Small RNAs derived from tRNAs and rRNAs are highly enriched in exosomes from both old and new world Leishmania providing evidence for conserved exosomal RNA Packaging

Abstract

BackgroundLeishmania use exosomes to communicate with their mammalian hosts and these secreted vesicles appear to contribute to pathogenesis by delivering protein virulence factors to macrophages. In other eukaryotes, exosomes were found to carry RNA cargo, such as mRNAs and small non-coding RNAs, capable of altering recipient cell phenotype. Whether leishmania exosomes also contain RNAs which they are able to deliver to bystander cells is not known. Here, we show that leishmania exosomes indeed contain RNAs and compare and contrast the RNA content of exosomes released by Leishmania donovani and Leishmania braziliensis.ResultsWe purified RNA from exosomes collected from axenic amastigote culture supernatant and found that when compared with total leishmania RNA, exosomes mainly contained short RNA sequences. Exosomes with intact membranes were capable of protecting their RNA cargo from degradation by RNase. Moreover, exosome RNA cargo was delivered to host cell cytoplasm in vitro. Sequencing of exosomal RNA indicated that the majority of cargo sequences were derived from non-coding RNA species such as rRNA and tRNA. In depth analysis revealed the presence of tRNA-derived small RNAs, a novel RNA type with suspected regulatory functions. Northern blotting confirmed the specific and selective enrichment of tRNA-derived small RNAs in exosomes. We also identified a number of novel transcripts, which appeared to be specifically enriched in exosomes compared to total cell RNA. In addition, we observed the presence of sequences mapping to siRNA-coding regions in L. braziliensis , but not in L. donovani exosomes.ConclusionsThese results show that leishmania exosomes are selectively and specifically enriched in small RNAs derived almost exclusively from non-coding RNAs. These exosomes are competent to deliver their cargo of novel, potential small regulatory RNAs to macrophages where they may influence parasite-host cell interactions. The remarkably high degree of congruence in exosomal RNA content between L. donovani and L. braziliensis, argues for the presence of a conserved mechanism for exosomal RNA packaging in leishmania. These findings open up a new avenue of research on non-canonical, small RNA pathways in this trypanosomatid, which may elucidate pathogenesis and identify novel therapeutic approaches.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1260-7) contains supplementary material, which is available to authorized users.

Tilting the balance between RNA interference and replication eradicates Leishmania RNA virus 1 and mitigates the inflammatory response

“…1A and Tables S1 and S2). The 33-nt peak reads mapped primarily to structural RNA loci (62% of mapped reads) (Table S2), similar to an sRNA class described in many eukaryotes, including trypanosomes and Leishmania lacking the RNAi pathway (41)(42)(43)(44). In contrast, reads from the 23-nt peak showed properties similar to AGO1-bound siRNAs (39), including their size and the presence of one to two untemplated nucleotides at the 3′ end in about 21% of the reads (Fig.…”

The languages of parasite communication

“…The definition of excreted molecules was rather vague, essentially encompassing all molecules that did not appear to follow classical secretion pathways. In the past few years, however, several parasites have been shown to produce membrane-bound vesicles that increase their chances of survival and transmission [10][11][12][29][30][31][32][33][34]. In theory, there are many ways in which the contents of these vesicles can be delivered to cells within the host.…”

“…One is the ability to recruit macrophages to the inoculation site; the other is to stimulate the conversion of arginine to polyamines, which are required for growth of Leishmania, rather than nitric oxide, which activates macrophages and promotes parasite killing [49]. Leishmania promastigotes were among the first examples of parasites that were shown to extrude exosomes in culture [31][32][33]. In common with extracellular vesicles from Trichomonas and T. cruzi, these are enriched for specific fragments of tRNAs and rRNAs [33].…”

“…Leishmania promastigotes were among the first examples of parasites that were shown to extrude exosomes in culture [31][32][33]. In common with extracellular vesicles from Trichomonas and T. cruzi, these are enriched for specific fragments of tRNAs and rRNAs [33]. With one exception, similar cargoes were identified in exosomes from L. donovani and L. brasiliensis; the latter is competent for RNA interference and also packages small interfering RNAs [33].…”

“…In common with extracellular vesicles from Trichomonas and T. cruzi, these are enriched for specific fragments of tRNAs and rRNAs [33]. With one exception, similar cargoes were identified in exosomes from L. donovani and L. brasiliensis; the latter is competent for RNA interference and also packages small interfering RNAs [33]. Very recently, it was demonstrated that exosomes with the same composition as culture-derived exosomes are produced by parasites in the sand fly and that these enhance the parasite load when co-injected with parasites [34].…”