Our laboratory has established that lysine acetylation is essential in the protozoan parasite Toxoplasma gondii, which is a life-threatening opportunistic pathogen in HIV/AIDS patients since the latent cyst form (bradyzoite) constantly reconverts into proliferating tachyzoites that cause rapid tissue damage in critical areas such as the brain and heart. Our work has not only demonstrated critical roles for Toxoplasma GCN5-family lysine acetyltransferases (KATs) in gene activation, but we have also discovered widespread acetylation on hundreds of non- histone proteins. The surprising frequency of acetylation modifications found on such a large and diverse group of proteins has heightened interest in KATs, how these enzymes are regulated, and what the biological consequences of their activity holds for target substrates. We propose a detailed study of the KAT complex designated GCN5b, which we have recently shown to be critical during both acute and chronic toxoplasmosis that endangers AIDS patients. Our studies have shown that (i) GCN5b is preferentially translated during bradyzoite induction, (ii) GCN5b protein is subject to post-translational modification (PTM), (iii) GCN5b associates with plant-like AP2 transcription factors, (iv) Expression of a dominant-negative GCN5b causes a rapid cessation in tachyzoite replication, and (v) GCN5b targets the specific histone residues known to be acetylated in bradyzoite promoters. Collectively, these findings have led to our hypothesis that multiple levels of regulation modulate the GCN5b complex to coordinate Toxoplasma replication and differentiation. The proposed studies to address this hypothesis investigate topical questions in cell biology, including mechanisms of translational control, the role of post-translational modifications, mapping of gene regulatory networks, and functions of KATs beyond histone acetylation. The results will expose a wealth of new opportunities for therapeutic intervention that may subvert not only tachyzoite proliferation, but also their ability to form latent tissue cyts.
Toxoplasma is an intracellular parasite that persists as an opportunistic latent cyst, which is frequently reactivated in HIV/AIDS patients causing serious and potentially fatal pathologies. Despite administration of HAART, the mortality rate of patients suffering from HIV-associated CNS disease, such as cerebral toxoplasmosis, remains high. Our proposal aims to study the function and regulation of a key parasite acetyltransferase, and the results promise to have far- reaching impact on other medically relevant infections, including other AIDS opportunist pathogens such as Cryptosporidium.
