title: A highly sensitive FRET-based approach reveals secretion of the actin-binding protein toxofilin during Toxoplasma gondii infection.
abstract: We have utilized a highly sensitive approach based on fluorescence resonance energy transfer (FRET) and beta-lactamase (BLA), which we adapted for the detection of Toxoplasma gondii secreted proteins. This assay revealed that the actin-binding protein toxofilin appears to be secreted into host cells during invasion. To determine the function of toxofilin during infection, we engineered a type I (RH strain) parasite with a targeted deletion of the toxofilin gene and compared the phenotypes of control and toxofilin knockout (Deltatxf) parasites in several in vitro assays, including invasion, growth, gliding motility, and egress of the Deltatxf parasites, as well as F-actin staining, phagocytosis and migration of cells infected with Deltatxf parasites or wild-type controls. Despite its apparent secretion into host cells and its ability to bind to and modulate host actin, we observed that toxofilin does not appear to play a role in these processes, under the conditions we examined, and we report these findings here.
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, CA 94305, USA.
authors.forename: Melissa B
authors.initials: MB
authors.lastname: Lodoen
authors.forename: Christiane
authors.initials: C
authors.lastname: Gerke
authors.forename: John C
authors.initials: JC
authors.lastname: Boothroyd
mesh_terms: D016475:3T3 Cells; D051344:Actin Capping Proteins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005455:Fluorescent Antibody Technique; D006801:Humans; D051379:Mice; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014122:Toxoplasma / Q000378:metabolism*; D014123:Toxoplasmosis / Q000378:metabolism* / Q000469:parasitology; D001618:beta-Lactamases / Q000378:metabolism
chemical_list: D051344:Actin Capping Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C406004:toxofilin protein, Toxoplasma gondii; D001618:beta-Lactamases