Extracts and Terpenoids from Stevia Species as Potential Anthelmintics for Neglected Tropical Diseases Caused by Cestode Parasites.

Cestodes are etiological agents of neglected diseases such as echinococcosis and cysticercosis, which are major public health problems. Antiparasitic treatment relies on a small number of approved drugs, which are often only partially effective, poorly tolerated and require prolonged administration. Thus, the discovery of novel potential treatments is critical. The Stevia genus (Asteraceae) includes species that are recognized as a source of bioactive compounds, with many species associated with medicinal uses. In this study, the cestocidal activity of four South American Stevia species that previously showed antiprotozoal activity was analyzed using a motility assay on the laboratory cestode model, Mesocestoides vogae. The four Stevia extracts showed cestocidal activity, with S. alpina var. alpina as the most active. The sesquiterpene lactones estafietin and eupatoriopicrin were purified from S. alpina var. alpina and S. maimarensis, respectively, and tested on M. vogae. Estafietin showed cestocidal activity, inhibiting parasite viability in a dose-dependent manner, even from the first day of incubation. Consistent with the motility effects, the extract of S. alpina var. alpina and estafietin induced marked alterations in the morphology of the parasite. The results of this report show that Stevia species represent a source of new molecules with potential for the treatment of neglected tropical diseases caused by cestodes.

In vitro system for the growth and asexual multiplication of Taenia crassiceps cysticerci.

Taenia solium is the aetiological agent of cysticercosis, a zoonosis that causes severe health and economic losses across Latin America, Africa and Asia. The most serious manifestation of the disease is neurocysticercosis, which occurs when the larval stage (cysticercus) establishes in the central nervous system. Using Taenia crassiceps as an experimental model organism for the study of cysticercosis, we aimed to identify the in vitro conditions necessary to allow parasite development at the short- and long terms. First, cysticerci were incubated for 15 days in different media and parasite densities. The number of buddings and cysticerci diameter were measured to evaluate asexual multiplication and parasite growth, respectively. Vitality was determined by trypan blue staining and morphology analysis. As a result, high cysticerci density and medium containing FBS and the excretion/secretion (E/S) products of feeder cells induced parasite survival, growth and multiplication. Then, the long-term (5 weeks) incubation of the parasites in co-culture with feeder cells was evaluated. Consequently, the mammalian cell lines induced a significant increase in total parasite volume while axenic cultures did not show any statistically significant change over time. In this study, the proper conditions to maintain T. crassiceps in vitro are described for the first time in a simpler and more controlled setting other than experimental infections. In addition, it was shown that cysticerci growth, survival and asexual multiplication depend on a complex network of secreted factors from both parasite and host.

Transcriptional effects of electroporation on Echinococcus multilocularis primary cell culture.

Echinococcus multilocularis is the etiological agent of alveolar echinococcosis (AE), a serious parasitic disease in the Northern Hemisphere. The E. multilocularis primary cell cultivation system, together with E. multilocularis genome data and a range of pioneering molecular-based tools have advanced the research on this and other cestodes. RNA interference (RNAi) and microRNA knock-down have recently contributed to the study of the cellular and molecular basis of tapeworm development and host-parasite interaction. These, as well as other techniques, normally involve an electroporation step for the delivery of RNA, DNA, peptides, and small molecules into cells. Using transcriptome data and bioinformatic analyses, we herein report a genome-wide comparison between primary cells of E. multilocularis and primary cells under electroporated conditions after 48 h of culture. We observed that ~ 15% of genes showed a significant variation in expression level, including highly upregulated genes in electroporated cells, putatively involved in detoxification and membrane remodeling. Furthermore, we found genes related to carbohydrate metabolism, proteolysis, calcium ion binding and microtubule processing significantly altered, which could explain the cellular dispersion and the reduced formation of cellular aggregates observed during the first 48 h after electroporation.

The Role of MicroRNAs in HIV Infection.

MicroRNAs (miRNAs), a class of small, non-coding RNAs, play a pivotal role in regulating gene expression at the post-transcriptional level. These regulatory molecules are integral to many biological processes and have been implicated in the pathogenesis of various diseases, including Human Immunodeficiency Virus (HIV) infection. This review aims to cover the current understanding of the multifaceted roles miRNAs assume in the context of HIV infection and pathogenesis. The discourse is structured around three primary focal points: (i) elucidation of the mechanisms through which miRNAs regulate HIV replication, encompassing both direct targeting of viral transcripts and indirect modulation of host factors critical for viral replication; (ii) examination of the modulation of miRNA expression by HIV, mediated through either viral proteins or the activation of cellular pathways consequent to viral infection; and (iii) assessment of the impact of miRNAs on the immune response and the progression of disease in HIV-infected individuals. Further, this review delves into the potential utility of miRNAs as biomarkers and therapeutic agents in HIV infection, underscoring the challenges and prospects inherent to this line of inquiry. The synthesis of current evidence positions miRNAs as significant modulators of the host-virus interplay, offering promising avenues for enhancing the diagnosis, treatment, and prevention of HIV infection.

