Analysis of two potential membrane proteins in Toxoplasma gondii

Toxoplasma gondii is an obligate, intracellular protozoan parasite of warm-blooded animals. T. gondii causes encephalitis in immunocompromised patients and multiple fetal complications, including abortion, in women whose primary exposure to T. gondii is during pregnancy. T. gondii has two developmental stages in its asexual life cycle, the rapidly replicating tachyzoites and the slow-growing, encysted bradyzoites. Tachyzoites are responsible for dissemination of the parasite throughout the host's body, marking the acute phase of infection. The chronic stage of infection is characterized by the appearance of cysts, containing bradyzoites, in the brain and muscle tissues. In these studies, a gene suspected of impacting persistent infection was investigated. Also, the expression pattern of a glycosylphosphatidylinositol (GPI)-anchored surface antigen was determined.; The 3xD4 locus was isolated in a signature-tagged mutagenesis screen for genes essential to establishment of chronic infection in mice, but not required for survival in tissue culture. The 3xD4 signature-tagged mutagenesis strain (3xD4 STM) shows greater than 100-fold decrease in virulence in mice compared to the wild-type Prugniaud (Pru) strain. To determine whether 3xD4 is responsible for the decreased virulence in mice, the gene was re-expressed in the 3xD4 STM strain and the locus was disrupted in a wild-type parasite. Surprisingly, the genetic complementation studies did not restore the 3xD4 STM strain to wild-type levels of cyst formation in vivo. Similarly, disruption of the 3xD4 locus in a wild-type background did not result in decreased lethality and cyst burden compared to wild-type in vivo. Current studies show that the insertion of the signature-tag in the D4 strain disrupts a patatin-like phospholipase. This disruption is likely the cause of the virulence defect.; In a separate study, stage specificity of a GPI-anchored surface antigen was investigated. BSR4 was previously shown by immunofluorescence microscopy to react to the bradyzoite-specific P36 monoclonal antibody (P36 mAb). Data generated in this thesis showed the P36 mAb predominantly recognizes SRS9, a related bradyzoite-specific surface antigen. The lack of SRS9 expression is the major contributor to the loss of P36 mAb reactivity in the BSR4 mutants. Further analysis suggested that the BSR4 mutant strains are defective in bradyzoite differentiation.