MicroRNA-Transcription factor regulatory networks in the early strobilar development of Echinococcus granulosus protoscoleces.

BACKGROUND: Echinococcus granulosus sensu lato has a complex developmental biology with a variety of factors relating to both intermediate and final hosts. To achieve maximum parasite adaptability, the development of the cestode is dependent on essential changes in transcript regulation. Transcription factors (TFs) and miRNAs are known as master regulators that affect the expression of downstream genes through a wide range of metabolic and signaling pathways. In this study, we aimed to develop a regulatory miRNA-Transcription factor (miRNA-TF) network across early developmental stages of E. granulosus protoscoleces by performing in silico analysis, and to experimentally validate TFs expression in protoscoleces obtained from in vitro culture, and from in vivo experiments. RESULTS: We obtained list of 394 unique E. granulosus TFs and matched them with 818 differentially expressed genes which identified 41 predicted TFs with differential expression. These TFs were used to predict the potential targets of 31 differentially expressed miRNAs. As a result, eight miRNAs and eight TFs were found, and the predicted network was constructed using Cytoscape. At least four miRNAs (egr-miR-124a, egr-miR-124b-3p, egr-miR-745-3p, and egr-miR-87-3p) and their corresponding differentially expressed TFs (Zinc finger protein 45, Early growth response protein 3, Ecdysone induced protein 78c and ETS transcription factor elf 2) were highlighted in this investigation. The expression of predicted differentially expressed TFs obtained from in vitro and in vivo experiments, were experimentally validated by quantitative polymerase chain reaction. This confirmed findings of RNA-seq data. CONCLUSION: miRNA-TF networks presented in this study control some of the most important metabolic and signaling pathways in the development and life cycle of E. granulosus, providing a potential approach for disrupting the early hours of dog infection and preventing the development of the helminth in the final host.

Ecological and molecular associations between neotropical wild felids and Taenia (Cestoda: Taeniidae) in the Atlantic Forest: a new report for Taenia omissa.

Ecological associations between wild felids and parasites from the Taeniidae family are related to predator-prey interactions, where felids act as definitive hosts while their prey, herbivores and/or omnivores, act as intermediate hosts. In the Atlantic Forest, six neotropical felid species coexist in sympatry, but the ecological parasite-host interactions remain poorly studied. Taenia omissa is a tapeworm that parasitizes cougars (Puma concolor) as its only definitive host and their ungulate prey as intermediate hosts. The aim of this study was to identify tapeworms present in road-killed fauna using both molecular and morphological characteristics and their predator-prey relationship. Adult tapeworms found in a cougar, a jaguarundi (Herpailurus yagouaroundi), and two ocelots (Leopardus pardalis); and metacestodes found in a red brocket deer (Mazama americana) and a wild guinea pig (Cavia aperea) were analyzed. Through morphological analysis of rostellar hooks and molecular analysis of the mitochondrial genetic marker cox1, Taenia omissa adult individuals were identified in the cougar, and metacestodes in the red brocket deer, proving the existence of a full host-parasite life cycle in the Atlantic Forest region. This new report reveals the southernmost record of T. omissa and broadens its geographic distribution. In addition, isolates of the Taenia genus divergent from those described so far in molecular databases were reported and suggested a wild cycle that involves the jaguarundi and agouti (Dasyprocta asarae) as definitive and intermediate hosts, respectively. These results highlight the complexity of the tapeworm population in the region and the need to study it with both morphological and molecular approaches.

MicroRNA profile of the strobilated worms of Echinococcus granulosus derived from in vivo and in vitro systems by using high-throughput approach.