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.

Extracellular vesicles from Trypanosoma cruzi-dendritic cell interaction show modulatory properties and confer resistance to lethal infection as a cell-free based therapy strategy.

Extracellular vesicles (EVs) include a heterogeneous group of particles. Microvesicles, apoptotic bodies and exosomes are the most characterized vesicles. They can be distinguished by their size, morphology, origin and molecular composition. To date, increasing studies demonstrate that EVs mediate intercellular communication. EVs reach considerable interest in the scientific community due to their role in diverse processes including antigen-presentation, stimulation of anti-tumoral immune responses, tolerogenic or inflammatory effects. In pathogens, EV shedding is well described in fungi, bacteria, protozoan and helminths parasites. For Trypanosoma cruzi EV liberation and protein composition was previously described. Dendritic cells (DCs), among other cells, are key players promoting the immune response against pathogens and also maintaining self-tolerance. In previous reports we have demonstrate that T. cruzi downregulates DCs immunogenicity in vitro and in vivo. Here we analyze EVs from the in vitro interaction between blood circulating trypomastigotes (Tp) and bone-marrow-derived DCs. We found that Tp incremented the number and the size of EVs in cultures with DCs. EVs displayed some exosome markers and intracellular RNA. Protein analysis demonstrated that the parasite changes the DC protein-EV profile. We observed that EVs from the interaction of Tp-DCs were easily captured by unstimulated-DCs in comparison with EVs from DCs cultured without the parasite, and also modified the activation status of LPS-stimulated DCs. Noteworthy, we found protection in animals treated with EVs-DCs+Tp and challenged with T. cruzi lethal infection. Our goal is to go deep into the molecular characterization of EVs from the DCs-Tp interaction, in order to identify mediators for therapeutic purposes.

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.

The potential for histone deacetylase (HDAC) inhibitors as cestocidal drugs.

BACKGROUND: Echinococcosis and cysticercosis are neglected tropical diseases caused by cestode parasites (family Taeniidae). Not only there is a small number of approved anthelmintics for the treatment of these cestodiases, but also some of them are not highly effective against larval stages, such that identifying novel drug targets and their associated compounds is critical. Histone deacetylase (HDAC) enzymes are validated drug targets in cancers and other diseases, and have been gaining relevance for developing new potential anti-parasitic treatments in the last years. Here, we present the anthelmintic profile for a panel of recently developed HDAC inhibitors against the model cestode Mesocestoides vogae (syn. M. corti). METHODOLOGY/PRINCIPAL FINDINGS: Phenotypic screening was performed on M. vogae by motility measurements and optical microscopic observations. Some HDAC inhibitors showed potent anthelmintic activities; three of them -entinostat, TH65, and TH92- had pronounced anthelmintic effects, reducing parasite viability by ~100% at concentrations of ≤ 20 µM. These compounds were selected for further characterization and showed anthelmintic effects in the micromolar range and in a time- and dose-dependent manner. Moreover, these compounds induced major alterations on the morphology and ultrastructural features of M. vogae. The potencies of these compounds were higher than albendazole and the anthelmintic effects were irreversible. Additionally, we evaluated pairwise drug combinations of these HDAC inhibitors and albendazole. The results suggested a positive interaction in the anthelmintic effect for individual pairs of compounds. Due to the maximum dose approved for entinostat, adjustments in the dose regime and/or combinations with currently-used anthelmintic drugs are needed, and the selectivity of TH65 and TH92 towards parasite targets should be assessed. CONCLUSION, SIGNIFICANCE: The results presented here suggest that HDAC inhibitors represent novel and potent drug candidates against cestodes and pave the way to understanding the roles of HDACs in these parasites.

Extracellular non-coding RNA signatures of the metacestode stage of Echinococcus multilocularis.

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.

Development of a copro-LAMP assay for detection of several species of Echinococcus granulosus sensu lato complex.

Cystic echinococcosis represents a significant problem in human and animal health and constitutes one of the most severe Neglected Tropical Diseases prioritized by the World Health Organization. The etiological agent is the complex Echinococcus granulosus sensu lato (s. l.), composed of several species/genotypes. Diagnosis in the definitive host and molecular epidemiology studies are important points for cystic echinococcosis control. Here we developed a new copro-LAMP assay, LAMP EGSL, for diagnosis in the definitive host for simultaneous detection of Echinococcus granulosus sensu stricto (s. s.), Echinococcus ortleppi, and Echinococcus canadensis species. Also, the analytical sensitivity, specificity and plausibility of performance in a rural context of a previously reported species-specific LAMP reaction, was evaluated. Both reactions showed high analytical sensitivity values (10 fg-100 fg DNA) and did not show cross reaction with DNA from host or other helminthic parasites. LAMP EGSL was performed with samples from an endemic area. In addition, the alkaline hydrolysis of one E. granulosus s. s. adult parasite followed by specific LAMP to E. granulosus s. s. was performed in a laboratory with low resources from another cystic echinococcosis endemic area. The results obtained suggest that LAMP EGSL represents a potential tool for canine diagnosis that could be useful for cystic echinococcosis control programs. In addition, we showed that LAMP reaction for E. granulous s. s., E. ortleppi and E. canadensis specific detection, could be useful for molecular epidemiology studies applicable to the definitive host. Both reactions were performed in endemic, rural areas without sophisticated equipment.

