title: Chemical genetic screen identifies Toxoplasma DJ-1 as a regulator of parasite secretion, attachment, and invasion.
abstract: Toxoplasma gondii is a member of the phylum Apicomplexa that includes several important human pathogens, such as Cryptosporidium and Plasmodium falciparum, the causative agent of human malaria. It is an obligate intracellular parasite that can cause severe disease in congenitally infected neonates and immunocompromised individuals. Despite the importance of attachment and invasion to the success of the parasite, little is known about the underlying mechanisms that drive these processes. Here we describe a screen to identify small molecules that block the process of host cell invasion by the T. gondii parasite. We identified a small molecule that specifically and irreversibly blocks parasite attachment and subsequent invasion of host cells. Using tandem orthogonal proteolysis-activity-based protein profiling, we determined that this compound covalently modifies a single cysteine residue in a poorly characterized protein homologous to the human protein DJ-1. Mutation of this key cysteine residue in the native gene sequence resulted in parasites that were resistant to inhibition of host cell attachment and invasion by the compound. Further analysis of the invasion phenotype confirmed that modification of Cys127 on TgDJ-1 resulted in a block of microneme secretion and motility, even in the presence of direct stimulators of calcium release. Together, our results suggest that TgDJ-1 plays an important role that is likely downstream of the calcium flux required for microneme secretion, parasite motility, and subsequent invasion of host cells.
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: Carolyn I
authors.initials: CI
authors.lastname: Hall
authors.forename: Michael L
authors.initials: ML
authors.lastname: Reese
authors.forename: Eranthie
authors.initials: E
authors.lastname: Weerapana
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Child
authors.forename: Paul W
authors.initials: PW
authors.lastname: Bowyer
authors.forename: Victoria E
authors.initials: VE
authors.lastname: Albrow
authors.forename: Jeralyn D
authors.initials: JD
authors.lastname: Haraldsen
authors.forename: MacDonald R
authors.initials: MR
authors.lastname: Phillips
authors.forename: Edgar Deu
authors.initials: ED
authors.lastname: Sandoval
authors.forename: Gary E
authors.initials: GE
authors.lastname: Ward
authors.forename: Benjamin F
authors.initials: BF
authors.lastname: Cravatt
authors.forename: John C
authors.initials: JC
authors.lastname: Boothroyd
authors.forename: Matthew
authors.initials: M
authors.lastname: Bogyo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003600:Cytosol / Q000378:metabolism; D017931:DNA Primers; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D014122:Toxoplasma / Q000187:drug effects / Q000235:genetics / Q000502:physiology*
chemical_list: D017931:DNA Primers; D015800:Protozoan Proteins