MicroRNAs are critical gene regulators at the post-transcriptional level and play essential roles in numerous developmental processes in metazoan parasites including the causative agent of cystic echinococcosis, Echinococcus granulosus. The molecular basis of different patterns of E. granulosus development in the canine definitive host and in in vitro culture systems is poorly understood. In the present study, miRNA transcriptomes of the strobilated worms derived from experimental infection in the definitive host were compared with those from diphasic culture system after 60-day protoscoleces cultivation. Total RNA was extracted from in vivo- and in vitro-derived strobilated worms. Small RNA libraries were constructed, and deep sequencing was performed. Subsequently, differential miRNA expressions and target predictions were obtained, and pathway analysis was performed by gene ontology and KEGG. Seven miRNAs were differentially expressed between the in vivo- and in vitro-derived worms. In addition, we reported 13 novel miRNA candidates and 42 conserved miRNAs. Four out of five top miRNAs with the highest read counts were shared between the in vivo and in vitro-derived worms, i.e., egr-miR-10a-5p, egr-let-7-5p, egr-bantam-3p, and egr-miR-71-5p. Target prediction of the differential miRNAs between the two systems showed significant differences in the membrane-enclosed lumen, membrane part, and an intrinsic component of the membrane. Findings of KEGG analysis indicated that differentially expressed miRNAs were involved in hippo, MAPK, and WNT signaling pathways. The study demonstrated a significant difference in miRNA transcriptomes and related signaling pathways between the two systems, suggesting the importance of host-parasite interplay in the fate of protoscoleces development in in vivo and in vitro systems.

The genomes of four tapeworm species reveal adaptations to parasitism.

Tapeworms (Cestoda) cause neglected diseases that can be fatal and are difficult to treat, owing to inefficient drugs. Here we present an analysis of tapeworm genome sequences using the human-infective species Echinococcus multilocularis, E. granulosus, Taenia solium and the laboratory model Hymenolepis microstoma as examples. The 115- to 141-megabase genomes offer insights into the evolution of parasitism. Synteny is maintained with distantly related blood flukes but we find extreme losses of genes and pathways that are ubiquitous in other animals, including 34 homeobox families and several determinants of stem cell fate. Tapeworms have specialized detoxification pathways, metabolism that is finely tuned to rely on nutrients scavenged from their hosts, and species-specific expansions of non-canonical heat shock proteins and families of known antigens. We identify new potential drug targets, including some on which existing pharmaceuticals may act. The genomes provide a rich resource to underpin the development of urgently needed treatments and control.

The Echinococcus canadensis (G7) genome: a key knowledge of parasitic platyhelminth human diseases.

BACKGROUND: The parasite Echinococcus canadensis (G7) (phylum Platyhelminthes, class Cestoda) is one of the causative agents of echinococcosis. Echinococcosis is a worldwide chronic zoonosis affecting humans as well as domestic and wild mammals, which has been reported as a prioritized neglected disease by the World Health Organisation. No genomic data, comparative genomic analyses or efficient therapeutic and diagnostic tools are available for this severe disease. The information presented in this study will help to understand the peculiar biological characters and to design species-specific control tools. RESULTS: We sequenced, assembled and annotated the 115-Mb genome of E. canadensis (G7). Comparative genomic analyses using whole genome data of three Echinococcus species not only confirmed the status of E. canadensis (G7) as a separate species but also demonstrated a high nucleotide sequences divergence in relation to E. granulosus (G1). The E. canadensis (G7) genome contains 11,449 genes with a core set of 881 orthologs shared among five cestode species. Comparative genomics revealed that there are more single nucleotide polymorphisms (SNPs) between E. canadensis (G7) and E. granulosus (G1) than between E. canadensis (G7) and E. multilocularis. This result was unexpected since E. canadensis (G7) and E. granulosus (G1) were considered to belong to the species complex E. granulosus sensu lato. We described SNPs in known drug targets and metabolism genes in the E. canadensis (G7) genome. Regarding gene regulation, we analysed three particular features: CpG island distribution along the three Echinococcus genomes, DNA methylation system and small RNA pathway. The results suggest the occurrence of yet unknown gene regulation mechanisms in Echinococcus. CONCLUSIONS: This is the first work that addresses Echinococcus comparative genomics. The resources presented here will promote the study of mechanisms of parasite development as well as new tools for drug discovery. The availability of a high-quality genome assembly is critical for fully exploring the biology of a pathogenic organism. The E. canadensis (G7) genome presented in this study provides a unique opportunity to address the genetic diversity among the genus Echinococcus and its particular developmental features. At present, there is no unequivocal taxonomic classification of Echinococcus species; however, the genome-wide SNPs analysis performed here revealed the phylogenetic distance among these three Echinococcus species. Additional cestode genomes need to be sequenced to be able to resolve their phylogeny.

