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.

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.

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.

First genetic characterization of human cystic echinococcosis in Uruguay.

Cystic echinococcosis is endemic and hyperendemic in Uruguay. The objective of this study was to determine the species and genotype of Echinococcus granulosus sensu lato in symptomatic patients with cystic echinococcosis who underwent surgery, together with the location and stage of the cysts. The study included 13 patients aged between 6 and 57 years old. Samples of cysts from these cases were analyzed using DNA extraction, polymerase chain reaction amplification and sequencing. The results revealed the presence of E. granulosus sensu stricto in all cases, with 12 samples belonging to the G1 genotype and 1 to the G3 genotype, suggesting that disease persistence might be related to the dog-sheep cycle. However, other intermediate hosts, such as cattle, could also be involved. Cysts were most frequently found in the liver, followed by muscle and other sites (e.g. pulmonary, vertebral, pelvic and cardiac); and stage CE1 was most frequently found, followed by CE2 and CE3b. Three cases occurred in children or adolescents, suggesting an active parasite cycle in at least some areas of the country. Since there is considerable diversity of E. granulosus sensu lato species and genotypes in South America, it is important to continue the present study in order to draw stronger epidemiological conclusions.

La equinococosis quística es endémica e hiperendémica en Uruguay. El objetivo de este estudio fue determinar la especie y el genotipo de Echinococcus granulosus sensu lato en pacientes sintomáticos con equinococosis quística que fueron sometidos a cirugía, además de la localización y estadio de los quistes. En el estudio participaron 13 pacientes de entre 6 y 57 años. Las muestras de quistes de estos casos se analizaron mediante la extracción de ADN, la amplificación de la reacción en cadena de la polimerasa y la secuenciación. Los resultados revelaron la presencia de E. granulosus sensu stricto en todos los casos, con 12 muestras pertenecientes al genotipo G1 y 1 muestra de genotipo G3, lo que sugiere que la persistencia de la enfermedad podría estar relacionada con el ciclo perro/ovinos. Sin embargo, otros huéspedes intermedios, como el ganado, también podrían estar involucrados. Los quistes se encontraron con mayor frecuencia en el hígado, seguido por los músculos y otros sitios (por ejemplo, quistes pulmonares, vertebrales, pélvicos y cardíacos); y el estadio más frecuente fue CE1, seguido por CE2 y CE3b. Tres de los casos ocurrieron en niños o adolescentes, lo que sugiere un ciclo activo de parásitos al menos en algunas áreas del país. Dado que existe una considerable diversidad de especies y genotipos de E. granulosus sensu lato en América del Sur, es importante continuar con este estudio para extraer conclusiones epidemiológicas más sólidas.

A equinococose cística é endêmica e hiperendêmica no Uruguai. O objetivo do estudo foi determinar a espécie e o genótipo do Echinococcus granulosus sensu lato em pacientes sintomáticos com equinococose cística que foram submetidos a cirurgia, juntamente com a localização e o estágio dos cistos. O estudo incluiu 13 pacientes entre 6 e 57 anos de idade. As amostras de cistos foram analisadas utilizando extração do DNA, amplificação pela reação em cadeia da polimerase e sequenciamento. Os resultados revelaram a presença de E. granulosus sensu stricto em todos os casos, com 12 amostras pertencentes ao genótipo G1 e uma ao genótipo G3, sugerindo que a persistência da doença pode estar relacionada ao ciclo cão-ovelha. Entretanto, outros hospedeiros intermediários como gado também poderiam estar envolvidos. Os cistos foram mais frequentemente encontrados no fígado, seguido por músculos e outros locais (por exemplo, pulmão, vértebras, pelve e coração), e o estágio mais frequentemente encontrado foi o CE1, seguido por CE2 e CE3b. Três casos ocorreram em crianças ou adolescentes, o que sugere um ciclo parasitário ativo em pelo menos algumas áreas do país. Devido à considerável diversidade de espécies e genótipos de E. granulosus sensu lato na América do Sul, é importante dar continuidade do presente estudo a fim de se chegar a conclusões epidemiológicas mais sólidas.

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.

Fatty acid-binding proteins in Echinococcus spp.: the family has grown.

