Transcriptomic Features of Echinococcus granulosus Protoscolex during the Encystation Process.
ABSTRACT: Cystic echinococcosis (CE) is a zoonotic infection caused by Echinococcus granulosus larvae. It seriously affects the development of animal husbandry and endangers human health. Due to a poor understanding of the cystic fluid formation pathway, there is currently a lack of innovative methods for the prevention and treatment of CE. In this study, the protoscoleces (PSCs) in the encystation process were analyzed by high-throughput RNA sequencing. A total of 32,401 transcripts and 14,903 cDNAs revealed numbers of new genes and transcripts, stage-specific genes, and differently expressed genes. Genes encoding proteins involved in signaling pathways, such as putative G-protein coupled receptor, tyrosine kinases, and serine/threonine protein kinase, were predominantly up-regulated during the encystation process. Antioxidant enzymes included cytochrome c oxidase, thioredoxin glutathione, and glutathione peroxidase were a high expression level. Intriguingly, KEGG enrichment suggested that differentially up-regulated genes involved in the vasopressin-regulated water reabsorption metabolic pathway may play important roles in the transport of proteins, carbohydrates, and other substances. These results provide valuable information on the mechanism of cystic fluid production during the encystation process, and provide a basis for further studies on the molecular mechanisms of growth and development of PSCs.
Project description:Background:Cystic echinococcosis is a life-threatening disease caused by the larval stages of the dog tapeworm Echinococcus granulosus. Protoscoleces (PSCs) of this worm have the ability of bi-directional development to either larval cysts or strobilar adult worms. However, the molecular mechanisms underlying this development process are unknown. Results:RNA and small RNAs sequencing was employed to characterize the gene and miRNA expression at 0-24 h and 7-14 days in the bi-directional development of PSCs. A total of 963 genes and 31 miRNAs were differentially expressed in the early development of PSCs to adult worms whereas 972 genes and 27 miRNAs were differentially expressed in the early development of PSCs to cysts. Pairwise comparison between the two developmental patterns showed that 172 genes and 15 miRNAs were differentially expressed at three time-points. Most of these genes were temporally changed at 24 h or 7 days. GO enrichment analysis revealed that the differentially expressed genes in early adult worm development are associated with nervous system development and carbohydrate metabolic process; whereas, the differentially expressed genes in early cystic development are associated with transmembrane transporter activity and nucleoside triphosphatase activity. In addition, miR-71 and miR-219 regulated genes are likely involved in oxidation reduction in adult worm development. Conclusion:The early stages of bi-directional development in E. granulosus PSCs are controlled by miRNAs and genes likely associated with nervous system development and carbohydrate metabolic process. ATP-dependent transporter genes are associated with cystic development. These results may be important for exploring the mechanisms underlying early development in E. granulosus providing novel information that can be used to discover new therapeutics for controlling cystic echinococcosis.
Project description:Entamoeba histolytica is an intestinal parasite that infects 50-100 million people and causes up to 55,000 deaths annually. The transmissive form of E. histolytica is the cyst, with a single infected individual passing up to 45 million cysts per day, making cyst production an attractive target for infection control. Lectins and chitin are secreted to form the cyst wall, although little is known about the underlying membrane trafficking processes supporting encystation. As E. histolytica does not readily form cysts in vitro, we assessed membrane trafficking gene expression during encystation in the closely related model Entamoeba invadens. Genes involved in secretion are up-regulated during cyst formation, as are some trans-Golgi network-to-endosome trafficking genes. Furthermore, endocytic and general trafficking genes are up-regulated in the mature cyst, potentially preserved as mRNA in preparation for excystation. Two divergent dynamin-related proteins found in Entamoeba are predominantly expressed during cyst formation. Phylogenetic analyses indicate that they are paralogous to, but quite distinct from, classical dynamins found in human, suggesting that they may be potential drug targets to block encystation. The membrane-trafficking machinery is clearly regulated during encystation, providing an additional facet to understanding this crucial parasitic process.
