Translational control and latent Toxoplasma infection

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DESCRIPTION (provided by applicant): Toxoplasma gondii is an intracellular protozoan parasite that causes congenital birth defects, as well as life-threatening opportunistic disease in the immunocompromised, such as AIDS patients or patients undergoing cancer chemotherapy. Central to pathogenesis is the ability of Toxoplasma to convert from a rapidly growing form, referred to as the tachyzoite, to a latent cyst form, called the bradyzoite. Currently, we know little about the molecular events underlying how the parasite develops into cysts, and no drugs exist that can rid the body of the cyst form. It is well established that the development of bradyzoites from tachyzoites is initiated by cellular stress. We discovered that the same cellular stresses that induce cyst development also induce the phosphorylation of the Toxoplasma version of eukaryotic initiation factor -2 (TgIF2). Phosphorylation of TgIF2 correlates with lowered global translation and induced expression of bradyzoite marker genes. TgIF2 remains phosphorylated in bradyzoites, suggesting that translational control is also important for maintenance of the quiescent state of bradyzoite cysts. Our hypothesis is that TgIF2 phosphorylation results in both repression of general protein synthesis and preferential translation of key transcription factors, which are critical for bradyzoite development in response to stress. To address this hypothesis, we propose two Aims. Aim 1 will address the role of TgIF2 phosphorylation in bradyzoite development. We will create a mutant Toxoplasma parasite that substitutes the phosphorylated site, serine-71 in TgIF2 to alanine, therefore blocking phosphorylation and translational control during stress. We propose that such a mutant will be sensitized to stress and be unable to elicit translational control mechanisms required for Toxoplasma to convert from the tachyzoite to the bradyzoite cyst. Aim 2 will identify and characterize preferentially translated mRNAs involved in cyst development. We will use microarray analyses of polysome-associated mRNA from stressed and unstressed parasites to identify key regulatory factors subject to preferential translation. This study will address the requirement of TgIF2 phosphorylation and translational control for the clinically relevant process of bradyzoite development. Knowledge of eIF2 kinase-directed pathways will provide new therapeutic targets for the treatment of Toxoplasma infections and related parasitic diseases. PUBLIC HEALTH RELEVANCE: Toxoplasma gondii is a protozoan parasite that causes significant disease in AIDS patients, cancer patients undergoing chemotherapy, and organ-transplant recipients taking immunosuppressants to minimize organ rejection. Chronic toxoplasmosis is currently incurable because the parasite is able to develop into cysts that remain latent until immunosuppression. We are taking an innovative approach focused on mechanisms controlling protein synthesis to elucidate the process of parasite cyst development. The regulation of gene expression plays a key role in this pathogenic process; therefore, our results will be important for new therapies to combat opportunistic infectious diseases, such as Toxoplasma.