Cystic echinococcosis in South America: systematic review of species and genotypes of Echinococcus granulosus sensu lato in humans and natural domestic hosts.

OBJECTIVE: To systematically review publications on Echinococcus granulosus sensu lato species/genotypes reported in domestic intermediate and definitive hosts in South America and in human cases worldwide, taking into account those articles where DNA sequencing was performed; and to analyse the density of each type of livestock that can act as intermediate host, and features of medical importance such as cyst organ location. METHODS: Literature search in numerous databases. We included only articles where samples were genotyped by sequencing since to date it is the most accurate method to unambiguously identify all E. granulosus s. l. genotypes. Also, we report new E. granulosus s. l. samples from Argentina and Uruguay analysed by sequencing of cox1 gene. RESULTS: In South America, five countries have cystic echinococcosis cases for which sequencing data are available: Argentina, Brazil, Chile, Peru and Uruguay, adding up 1534 cases. E. granulosus s. s. (G1) accounts for most of the global burden of human and livestock cases. Also, E. canadensis (G6) plays a significant role in human cystic echinococcosis. Likewise, worldwide analysis of human cases showed that 72.9% are caused by E. granulosus s. s. (G1) and 12.2% and 9.6% by E. canadensis G6 and G7, respectively. CONCLUSIONS: E. granulosus s. s. (G1) accounts for most of the global burden followed by E. canadensis (G6 and G7) in South America and worldwide. This information should be taken into account to suit local cystic echinococcosis control and prevention programmes according to each molecular epidemiological situation.

[First report of Echinococcus vogeli in a paca in Misiones province, Argentina]. / Primera identificación de Echinococcus vogeli en una paca en la provincia de Misiones, Argentina.

We report the first finding of Echinococcus vogeli in a paca, Cuniculus paca, in the tropical forest of Misiones, in the north of Argentina. The presence of the bush dog, Speothos venaticus, E. vogelís only natural definitive host, was also reported. The polycystic hydatids, 2 to 3 cm in diameter, were only found in the liver of an adult paca. The size range of the hooks and the relative proportion blade/handle did not show significant differences with respect to the ones reported for E. vogeli. The size of E. granulosus hooks, measured for comparison purposes, was significantly smaller (p E. vogeli in Argentina. The probability of finding neotropical echinococcosis in humans reinforces the need to expand the search for E. vogeli in Argentina. Echinococcosis due to E. vogeli is very aggressive and may cause death in about a third of the human population affected.

microRNA silencing in a whole worm cestode model provides insight into miR-71 function.

Parasites belonging to the class Cestoda include zoonotic species such as Echinococcus spp. and Taenia spp. that cause morbidity and mortality in endemic areas, mainly affecting pastoral and rural communities in low income countries but also upper middle income countries. Cestodes show remarkable developmental plasticity, implying tight regulation of gene expression throughout their complex life cycles. Despite the recent availability of genomic data for cestodes, little progress was made on postgenomic functional studies. MicroRNAs (miRNAs) are key components of gene regulatory systems that guide diverse developmental processes in multicellular organisms. miR-71 is a highly expressed miRNA in cestodes, which is absent in vertebrates and targets essential parasite genes, representing a potential key player in understanding the role of miRNAs in cestodes biology. Here we used transfection with antisense oligonucleotides to perform whole worm miRNA knockdown in tetrathyridia of Mesocestoides vogae (syn. Mesocestoides corti), a laboratory model of cestodes. We believe this is the first report of miRNA knockdown at the organism level in these parasites. Our results showed that M. vogae miR-71 is involved in the control of strobilation in vitro and in the establishment of murine infection. In addition, we identified miR-71 targets in M. vogae, several of them being de-repressed upon miR-71 knockdown. This study provides new knowledge on gene expression regulation in cestodes and suggests that miRNAs could be evaluated as new selective therapeutic targets for treating Neglected Tropical Diseases prioritised by the World Health Organization.