Project description:Encystation is an essential differentiation process for the completion of the life cycle of a group of intestinal protozoa including Entamoeba histolytica, the causative agent of intestinal and extraintestinal amebiasis. However, regulation of gene expression during encystation is poorly understood. To comprehensively understand the process at the molecular level, the transcriptomic profiles of E. invadens, which is a related reptilian species that causes an invasive disease similar to that of E. histolytica, was investigated during encystation. Using a custom-generated Affymetrix platform microarray, we performed time course (0.5, 2, 8, 24, 48, and 120 h) gene expression analysis of encysting E. invadens. ANOVA analysis revealed that a total of 1,528 genes showed ?3 fold up-regulation at one or more time points, relative to the trophozoite stage. Of these modulated genes, 8% (116 genes) were up-regulated at the early time points (0.5, 2 and 8h), while 63% (962 genes) were up-regulated at the later time points (24, 48, and 120 h). Twenty nine percent (450 genes) are either up-regulated at 2 to 5 time points or constitutively up-regulated in both early and late stages. Among the up-regulated genes are the genes encoding transporters, cytoskeletal proteins, proteins involved in vesicular trafficking (small GTPases), Myb transcription factors, cysteine proteases, components of the proteasome, and enzymes for chitin biosynthesis. This study represents the first kinetic analysis of gene expression during differentiation from the invasive trophozoite to the dormant, infective cyst stage in Entamoeba. Functional analysis on individual genes and their encoded products that are modulated during encystation may lead to the discovery of targets for the development of new chemotherapeutics that interfere with stage conversion of the parasite.
Project description:Differentiation into infectious cysts through the process of encystation is crucial for transmission and survival of the intestinal protozoan parasite Giardia intestinalis. Hitherto the majority of studies have focused on the early events, leaving late encystation poorly defined. In order to further study encystation, focusing on the later events, we developed a new encystation protocol that generates a higher yield of mature cysts compared to standard methods. Transcriptome changes during the entire differentiation from trophozoites to cysts were thereafter studied using RNA sequencing (RNA-seq). A high level of periodicity was observed for up- and down-regulated genes, both at the level of the entire transcriptome and putative regulators. This suggests the trajectory of differentiation to be coordinated through developmentally linked gene regulatory activities. Our study identifies a core of 13 genes that are consistently up-regulated during initial encystation. Of these, two constitute previously uncharacterized proteins that we were able to localize to a new type of encystation-specific vesicles. Interestingly, the largest transcriptional changes were seen in the late phase of encystation with the majority of the highly up-regulated genes encoding hypothetical proteins. Several of these were epitope-tagged and localized to further characterize these previously unknown genetic components of encystation and possibly excystation. Finally, we also detected a switch of variant specific surface proteins (VSPs) in the late phase of encystation. This occurred at the same time as nuclear division and DNA replication, suggesting a potential link between the processes.
Project description:In this study we analyzed how gene expression is regulated through the process of encystation in Giardia Overall design: Examination of differential gene expression during the encystation process of Giardia intestinalis
Project description:Cystic echinococcosis (CE) is a cosmopolitan parasitic disease caused by infection with the larval stage of Echinococcus granulosus sensu lato. Thioredoxin peroxidase (TPx) may play an essential role in the antioxidant defence system of E. granulosus s.l. as neither catalase nor glutathione peroxidase activities have been detected in the parasite. However, it is not known whether TPx affects the survival and growth of E. granulosus s.l. during development. In this study, three fragments of siRNA specific for EgTPx (siRNA-1/2/3) were designed and transfected into protoscoleces of E. granulosus sensu stricto by electroporation. Quantitative real-time PCR and Western blotting analysis showed that siRNA-3 significantly reduced the expression of EgTPx. Coincidentally, knockdown of EgTPx expression in protoscoleces with siRNA-3 significantly reduced the viability of the parasite under oxidative stress induced by 0.6 mM H2O2. In vitro culture studies showed that protoscoleces treated with siRNA-3 reduced pre-microcyst formation. In vivo experiments showed that injecting mice intraperitoneally with protoscoleces treated with siRNA-3 resulted in a significant reduction in the number, size and weight of CE cysts compared with those of control animals. Silencing of EgTPx led to the impairment of growth of E. granulosus s.s. both in vitro and in vivo, indicating that EgTPx is an important factor for protoscoleces survival and plays an important role in the antioxidant defence against the host during development.
