Autophagy and Ocular Toxoplasmosis

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The intracellular protozoan Toxoplasma gondii is the most common cause of infectious retinitis in the world. Ocular toxoplasmosis tends to recur and leads to vision loss in 25% of patients, especially in children with congenital infection, the elderly and the immunosuppressed. Current treatment does not improve visual function or prevent relapses. A better understanding of the mechanisms that control ocular toxoplasmosis may result in novel therapeutic approaches against this disease. Autophagy is a constitutive process of lysosomal degradation. T. gondii must avoid targeting by autophagy in order to survive within host cells. We showed that during invasion of host cells, T. gondii induces EGFR signaling that results in avoidance of initial autophagic targeting. Recently, we found that T. gondii causes sustained Src signaling that maintains activation of EGFR and Akt (inhibitor of autophagy). Pharmacologic inhibition of EGFR triggers autophagic killing of T. gondii in previously infected cells and protects against ocular toxoplasmosis. However, the protection is partial (EGFR expression is restricted; EGFR is only partially responsible for Akt activation). In contrast, Src is ubiquitous and low concentrations of a Src inhibitor ablates Akt activation and kills T. gondii. How autophagosomes selectively target T. gondii (required for effective pathogen elimination) is unknown. The objective of this application is to examine the role of Src in avoidance of autophagic killing of T. gondii, understand how autophagy selectively targets the parasite and determine the relevance of this mechanism in resistance against ocular toxoplasmosis. The central hypothesis is that inhibition of Src enables the activation of a specific protein kinase that triggers selective autophagic targeting and killing of T. gondii promoting protection against ocular toxoplasmosis. In the first aim we will examine how inhibition of Src triggers activation of this protein kinase in T. gondii-infected cells. This aim will be pursued using genetic and pharmacologic approaches that block specific signaling pathways. In the second aim we will examine the role of this kinase in selective vs bulk autophagy in T. gondii- infected cells. In the third aim, we will examine the molecular events controlled by this protein kinase that explain how autophagosomes selectively target the parasite. Both aims will be pursued using a combined approach of confocal microscopy using antibodies against endogenous proteins, live-cell microscopy using fluorescently-tagged proteins and electron microscopy. In the fourth aim we will use an animal model of ocular toxoplasmosis and transgenic mice to examine the role of Src, the protein kinase controlled by Src and autophagy in ocular toxoplasmosis. The proposed work will further our understanding of how host cell signaling regulates autophagic targeting of T. gondii and the outcome of the infection, and may lead to adjunctive approaches to improve the treatment of toxoplasmosis.