Identification and characterization of sirtuin enzymes in cestodes and evaluation of sirtuin inhibitors as new cestocidal molecules.

Anti-parasitic treatment of neglected tropical diseases caused by cestodes such as echinococcosis and cysticercosis relies on a small number of approved anthelmintic drugs. Furthermore, the treatment is usually prolonged and often partially effective and not well tolerated by some patients. Therefore, the identification of novel drug targets and their associated compounds is critical. In this study, we identified and characterised sirtuin enzymes in cestodes and evaluated the cestocidal potential of sirtuin inhibitors as new cestocidal molecules. Sirtuins are a highly conserved family of nicotinamide-adenine dinucleotide-lysine deacylases involved in multiple cellular functions. Here, we described the full repertoire of sirtuin-encoding genes in several cestode species. We identified six sirtuin-encoding genes that were classified into sirtuins Class I (SIRT1, SIRT2, and SIRT3), Class III (SIRT5), and Class IV (SIRT6 and SIRT7). In Echinococcus spp., sirtuin genes showed transcriptional expression throughout several developmental stages, sirtuin 2 (SIRT2) being the most expressed. To evaluate the potential of sirtuin inhibitors as new cestocidal molecules, we determined the in vitro effect of several Class I sirtuin inhibitors by motility assay. Of those, the selective SIRT2 inhibitor Mz25 showed a strong cestocidal activity in Mesocestoides vogae (syn. Mesocestoides corti) tetrathyridia at various concentrations. The Mz25 cestocidal activity was time- and dose-dependent with a half-maximal inhibitory concentration value significantly lower than that of albendazole. Additionally, Mz25 induced extensive damage in the general morphology with marked alterations in the tegument and ultrastructural features. By homology modelling, we found that cestode SIRT2s showed a high conservation of the canonical sirtuin structure as well as in the residues related to Mz25 binding. Interestingly, some non-conservative mutations were found on the selectivity pocket (an Mz25-induced structural rearrangement on the active site), which represent a promising lead for developing selective cestode SIRT2 inhibitors derived from Mz25. Nevertheless, the Mz25 molecular target in M. vogae is unknown and remains to be determined. This report provides the basis for further studies of sirtuins to understand their roles in cestode biology and to develop selective sirtuin inhibitors to treat these neglected tropical diseases.

Expression profiling of Echinococcus multilocularis miRNAs throughout metacestode development in vitro.

The neglected zoonotic disease alveolar echinococcosis (AE) is caused by the metacestode stage of the tapeworm parasite Echinococcus multilocularis. MicroRNAs (miRNAs) are small non-coding RNAs with a major role in regulating gene expression in key biological processes. We analyzed the expression profile of E. multilocularis miRNAs throughout metacestode development in vitro, determined the spatial expression of miR-71 in metacestodes cultured in vitro and predicted miRNA targets. Small cDNA libraries from different samples of E. multilocularis were sequenced. We confirmed the expression of 37 miRNAs in E. multilocularis being some of them absent in the host, such as miR-71. We found a few miRNAs highly expressed in all life cycle stages and conditions analyzed, whereas most miRNAs showed very low expression. The most expressed miRNAs were miR-71, miR-9, let-7, miR-10, miR-4989 and miR-1. The high expression of these miRNAs was conserved in other tapeworms, suggesting essential roles in development, survival, or host-parasite interaction. We found highly regulated miRNAs during the different transitions or cultured conditions analyzed, which might suggest a role in the regulation of developmental timing, host-parasite interaction, and/or in maintaining the unique developmental features of each developmental stage or condition. We determined that miR-71 is expressed in germinative cells and in other cell types of the germinal layer in E. multilocularis metacestodes cultured in vitro. MiRNA target prediction of the most highly expressed miRNAs and in silico functional analysis suggested conserved and essential roles for these miRNAs in parasite biology. We found relevant targets potentially involved in development, cell growth and death, lifespan regulation, transcription, signal transduction and cell motility. The evolutionary conservation and expression analyses of E. multilocularis miRNAs throughout metacestode development along with the in silico functional analyses of their predicted targets might help to identify selective therapeutic targets for treatment and control of AE.