Project description:Giardia lamblia is a protozoan parasite and the earliest branching clade of eukaryota. The Giardia life cycle alternates between an asexually replicating vegetative form and an infectious cyst form. Encystation and excystation are crucial processes for the survival and transmission of Giardia. Cysteine proteases in Giardia have been implicated in proteolytic processing events that enable the continuance of the life cycle throughout encystation and excystation. Using quantitative real-time PCR, the expression of twenty-seven clan CA cysteine protease genes in the Giardia genome was measured during both vegetative growth and encystation. Giardia cysteine protease 2 was the most highly expressed cysteine protease during both life cycle stages measured, with a dramatic expression increase during encystation. The mRNA transcript for Giardia cysteine protease 2 was 7-fold up-regulated during encystation and was greater than 3-fold higher than any other Giardia protease gene product. Recombinant Giardia cysteine protease 2 was expressed, purified, and biochemically characterized. The activity of the recombinant cysteine protease 2 protein was confirmed to be identical to the dominant cysteine protease activity found in G. lamblia lysates. Giardia cysteine protease 2 was co-localized with cyst wall protein in encystation-specific vesicles during encystation and processed cyst wall protein 2 to the size found in Giardia cyst walls. These data suggest that Giardia cysteine protease 2 is not only the major cysteine endoprotease expressed in Giardia, but is also central to the encystation process.
Project description:Several eukaryotic parasites form cysts that transmit infection. The process is found in diverse organisms such as Toxoplasma, Giardia, and nematodes. In Entamoeba histolytica this process cannot be induced in vitro, making it difficult to study. In Entamoeba invadens, stage conversion can be induced, but its utility as a model system to study developmental biology has been limited by a lack of genomic resources. We carried out genome and transcriptome sequencing of E. invadens to identify molecular processes involved in stage conversion.We report the sequencing and assembly of the E. invadens genome and use whole transcriptome sequencing to characterize changes in gene expression during encystation and excystation. The E. invadens genome is larger than that of E. histolytica, apparently largely due to expansion of intergenic regions; overall gene number and the machinery for gene regulation are conserved between the species. Over half the genes are regulated during the switch between morphological forms and a key signaling molecule, phospholipase D, appears to regulate encystation. We provide evidence for the occurrence of meiosis during encystation, suggesting that stage conversion may play a key role in recombination between strains.Our analysis demonstrates that a number of core processes are common to encystation between distantly related parasites, including meiosis, lipid signaling and RNA modification. These data provide a foundation for understanding the developmental cascade in the important human pathogen E. histolytica and highlight conserved processes more widely relevant in enteric pathogens.
Project description:Giardia lamblia is an intestinal protozoan parasite required to survive in the environment in order to be transmitted to a new host. To ensure parasite survival, flagellated trophozoites colonizing the small intestine differentiate into non-motile environmentally-resistant cysts which are then shed in the environment. This cell differentiation process called encystation is characterized by significant morphological remodeling which includes secretion of large amounts of cyst wall material. Although much is known about the transcriptional regulation of encystation and the synthesis and trafficking of cyst wall material, the investigation of global changes in protein content and abundance during G. lamblia encystation is still unaddressed. In this study, we report on the quantitative analysis of the G. lamblia proteome during encystation using tandem mass spectrometry. Quantification of more than 1000 proteins revealed major changes in protein abundance in early, mid and late encystation, notably in constitutive secretory protein trafficking. Early stages of encystation were marked by a striking decrease of endoplasmic reticulum-targeted variant-specific surface proteins and significant increases in cytoskeleton regulatory components, NEK protein kinases and proteins involved in protein folding and glycolysis. This was in stark contrast to cells in the later stages of encystation which presented a surprisingly similar proteome composition to non-encysting trophozoites. Altogether these data constitute the first quantitative atlas of the Giardia proteome covering the whole process of encystation and point towards an important role for post-transcriptional control of gene expression in Giardia differentiation. Furthermore, our data provide a valuable resource for the community-based annotation effort of the G. lamblia genome, where almost 70% of all predicted gene models remains "hypothetical".
Project description:Encystation is an essential process for Acanthamoeba survival under nutrient-limiting conditions and exposure to drugs. The expression of several genes has been observed to increase or decrease during encystation. Epigenetic processes involved in regulation of gene expression have been shown to play a role in several pathogenic parasites. In the present study, we identified the protein arginine methyltransferase 5 (PRMT5), a known epigenetic regulator, in Acanthamoeba castellanii. PRMT5 of A. castellanii (AcPRMT5) contained domains found in S-adenosylmethionine-dependent methyltransferases and in PRMT5 arginine-N-methyltransferase. Expression levels of AcPRMT5 were increased during encystation of A. castellanii. The EGFP-PRMT5 fusion protein was mainly localized in the nucleus of trophozoites. A. castellanii transfected with siRNA designed against AcPRMT5 failed to form mature cysts. The findings of this study lead to a better understanding of epigenetic mechanisms behind the regulation of encystation in cyst-forming pathogenic protozoa.