title: Destabilization domain approach adapted for regulated protein expression in the protozoan parasite Entamoeba histolytica.
abstract: A plethora of information has been gained by sequencing the genome of the human parasite Entamoeba histolytica, however a lack of robust genetic tools hampers experimental elucidation of gene functions. We adapted the destabilization domain approach for modulation of protein levels in E. histolytica using the destabilization domains of FK506 binding protein (ddFKBP) and dihydrofolate reductase (ddDHFR), respectively. In our studies, the ddFKBP appears to be more tightly regulated than ddDHFR, with minimal detectable protein in trophozoites in the absence of the stabilizing compound. The on- and off-rate kinetics for ddFKBP were rapid, with stabilization and degradation within 3h of addition or removal of stabilizing compound, respectively. The kinetics for ddDHFR was different, with rapid stabilization (within 3h of stabilizing compound being added) but much slower degradation (protein not destabilized until 24h after compound removal). Furthermore, we demonstrated that for the ddFKBP, the standard stabilizing compound Shield-1 could be effectively replaced by two cheaper alternatives (rapamycin and FK506), indicating that the more cost-effective alternatives are viable options for use with E. histolytica. Thus, the destabilization domain approach represents a powerful method to study protein functions in E. histolytica and adds to the catalog of genetic tools that could be used to study this important human pathogen.
authors.affiliation: Division of Infectious Diseases, Department of Internal Medicine, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA.
authors.forename: Yuk-Chien
authors.initials: YC
authors.lastname: Liu
authors.affiliation: Division of Infectious Diseases, Department of Internal Medicine, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address: usingh@stanford.edu.
authors.forename: Upinder
authors.initials: U
authors.lastname: Singh
mesh_terms: D004748:Entamoeba histolytica / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000502:physiology*; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D015800:Protozoan Proteins
keywords: Amebiasis; Destabilized protein; Enteric pathogen; Inducible expression; Protein degradation