified from Illumina TrueseqTM small RNA preparation kit.

This research article elaborates the analysis and predictive data mining of high throughput transcriptomic data produced by aforementioned RNA-Seq study carried out in 2014. The analysis included important steps of filtration of raw reads, mapping reads to transcriptome and detection of variants. Further, these variants were classified according to their types (Single Nucleotide Polymorphisms (SNPs), Multiple Nucleotide Polymorphisms (MNPs), indels and complex events) and functionally analyzed. It was found that the transcripts consisting of these variants encode various important proteins that have vital role in the pathogenesis, prognosis and diagnosis of Chagas disease. Some of the putative candidate variants were found to be present only in specific stage of T. cruzi that strengthens the hope of finding stage specific biomarkers. Since EVs have a pivotal role in host-pathogen interaction, this study would help researchers to have a better understanding of the roles and significance of EVs in Chagas disease.