MicroRNAs in parasitic tapeworms: discovery, functional analysis and potential role in host-parasite communication

NATALIA MACCHIAROLI; MATIAS PEREZ; EUGENIA ANCAROLA; LUCAS MALDONADO; LAURA KAMENETZKY; ANTONIO MARCILLA; KLAUS BREHM; MARCELA CUCHER; MARA ROSENZVIT

Conferencia; Molecular Helminthology: An Integrated Approach; 2017

MicroRNAs in parasitic tapeworms: discovery, functional analysis and potential role in host-parasite communicationMacchiaroli N1, Pérez M1, Ancarola M E1, Maldonado L1, Kamenetzky L1, Marcilla A2, Brehm K3, Cucher M1 and Rosenzvit M C11IMPaM CONICET-UBA, University of Buenos Aires, Argentina. 2Departament of Cellular Biology and Parasitology, University of Valencia, Spain, 3Institute of Hygiene and Microbiology, University of Würzburg, Würzburg, GermanyIntroductionTapeworms (Cestoda) are etiological agents of neglected diseases such as echinococcosis and neurocysticercosis. MicroRNAs (miRNAs) are small silencing RNAs with key roles in multiple cellular processes. We identified miRNAs in cestodes for the first time and reported differential expression among developmental stages. The objectives of this work are to compare miRNA repertoire and expression profile among tapeworms and to start addressing their functions. MethodsMiRNA repertoire and expression profiles of were determined by smallRNAseq, Northern blot and poly-A RT-(q)PCR. MiRNA targets were predicted by an integrated bioinformatics pipeline that included 3?UTR identification using the recently sequenced Echinococcus canadensis genome (Maldonado et al., under review). Extracellular vesicles (EVs) were identified by transmission electron microscopy and proteomics (LC-MS/MS). ResultsmiRNA repertoire is highly conserved among tapeworms being miRNA expression profile biased to few miRNAs (Figure 1), suggesting important functions of these miRNAs in cestode biology. A high proportion of genes were regulated by miRNAs including genes implicated in essential cellular functions such as HMG and bromodomain proteins (Figure 2). Signaling pathways such as MAPK and WNT were among the most represented, suggesting miRNA roles in parasite growth and development. In vitro secretion of EVs (Figure 3) with miRNA cargo was determined for Mesocestoides corti and Taenia crassiceps. EVs were also observed in the interface of the germinal and laminated layers of Echinococcus multilocularis metacestodes but apparently they were not secreted outwardly, suggesting specific roles in different tapeworms.DiscussionmiRNAs are the main smallRNA silencing molecules in cestodes being several of them absent/divergent in the host. Important pathways were predicted to be targeted by miRNAs. miRNAs are secreted in EVs, and could thus be involved in host-parasite and/or parasite-parasite communication. Altogether, these results suggest that miRNAs are candidates for novel diagnosis and therapeutic interventions and pave the way for further functional studies. Figure 1: Highly expressed miRNAs in tapeworms. Very abundant miRNAs, with 10% or higher expression with respect to total mature miRNAs are shown. The results were obtained by smallRNA seq analyses in Taenia crassiceps (this work), Echinococcus multilocularis, Echinococcus canadensis (Cucher et al., 2015) and Mesocestoides corti (Basika et al., 2016). Figure 2. Conserved miRNA targets and sites in Echinococcus. Predicted miRNA target sites in 3?UTRs of selected targets: High mobilty group (HMG) and a bromodomain containing protein. All sites are conserved in Echinococcus canadensis, Echinococcus multilocularis and Echinococcus granulosus. Figure 3: Extracellular vesicles secreted by cestodes. Transmission electron microscopy of culture supernatant (A), tegument surface (B), and tegument (C). Representative images of T. crassiceps and M. corti. Arrowheads indicate extracellular vesicles. BM: basal membrane; D: dense secretory body; MI: microthrix; MU: muscle; MVB: multivesicular body; PI: pinosome; SL: surface layer.