Identification and expression profiling of microRNAs in Hymenolepis.

Tapeworms (cestodes) of the genus Hymenolepis are the causative agents of hymenolepiasis, a neglected zoonotic disease. Hymenolepis nana is the most prevalent human tapeworm, especially affecting children. The genomes of Hymenolepis microstoma and H. nana have been recently sequenced and assembled. MicroRNAs (miRNAs), a class of small non-coding RNAs, are principle regulators of gene expression at the post-transcriptional level and are involved in many different biological processes. In previous work, we experimentally identified miRNA genes in the cestodes Echinococcus, Taenia and Mesocestoides. However, current knowledge about miRNAs in Hymenolepis is limited. In this work we described for the first known time the expression profile of the miRNA complement in H. microstoma, and discovered miRNAs in H. nana. We found a reduced complement of 37 evolutionarily conserved miRNAs, putatively reflecting their low morphological complexity and parasitic lifestyle. We found high expression of a few miRNAs in the larval stage of H. microstoma that are conserved in other cestodes, suggesting that these miRNAs may have important roles in development, survival and for host-parasite interplay. We performed a comparative analysis of the identified miRNAs across the Cestoda and showed that most of the miRNAs in Hymenolepis are located in intergenic regions, implying that they are independently transcribed. We found a Hymenolepis-specific cluster composed of three members of the mir-36 family. Also, we found that one of the neighboring genes of mir-10 was a Hox gene as in most bilaterial species. This study provides a valuable resource for further experimental research in cestode biology that might lead to improved detection and control of these neglected parasites. The comprehensive identification and expression analysis of Hymenolepis miRNAs can help to identify novel biomarkers for diagnosis and/or novel therapeutic targets for the control of hymenolepiasis.

Histone deacetylase enzymes as potential drug targets of Neglected Tropical Diseases caused by cestodes.

Cestode parasites cause neglected diseases, such as echinococcosis and cysticercosis, which represent a significant problem in human and animal health. Benzimidazoles and praziquantel are the only available drugs for chemotherapy and it is therefore important to identify new alternative drugs against cestode parasites. Histone deacetylases (HDACs) are validated drug targets for the treatment of cancer and other diseases, including neglected diseases. However, knowledge of HDACs in cestodes is very scarce. In this work, we investigated cestode HDACs as potential drug targets to develop new therapies against neglected diseases caused by cestodes. Here we showed the full repertoire of HDAC coding genes in several members of the class Cestoda. Between 6 and 7 zinc-dependent HDAC coding genes were identified in the genomes of species from Echinococcus, Taenia, Mesocestoides and Hymenolepis genera. We classified them as Class I and II HDACs and analyzed their transcriptional expression levels throughout developmental stages of Echinococcus spp. We confirmed for the first time the complete HDAC8 nucleotide sequences from Echinococcus canadensis G7 and Mesocestoides corti. Homology models for these proteins showed particular structural features which differentiate them from HDAC8 from Homo sapiens. Furthermore, we showed that Trichostatin A (TSA), a pan-HDAC inhibitor, decreases the viability of M. corti, alters its tegument and morphology and produces an increment of the total amount of acetylated proteins, including acetylated histone H4. These results suggest that HDAC from cestodes are functional and might play important roles on survival and development. The particular structural features observed in cestode HDAC8 proteins suggest that these enzymes could be selectively targeted. This report provides the basis for further studies on cestode HDAC enzymes and for discovery of new HDAC inhibitors for the treatment of neglected diseases caused by cestode parasites.

Deciphering the role of miR-71 in Echinococcus multilocularis early development in vitro.

