Reactivation of Chronic Toxoplasmosis

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Project Summary/Abstract Toxoplasma gondii is a widespread parasite of animals that cycles between cats, which shed infectious oocysts in their feces, and a variety of animals that harbor long-lived chronic infections. Humans are also commonly infected either by ingestion of oocysts in contaminated water, or by ingestion of tissue cysts in undercooked meat. Following infection, the parasite initially proliferates as fast-growing tachyzoites, which disseminate widely in the body. It then differentiates into semi-dormant bradyzoites that reside within tissue cysts, most commonly in muscle and brain. Although infections in healthy individuals are controlled by the immune system, tissue cysts persist in the face of immunity and are not eliminated by drug treatment. As such, chronic infections with T gondii pose a serious risk to HIV-infected AIDS patients due release of parasites from semi-dormant tissue cysts and re-emergence of the highly proliferative tachyzoite stage, which can lead to life threatening complications. Although once considered latent, newer studies reveal that bradyzoites replicate, albeit asynchronously and infrequently. Moreover, tissue cysts periodically rupture to release bradyzoites that infect new host cells and give rise to daughter tissue cysts. One barrier to egress of bradyzoites is their rigid cyst wall, comprised of proteins and carbohydrates, including the dominant antigen CST1, which is heavily glycosylated by O-linked sugars. Loss of CST1, or deletion of its mucin domain, results in fragile cysts, implying that this glycoprotein provides rigidity to the cell wall. Consistent with the idea that carbohydrates form a critical part of the cyst wall, our studies demonstrate that exogenously added glucanase and chitinase enzymes digest the cyst wall and result in release of bradyzoites. To explore endogenous pathways that control this process, we will examine the roles of two parasite glycosyl hydrolases that are expressed in bradyzoites, secreted from the parasite, and localized to the cyst wall. Genetic disruption of glucanase (GLN1) and chitinase-like protein (CLP1) genes in a cystogenic strain of T. gondii results in reduced formation of daughter cysts, supporting the hypothesis that these enzymes contribute to cyst maturation and turnover. We will examine the substrate specificity of these enzymes in vitro and assess their roles in vivo in formation and turnover of tissue cysts. We will also examine the role of these glycosyl hydrolases in chronic infection of immunocompromised mouse models that closely mimic impaired immunity seen in HIV-infected AIDS patients. The proposed studies will explore the hypothesis that glycosyl hydrolase enzymes contribute to the turnover of tissue cysts resulting in reactivation of toxoplasmosis, which poses a major risk for severe disease in HIV-infected AIDS patients.