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.

Revisiting the Phylogenetic History of Helminths Through Genomics, the Case of the New Echinococcus oligarthrus Genome.

The first parasitic helminth genome sequence was published in 2007; since then, only ∼200 genomes have become available, most of them being draft assemblies. Nevertheless, despite the medical and economical global impact of helminthic infections, parasite genomes in public databases are underrepresented. Recently, through an integrative approach involving morphological, genetic, and ecological aspects, we have demonstrated that the complete life cycle of Echinococcus oligarthrus (Cestoda: Taeniidae) is present in South America. The neotropical E. oligarthrus parasite is capable of developing in any felid species and producing human infections. Neotropical echinococcosis is poorly understood yet and requires a complex medical examination to provide the appropriate intervention. Only a few cases of echinococcosis have been unequivocally identified and reported as a consequence of E. oligarthrus infections. Regarding phylogenetics, the analyses of mitogenomes and nuclear datasets have resulted in discordant topologies, and there is no unequivocal taxonomic classification of Echinococcus species so far. In this work, we sequenced and assembled the genome of E. oligarthrus that was isolated from agoutis (Dasyprocta azarae) naturally infected and performed the first comparative genomic study of a neotropical Echinococcus species. The E. oligarthrus genome assembly consisted of 86.22 Mb which showed ∼90% identity and 76.3% coverage with Echinococcus multilocularis and contained the 85.0% of the total expected genes. Genetic variants analysis of whole genome revealed a higher rate of intraspecific genetic variability (23,301 SNPs; 0.22 SNPs/kb) rather than for the genomes of E. multilocularis and Echinococcus canadensis G7 but lower with respect to Echinococcus granulosus G1. Comparative genomics against E. multilocularis, E. granulosus G1, and E. canadensis G7 revealed 38,762, 125,147, and 170,049 homozygous polymorphic sites, respectively, indicating a higher genetic distance between E. oligarthrus and E. granulosus sensu lato species. The SNP distribution in chromosomes revealed a higher SNP density in the longest chromosomes. Phylogenetic analysis using whole-genome SNPs demonstrated that E. oligarthrus is one of the basal species of the genus Echinococcus and is phylogenetically closer to E. multilocularis. This work sheds light on the Echinococcus phylogeny and settles the basis to study sylvatic Echinococcus species and their developmental evolutionary features.

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.

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.

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.

High-throughput characterization of Echinococcus spp. metacestode miRNomes.

Echinococcosis is a worldwide zoonosis of great public health concern, considered a neglected disease by the World Health Organisation. The cestode parasites Echinococcus granulosus sensu lato (s. l.) and Echinococcus multilocularis are the main aetiological agents. In the intermediate host, these parasites display particular developmental traits that lead to different patterns of disease progression. In an attempt to understand the causes of these differences, we focused on the analysis of microRNAs (miRNAs), small non-coding regulatory RNAs with major roles in development of animals and plants. In this work, we analysed the small RNA expression pattern of the metacestode, the stage of sanitary relevance, and provide a detailed description of Echinococcus miRNAs. Using high-throughput small RNA sequencing, we believe that we have carried out the first experimental identification of miRNAs in E. multilocularis and have expanded the Echinococcus miRNA catalogue to 38 miRNA genes, including one miRNA only present in E. granulosus s. l. Our findings show that although both species share the top five highest expressed miRNAs, 13 are differentially expressed, which could be related to developmental differences. We also provide evidence that uridylation is the main miRNA processing mechanism in Echinococcus spp. These results provide detailed information on Echinococcus miRNAs, which is the first step in understanding their role in parasite biology and disease establishment and/or progression, and their future potential use as drug or diagnostic targets.

Echinococcus granulosus antigen B gene family: further studies of strain polymorphism at the genomic and transcriptional levels.

Echinococcus granulosus AgB gene family is constituted by five gene loci. In a previous study, we analyzed the strain variation of EgAgB2/B4 sequences. Here, we have analyzed, by SSCP and sequencing, 250 genomic clones of EgAgB1/B3/B5 gene cluster from five E. granulosus strains. Several new EgAgB genomic variants were found. EgAgB1 and EgAgB3 genomic sequences grouped E. granulosus strains by phylogenetic tools in two clusters: one formed by G1/G2 and the other by G5, G6/G7 strains, in accordance with other molecular markers. EgAgB5 genomic and cDNA sequences were only found in G1/G2 cluster. Reverse transcription-PCR analysis using subunit specific primers revealed that all the EgAgB genes were transcribed in G1 and G7 strains with the exception of EgAgB5 transcripts that were not detected in G7 strain. Interestingly, AgB2 transcripts that were probably processed by an aberrant splicing mechanism leading to a non-functional B2 protein were found in G7 strain.