Fatty acid-binding proteins (FABPs) are small intracellular proteins that reversibly bind fatty acids and other hydrophobic ligands. In cestodes, due to their inability to synthesise fatty acids de novo, FABPs have been proposed as essential proteins, and thus, as possible drug targets and/or carriers against these parasites. We performed data mining in Echinococcus multilocularis and Echinococcus granulosus genomes in order to test whether this family of proteins is more complex than previously reported. By exploring the genomes of E. multilocularis and E. granulosus, six genes coding for FABPs were found in each organism. In the case of E. granulosus, all of them have different coding sequences, whereas in E. multilocularis, two of the genes code for the same protein. Remarkably, one of the genes (in both cestodes) encodes a FABP with a C-terminal extension unusual for this family of proteins. The newly described genes present variations in their structure in comparison with previously described FABP genes in Echinococcus spp. The coding sequences for E. multilocularis were validated by cloning and sequencing. Moreover, differential expression patterns of FABPs were observed at different stages of the life cycle of E. multilocularis by exploring transcriptomic data from several sources. In summary, FABP family in cestodes is far more complex than previously thought and includes new members that seem to be only present in flatworms.

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.

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.

[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.

Circulating Small RNA Profiling of Patients with Alveolar and Cystic Echinococcosis.

Alveolar (AE) and cystic (CE) echinococcosis are two parasitic diseases caused by the tapeworms Echinococcus multilocularis and E. granulosus sensu lato (s. l.), respectively. Currently, AE and CE are mainly diagnosed by means of imaging techniques, serology, and clinical and epidemiological data. However, no viability markers that indicate parasite state during infection are available. Extracellular small RNAs (sRNAs) are short non-coding RNAs that can be secreted by cells through association with extracellular vesicles, proteins, or lipoproteins. Circulating sRNAs can show altered expression in pathological states; hence, they are intensively studied as biomarkers for several diseases. Here, we profiled the sRNA transcriptomes of AE and CE patients to identify novel biomarkers to aid in medical decisions when current diagnostic procedures are inconclusive. For this, endogenous and parasitic sRNAs were analyzed by sRNA sequencing in serum from disease negative, positive, and treated patients and patients harboring a non-parasitic lesion. Consequently, 20 differentially expressed sRNAs associated with AE, CE, and/or non-parasitic lesion were identified. Our results represent an in-depth characterization of the effect E. multilocularis and E. granulosus s. l. exert on the extracellular sRNA landscape in human infections and provide a set of novel candidate biomarkers for both AE and CE detection.

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.

Characterization of a new type of neuronal 5-HT G- protein coupled receptor in the cestode nervous system.

Cestodes are platyhelminth parasites with a wide range of hosts that cause neglected diseases. Neurotransmitter signaling is of critical importance for these parasites which lack circulatory, respiratory and digestive systems. For example, serotonin (5-HT) and serotonergic G-protein coupled receptors (5-HT GPCRs) play major roles in cestode motility, development and reproduction. In previous work, we deorphanized a group of 5-HT7 type GPCRs from cestodes. However, little is known about another type of 5-HT GPCR, the 5-HT1 clade, which has been studied in several invertebrate phyla but not in platyhelminthes. Three putative 5-HT GPCRs from Echinococcus canadensis, Mesocestoides vogae (syn. M. corti) and Hymenolepis microstoma were cloned, sequenced and bioinformatically analyzed. Evidence grouped these new sequences within the 5-HT1 clade of GPCRs but differences in highly conserved GPCR motifs were observed. Transcriptomic analysis, heterologous expression and immunolocalization studies were performed to characterize the E. canadensis receptor, called Eca-5-HT1a. Functional heterologous expression studies showed that Eca-5-HT1a is highly specific for serotonin. 5-Methoxytryptamine and α-methylserotonin, both known 5-HT GPCR agonists, give stimulatory responses whereas methysergide, a known 5-HT GPCR ligand, give an antagonist response in Eca-5-HT1a. Mutants obtained by the substitution of key predicted residues resulted in severe impairment of receptor activity, confirming that indeed, these residues have important roles in receptor function. Immunolocalization studies on the protoscolex stage from E. canadensis, showed that Eca-5-HT1a is localized in branched fibers which correspond to the nervous system of the parasite. The patterns of immunoreactive fibers for Eca-5-HT1a and for serotonin were intimately intertwined but not identical, suggesting that they are two separate groups of fibers. These data provide the first functional, pharmacological and localization report of a serotonergic receptor that putatively belongs to the 5-HT1 type of GPCRs in cestodes. The serotonergic GPCR characterized here may represent a new target for antiparasitic intervention.

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.