Echinococcosis represents a major public health problem worldwide and is considered a neglected disease by the World Health Organization. The etiological agents are Echinococcus tapeworms, which display elaborate developmental traits that imply a complex control of gene expression. MicroRNAs (miRNAs), a class of small regulatory RNAs, are involved in the regulation of many biological processes such as development and metabolism. They act through the repression of messenger RNAs (mRNAs) usually by binding to the 3' untranslated region (3'UTR). Previously, we described the miRNome of several Echinococcus species and found that miRNAs are highly expressed in all life cycle stages, suggesting an important role in gene expression regulation. However, studying the role of miRNAs in helminth biology remains a challenge. To develop methodology for functional analysis of miRNAs in tapeworms, we performed miRNA knockdown experiments in primary cell cultures of Echinococcus multilocularis, which mimic the development of metacestode vesicles from parasite stem cells in vitro. First, we analysed the miRNA repertoire of E. multilocularis primary cells by small RNA-seq and found that miR-71, a bilaterian miRNA absent in vertebrate hosts, is one of the top five most expressed miRNAs. Using genomic information and bioinformatic algorithms for miRNA binding prediction, we found a high number of potential miR-71 targets in E. multilocularis. Inhibition of miRNAs can be achieved by transfection of antisense oligonucleotides (anti-miRs) that block miRNA function. To this end, we evaluated a variety of chemically modified anti-miRs for miR-71 knockdown. Electroporation of primary cells with 2'-O-methyl modified anti-miR-71 led to significantly reduced miR-71 levels. Transcriptomic analyses showed that several predicted miR-71 targets were up-regulated in anti-miR-treated primary cells, including genes potentially involved in parasite development, host parasite interaction, and several genes of as yet unknown function. Notably, miR-71-silenced primary cell cultures showed a strikingly different phenotype from control cells and did not develop into fully mature metacestodes. These findings indicate an important function of miR-71 in Echinococcus development and provide, for the first time, methodology to functionally study miRNAs in a tapeworm.

Computational Prediction of Novel miRNAs from Genome-Wide Data.

The computational prediction of novel microRNAs (miRNAs) within a full genome involves identifying sequences having the highest chance of being bona fide miRNA precursors (pre-miRNAs). These sequences are usually named candidates to miRNA. The well-known pre-miRNAs are usually only a few in comparison to the hundreds of thousands of potential candidates to miRNA that have to be analyzed. Although the selection of positive labeled examples is straightforward, it is very difficult to build a set of negative examples in order to obtain a good set of training samples for a supervised method. In this chapter we describe an approach to this problem, based on the unsupervised clustering of unlabeled sequences from genome-wide data, and the well-known miRNA precursors for the organism under study. Therefore, the protocol developed allows for quick identification of the best candidates to miRNA as those sequences clustered together with known precursors.

Genome-wide identification of microRNA targets in the neglected disease pathogens of the genus Echinococcus.

MicroRNAs (miRNAs), a class of small non-coding RNAs, are key regulators of gene expression at post-transcriptional level and play essential roles in biological processes such as development. MiRNAs silence target mRNAs by binding to complementary sequences in the 3'untranslated regions (3'UTRs). The parasitic helminths of the genus Echinococcus are the causative agents of echinococcosis, a zoonotic neglected disease. In previous work, we performed a comprehensive identification and characterization of Echinococcus miRNAs. However, current knowledge about their targets is limited. Since target prediction algorithms rely on complementarity between 3'UTRs and miRNA sequences, a major limitation is the lack of accurate sequence information of 3'UTR for most species including parasitic helminths. We performed RNA-seq and developed a pipeline that integrates the transcriptomic data with available genomic data of this parasite in order to identify 3'UTRs of Echinococcus canadensis. The high confidence set of 3'UTRs obtained allowed the prediction of miRNA targets in Echinococcus through a bioinformatic approach. We performed for the first time a comparative analysis of miRNA targets in Echinococcus and Taenia. We found that many evolutionarily conserved target sites in Echinococcus and Taenia may be functional and under selective pressure. Signaling pathways such as MAPK and Wnt were among the most represented pathways indicating miRNA roles in parasite growth and development. Genome-wide identification and characterization of miRNA target genes in Echinococcus provide valuable information to guide experimental studies in order to understand miRNA functions in the parasites biology. miRNAs involved in essential functions, especially those being absent in the host or showing sequence divergence with respect to host orthologs, might be considered as novel therapeutic targets for echinococcosis control.

microRNA analysis of Taenia crassiceps cysticerci under praziquantel treatment and genome-wide identification of Taenia solium miRNAs.

MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as important regulators of gene expression and perform critical functions in development and disease. In spite of the increased interest in miRNAs from helminth parasites, no information is available on miRNAs from Taenia solium, the causative agent of cysticercosis, a neglected disease affecting millions of people worldwide. Here we performed a comprehensive analysis of miRNAs from Taenia crassiceps, a laboratory model for T. solium studies, and identified miRNAs in the T. solium genome. Moreover, we analysed the effect of praziquantel, one of the two main drugs used for cysticercosis treatment, on the miRNA expression profile of T. crassiceps cysticerci. Using small RNA-seq and two independent algorithms for miRNA prediction, as well as northern blot validation, we found transcriptional evidence of 39 miRNA loci in T. crassiceps. Since miRNAs were mapped to the T. solium genome, these miRNAs are considered common to both parasites. The miRNA expression profile of T. crassiceps was biased to the same set of highly expressed miRNAs reported in other cestodes. We found a significant altered expression of miR-7b under praziquantel treatment. In addition, we searched for miRNAs predicted to target genes related to drug response. We performed a detailed target prediction for miR-7b and found genes related to drug action. We report an initial approach to study the effect of sub-lethal drug treatment on miRNA expression in a cestode parasite, which provides a platform for further studies of miRNA involvement in drug effects. The results of our work could be applied to drug development and provide basic knowledge of cysticercosis and other neglected helminth infections.

Cestode parasites release extracellular vesicles with microRNAs and immunodiagnostic protein cargo.

Intercellular communication is crucial in multiple aspects of cell biology. This interaction can be mediated by several mechanisms including extracellular vesicle (EV) transfer. EV secretion by parasites has been reported in protozoans, trematodes and nematodes. Here we report that this mechanism is present in three different species of cestodes, Taenia crassiceps, Mesocestoides corti and Echinococcus multilocularis. To confirm this we determined, in vitro, the presence of EVs in culture supernatants by transmission electron microscopy. Interestingly, while T. crassiceps and M. corti metacestodes secrete membranous structures into the culture media, similar vesicles were observed in the interface of the germinal and laminated layers of E. multilocularis metacestodes and were hardly detected in culture supernatants. We then determined the protein cargo in the EV-enriched secreted fractions of T. crassiceps and M. corti conditioned media by LC-MS/MS. Among the identified proteins, eukaryotic vesicle-enriched proteins were identified as expected, but also proteins used for cestode disease diagnosis, proteins related to neurotransmission, lipid binding proteins as well as host immunoglobulins and complement factors. Finally, we confirmed by capillary electrophoresis the presence of intravesicular RNA for both parasites and detected microRNAs by reverse transcription-PCR. This is the first report of EV secretion in cestode parasites and of an RNA secretion mechanism. These findings will provide valuable data not only for basic cestode biology but also for the rational search for new diagnostic targets.

Implementation of new tools in molecular epidemiology studies of Echinococcus granulosus sensu lato in South America.

The aim of this work was to determine Echinococcus granulosus sensu lato species and genotypes in intermediate and definitive hosts and in human isolates from endemic regions of Argentina and Brazil including those where no molecular data is available by a combination of classical and alternative molecular tools. A total of 227 samples were isolated from humans, natural intermediate and definitive hosts. Amplification of cytochrome c oxidase subunit I gene fragment was performed and a combination of AluI digestion assay, High Resolution Melting analysis (HRM) assay and DNA sequencing was implemented for Echinococcus species/genotype determination. E. granulosus sensu stricto (G1) was found in sheep (n=35), cattle (n=67) and dogs (n=5); E. ortleppi (G5) in humans (n=3) and cattle (n=108); E. canadensis (G6) in humans (n=2) and E. canadensis (G7) in pigs (n=7). We reported for the first time the presence of E. ortleppi (G5) and E. canadensis (G6) in humans from San Juan and Catamarca Argentinean provinces and E. canadensis (G7) in pigs from Cordoba Argentinean province. In this work, we widened molecular epidemiology studies of E. granulosus s. l. in South America by analyzing several isolates from definitive and intermediate hosts, including humans from endemic regions were such information was scarce or unavailable. The presence of different species/genotypes in the same region and host species reinforce the need of rapid and specific techniques for accurate determination of Echinococcus species such as the ones proposed in this work.

