Project description:RNA-seq was performed on encysting and excysting E. invadens, a parasite of reptiles that is used as a model system to study stage conversion in Entamoeba species, including the human pathogen E. histolytica. The goal of the project was to identify changes to the transcriptome during development, in order to better understand the mechanisms of development.
Project description:RNA-seq was performed on encysting and excysting E. invadens, a parasite of reptiles that is used as a model system to study stage conversion in Entamoeba species, including the human pathogen E. histolytica. The goal of the project was to identify changes to the transcriptome during development, in order to better understand the mechanisms of development. RNA was isolated from trophozoites, from encysting parasites (at 8, 24, 48 and 72 hours after transfer to encystation media) and from excysting parasites (2 and 8 hours after induction of excystation), converted to cDNA, and sequenced on a SOLiD platform. RNA-seq data was used to refine gene models, find potential unannotated genes, and identify genes that are developmentally regulated.
Project description:The developmental life cycle of the enteric parasite Entamoeba invadens: transcriptome analysis reveals a crucial role for phospholipase D in stage conversion
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:Although the cyst wall of Entamoeba invadens contains chitin, synthesis of this structural polymer during encystation has not been described before. Here we report that conditions which stimulate encystation of the parasite lead to increased chitin synthase (ChS) activity, measured by incorporation of [3H]GlcNAc ([3H]N-acetylglucosamine) from UDP-GlcNAc. The radiolabelled product was precipitable by trichloroacetic acid or ethanol and identified as chitin because it was digested by purified chitinase to radioactive chitobiose and GlcNAc. Cell fractionation indicated that approx. 60% of the enzyme is in the high-speed supernatant. pH-activity profiles showed that soluble ChS has an optimum at 6.0, whereas particulate ChS has a peak at pH 7.0-7.5. Both the activities were dependent on bivalent metal ions, especially Mn2+ and Mn2+ plus Co2+. In contrast with the ChS of other organisms, neither the particulate nor the soluble ChS of E. invadens was activated by trypsin treatment. Soluble and particulate ChS were also stimulated by digitonin and phosphatidylserine, whereas phosphatidylethanolamine stimulated only the soluble ChS. The enzyme activities were inhibited by UDP, UDP-glucose and UDP-GalNAc, but not by the analogues Polyoxin-D or Nikkomycin. This is the first report of an enzyme which is developmentally regulated during encystation of the primitive eukaryotic genus Entamoeba.
Project description:In this study, we used microarray analysis to determine genes involved in encystation. The expression profile more than 9,700 genes during encystation was compared.