Extracellular non-coding RNA signatures of the metacestode stage of Echinococcus multilocularis.
PLOS NEGLECTED TROPICAL DISEASES(2020)
Abstract
Extracellular RNAs (ex-RNAs) are secreted by cells through different means that may involve association with proteins, lipoproteins or extracellular vesicles (EV). In the context of parasitism, ex-RNAs represent new and exciting communication intermediaries with promising potential as novel biomarkers. In the last years, it was shown that helminth parasites secrete ex-RNAs, however, most work mainly focused on RNA secretion mediated by EV. Ex-RNA study is of special interest in those helminth infections that still lack biomarkers for early and/or follow-up diagnosis, such as echinococcosis, a neglected zoonotic disease caused by cestodes of the genus Echinococcus. In this work, we have characterised the ex-RNA profile secreted by in vitro grown metacestodes of Echinococcus multilocularis, the casuative agent of alveolar echinococcosis. We have used high throughput RNA-sequencing together with RT-qPCR to characterise the ex-RNA profile secreted towards the extra- and intra-parasite milieus in EV-enriched and EV-depleted fractions. We show that a polarized secretion of small RNAs takes place, with microRNAs mainly secreted to the extra-parasite milieu and rRNA- and tRNA-derived sequences mostly secreted to the intra-parasite milieu. In addition, we show by nanoparticle tracking analyses that viable metacestodes secrete EV mainly into the metacestode inner vesicular fluid (MVF); however, the number of nanoparticles in culture medium and MVF increases > 10-fold when metacestodes show signs of tegument impairment. Interestingly, we confirm the presence of host miRNAs in the intra-parasite milieu, implying their internalization and transport through the tegument towards the MVF. Finally, our assessment of the detection of Echinococcus miRNAs in patient samples by RT-qPCR yielded negative results suggesting the tested miRNAs may not be good biomarkers for this disease. A comprehensive study of the secretion mechanisms throughout the life cycle of these parasites will help to understand parasite interaction with the host and also, improve current diagnostic tools. Author summary Extracellular RNAs (ex-RNAs) are secreted by cells through association with proteins or extracellular vesicles (EV). In the context of parasitism, ex-RNAs represent novel communication intermediaries with promising potential as biomarkers. In order to better understand the role ex-RNAs may play in the context of the zoonotic disease echinococcosis, we have characterised the RNA profile secreted by the larval stage (metacestode) of Echinococcus multilocularis. By analysing the products secreted towards the extra- and intra-parasite milieus, we demonstrate that the metacestode displays a polarized secretion of different classes of small non-coding RNAs (sRNAs). In addition, we show that EV secretion occurs mainly towards the inner fluid of the metacestodes. Interestingly, we confirm the presence of host sRNAs in the intra-parasite milieu, implying their internalization and transport through the tegument. Finally, the detection of Echinococcus miRNAs in patient samples yielded negative results suggesting the tested miRNAs may not be good biomarkers for this disease. In summary, our results provide a detailed description of the ex-RNA landscape of the E. multilocularis metacestode together with information on the distribution of the detected RNA classes in different extracellular compartments. This information is of importance to better understand host-parasite interaction and also, to improve current diagnostic tools.
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Key words
rna,non-coding
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