Identification and profiling of microRNAs in two developmental stages of the model cestode parasite Mesocestoides corti.

MicroRNAs (miRNAs), a class of small non-coding RNAs, are key regulators of gene expression at post-transcriptional level and play essential roles in fundamental biological processes such as metabolism and development. The particular developmental characteristics of cestode parasites highlight the importance of studying miRNA gene regulation in these organisms. Here, we performed a comprehensive analysis of miRNAs in two developmental stages of the model cestode Mesocestoides corti. Using a high-throughput sequencing approach, we found transcriptional evidence of 42 miRNA loci in tetrathyridia larvae and strobilated worms. Tetrathyridium and strobilated worm-specific miRNAs were found, as well as differentialy expressed miRNAs between these developmental stages, suggesting miRNA regulation of stage-specific features. Moreover, it was shown that uridylation is a differential mechanism of post-transcriptional modification of M. corti miRNAs. The whole set of M. corti miRNAs represent 33 unique miRNA families, and confirm the remarkable loss of conserved miRNA families within platyhelminth parasites, reflecting their relatively low morphological complexity and high adaptation to parasitism. Overall, the presented results provide a valuable platform to studies aiming to identify and characterize novel miRNA-based molecular mechanisms of post-transcriptional gene regulation in cestodes, necessary for the elucidation of developmental aspects of the complex biology of these parasites.

microRNA profiling in the zoonotic parasite Echinococcus canadensis using a high-throughput approach.

BACKGROUND: microRNAs (miRNAs), a class of small non-coding RNAs, are key regulators of gene expression at post-transcriptional level and play essential roles in fundamental biological processes such as development and metabolism. The particular developmental and metabolic characteristics of cestode parasites highlight the importance of studying miRNA gene regulation in these organisms. Here, we perform a comprehensive analysis of miRNAs in the parasitic cestode Echinococcus canadensis G7, one of the causative agents of the neglected zoonotic disease cystic echinococcosis. METHODS: Small RNA libraries from protoscoleces and cyst walls of E. canadensis G7 and protoscoleces of E. granulosus sensu stricto G1 were sequenced using Illumina technology. For miRNA prediction, miRDeep2 core algorithm was used. The output list of candidate precursors was manually curated to generate a high confidence set of miRNAs. Differential expression analysis of miRNAs between stages or species was estimated with DESeq. Expression levels of selected miRNAs were validated using poly-A RT-qPCR. RESULTS: In this study we used a high-throughput approach and found transcriptional evidence of 37 miRNAs thus expanding the miRNA repertoire of E. canadensis G7. Differential expression analysis showed highly regulated miRNAs between life cycle stages, suggesting a role in maintaining the features of each developmental stage or in the regulation of developmental timing. In this work we characterize conserved and novel Echinococcus miRNAs which represent 30 unique miRNA families. Here we confirmed the remarkable loss of conserved miRNA families in E. canadensis, reflecting their low morphological complexity and high adaptation to parasitism. CONCLUSIONS: We performed the first in-depth study profiling of small RNAs in the zoonotic parasite E. canadensis G7. We found that miRNAs are the preponderant small RNA silencing molecules, suggesting that these small RNAs could be an essential mechanism of gene regulation in this species. We also identified both parasite specific and divergent miRNAs which are potential biomarkers of infection. This study will provide valuable information for better understanding of the complex biology of this parasite and could help to find new potential targets for therapy and/or diagnosis.