title: Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens.
abstract: Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach inE. invadens We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5' or 3' end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes inE. invadens Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system.
authors.affiliation: Division of Infectious Diseases, Department of Internal Medicine, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Susmitha
authors.initials: S
authors.lastname: Suresh
authors.affiliation: Division of Infectious Diseases, Department of Internal Medicine, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Gretchen
authors.initials: G
authors.lastname: Ehrenkaufer
authors.affiliation: Division of Infectious Diseases, Department of Internal Medicine, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Hanbang
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Division of Infectious Diseases, Department of Internal Medicine, Stanford University School of Medicine, Stanford, California, USA usingh@stanford.edu.|Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Upinder
authors.initials: U
authors.lastname: Singh
mesh_terms: D003001:Cloning, Molecular; D004747:Entamoeba / Q000235:genetics / Q000502:physiology*; D008018:Life Cycle Stages / Q000502:physiology; D018384:Oxidative Stress / Q000502:physiology; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D016053:RNA, Protozoan / Q000378:metabolism*
chemical_list: D015800:Protozoan Proteins; D016053:RNA, Protozoan