Toxoplasma gondii is an intracellular parasite that has infected up to one-third of the population. The pathogencan cause congenital birth defects and life-threatening opportunistic infection in HIV/AIDS. The replicativestage (tachyzoite) develops into a latent stage (bradyzoite) that resides inside brain, heart, and skeletal muscletissues, and is impervious to immunity and currently approved antiparasitic drugs. Tissue cysts give rise torecurrent reactivation of infection in immunocompromised patients, creating chronic disease in HIV/AIDSpatients. Despite its clinical importance, we know very little about the molecular details orchestrating theconversion between tachyzoites and bradyzoites. Studies have revealed that the development of bradyzoites,which can be triggered in vitro by cellular stress, is a complex process subject to regulation at transcriptionaland post-transcriptional levels. Recently, it has been shown in other species that mRNA modifications,specifically post-transcriptional methylation of adenosines at position 6 (m6A), influence gene expression byaltering RNA processing or translation. Changes in m6A marks have been associated with modulating cellularstress, development, and differentiation. The discovery of m6A represents a new layer of gene regulationcalled epitranscriptomics that has not been investigated in protozoan parasites. In preliminary studies, weestablish that Toxoplasma RNA is subject to m6A, and we have begun characterizing the enzyme complex thatdelivers this RNA modification. The study of m6A in Toxoplasma in the context of HIV/AIDS is significant asthis modification has been linked to stress-induced changes in cells, making it highly likely that m6Aparticipates in bradyzoite development. In this new R21, we will address our hypothesis that m6A mRNAmodifications are required for tachyzoite differentiation into bradyzoites by identifying changes in the m6Aepitranscriptome during stage conversion (Aim 1) and determining the ?writer? protein complex that deliversm6A onto Toxoplasma mRNA (Aim 2). Our proposed studies will be the first analysis of m6A on mRNA inparasites, and will generate valuable datasets and reagents needed to interrogate this vital new area ofinvestigation relevant to the development of the tissue cysts, which give rise to chronic toxoplasmosis inHIV/AIDS patients.