Extracellular vesicles shed by Trypanosoma cruzi are linked to small RNA pathways, life cycle regulation, and susceptibility to infection of mammalian cells.

The protozoan parasite Trypanosoma cruzi has a complex life cycle characterized by intracellular and extracellular forms alternating between invertebrate and mammals. To cope with these changing environments, T. cruzi undergoes rapid changes in gene expression, which are achieved essentially at the posttranscriptional level. At present, expanding families of small RNAs are recognized as key players in novel forms of posttranscriptional gene regulation in most eukaryotes. However, T. cruzi lacks canonical small RNA pathways. In a recent work, we reported the presence of alternate small RNA pathways in T. cruzi mainly represented by a homogeneous population of tRNA-derived small RNAs (tsRNAs). In T. cruzi epimastigotes submitted to nutrient starvation, tsRNAs colocalized with an argonaute protein distinctive of trypanosomatids (TcPIWI-tryp) and were recruited to particular cytoplasmic granules. Using epifluorescence and electronic microscopy, we observed that tsRNAs and the TcPIWI-tryp protein were recruited mainly to reservosomes and other intracellular vesicles including endosome-like vesicles and vesicular structures resembling the Golgi complex. These data suggested that, in T. cruzi, tsRNA biogenesis is probably part of endocytic/exocytic routes. We also demonstrated that epimastigotes submitted to nutrient starvation shed high levels of vesicles to the extracellular medium, which carry small tRNAs and TcPIWI-tryp proteins as cargo. At least a fraction of extracellular vesicle cargo was transferred between parasites and to mammalian susceptible cells. Our data afford experimental evidence, indicating that extracellular vesicles shed by T. cruzi promote not only life cycle transition of epimastigotes to trypomastigote forms but also infection susceptibility of mammalian cells.

Gene expression changes induced by Trypanosoma cruzi shed microvesicles in mammalian host cells: relevance of tRNA-derived halves.

At present, noncoding small RNAs are recognized as key players in novel forms of posttranscriptional gene regulation in most eukaryotes. However, canonical small RNA pathways seem to be lost or excessively simplified in some unicellular organisms including Trypanosoma cruzi which lack functional RNAi pathways. Recently, we reported the presence of alternate small RNA pathways in T. cruzi mainly represented by homogeneous populations of tRNA- and rRNA-derived small RNAs, which are secreted to the extracellular medium included in extracellular vesicles. Extracellular vesicle cargo could be delivered to other parasites and to mammalian susceptible cells promoting metacyclogenesis and conferring susceptibility to infection, respectively. Here we analyzed the changes in gene expression of host HeLa cells induced by extracellular vesicles from T. cruzi. As assessed by microarray assays a large set of genes in HeLa cells were differentially expressed upon incorporation of T. cruzi-derived extracellular vesicles. The elicited response modified mainly host cell cytoskeleton, extracellular matrix, and immune responses pathways. Some genes were also modified by the most abundant tRNA-derived small RNAs included in extracellular vesicles. These data suggest that microvesicles secreted by T. cruzi could be relevant players in early events of the T. cruzi host cell interplay.

Cloning, characterization and subcellular localization of a Trypanosoma cruzi argonaute protein defining a new subfamily distinctive of trypanosomatids.

Over the last years an expanding family of small non-coding RNAs (sRNA) has been identified in eukaryotic genomes which behave as sequence-specific triggers for mRNA degradation, translation repression, heterochromatin formation and genome stability. To achieve their effectors functions, sRNAs associate with members of the Argonaute protein family. Argonaute proteins are segregated into three paralogous groups: the AGO-like subfamily, the PIWI-like subfamily, and the WAGO subfamily (for Worm specific AGO). Detailed phylogenetic analysis of the small RNA-related machinery components revealed that they can be traced back to the common ancestor of eukaryotes. However, this machinery seems to be lost or excessively simplified in some unicellular organisms such as Saccharomyces cerevisiae, Trypanosoma cruzi, Leishmania major and Plasmodium falciparum which are unable to utilize dsRNA to trigger degradation of target RNAs. We reported here a unique ORF encoding for an AGO/PIWI protein in T. cruzi which was expressed in all stages of its life cycle at the transcript as well as the protein level. Database search for remote homologues, revealed the presence of a divergent PAZ domain adjacent to the well supported PIWI domain. Our results strongly suggested that this unique AGO/PIWI protein from T. cruzi is a canonical Argonaute in terms of its domain architecture. We propose to reclassify all Argonaute members from trypanosomatids as a distinctive phylogenetic group representing a new subfamily of Argonaute proteins and propose the generic designation of AGO/PIWI-tryp to identify them. Inside the Trypanosomatid-specific node, AGO/PIWI-tryps were clearly segregated into two paralog groups designated as AGO-tryp and PIWI-tryp according to the presence or absence of a functional link with RNAi-related phenomena, respectively.