title: Toxoplasma growth in vitro is dependent on exogenous tyrosine and is independent of AAH2 even in tyrosine-limiting conditions.
abstract: Toxoplasma gondii is an obligate intracellular parasite capable of infecting virtually all nucleated cell types in almost all warm-blooded animals. Interestingly, Toxoplasma has a relatively full repertoire of amino acid biosynthetic machinery, perhaps reflecting its broad host range and, consequently, its need to adapt to a wide array of amino acid resources. Although Toxoplasma has been shown to be auxotrophic for tryptophan and arginine, it has not previously been determined if Toxoplasma is also auxotrophic for tyrosine. Toxoplasma tachyzoites and bradyzoites were recently found to express an amino acid hydroxylase (AAH2) that is capable of synthesizing tyrosine and dihydroxyphenylalanine (DOPA) from phenylalanine; however, the role of AAH2 in tachyzoite and bradyzoite infection has not yet been identified. To determine if Toxoplasma requires exogenous tyrosine for growth, we performed growth assays on tachyzoites and bradyzoites in nutrient-rich media titrated with varying amounts of tyrosine. We found that Toxoplasma tachyzoites form significantly smaller plaques in tyrosine-limiting media in a dose-dependent manner and that this phenotype is not affected by deletion of TgAAH2. To determine if bradyzoites require exogenous tyrosine for growth, we induced differentiation from tachyzoites in vitro in tyrosine-limiting media and found that replication and vacuole number are all decreased in tyrosine-deficient media. Importantly, culture of confluent human fibroblasts in tyrosine-deficient media does not affect their viability, indicating that, at least in vitro, the need for tyrosine is at the level of Toxoplasma, not the host cell supporting its growth.
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, CA 94305, USA.
authors.forename: Nicole D
authors.initials: ND
authors.lastname: Marino
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, CA 94305, USA. Electronic address: jboothr@stanford.edu.
authors.forename: John C
authors.initials: JC
authors.lastname: Boothroyd
mesh_terms: D024322:Amino Acids, Aromatic / Q000378:metabolism; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000469:parasitology / Q000648:ultrastructure; D006801:Humans; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism*; D012692:Serial Passage; D014122:Toxoplasma / Q000187:drug effects / Q000254:growth & development* / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism* / Q000494:pharmacology; D014617:Vacuoles / Q000187:drug effects / Q000469:parasitology
chemical_list: D024322:Amino Acids, Aromatic; D003470:Culture Media; D014443:Tyrosine; D006867:Hydrolases
keywords: Amino acid hydroxylase; Apicomplexan; Metabolism