Investigating differentiation, maintenance, and reactivation of the Toxoplasma chronic stage

7. PROJECT SUMMARY/ABSTRACT Toxoplasma gondii is one of the most ubiquitous parasites of humans, chronically infecting one quarter of the world’s population. Though typically asymptomatic in immunocompetent hosts, infection can give rise to severe clinical complications in the immunocompromised or during periods of weakened immune function. Parasites that maintain the chronic infection are transcriptionally distinct from the acute stages that cause pathology, and are resistant to treatment with antiparasitics. Understanding regulation of the T. gondii chronic stage is therefore necessary to prevent and eradicate persistent reservoirs of disease. Our lab recently identified two core components of the chronic differentiation program. First, we found that a single transcription factor, BFD1, is necessary and sufficient for stage conversion. More recently, I discovered a cytosolic RNA-binding protein (BFD2) that functions immediately upstream of BFD1 to regulate its expression. Parasites lacking either factor are not able to produce chronic stages, either in mice or in response to alkaline stress—a standard in vitro trigger of differentiation. My work suggests that BFD2 functions as a translational activator of BFD1 under stress, and that this requires binding of BFD2 to BFD1 mRNA. In turn, BFD1 promotes BFD2 transcription, outlining a feedback loop. This reciprocal positive regulation provides the driving force to commit parasites to their chronic differentiation program; yet, precisely how the BFD1-BFD2 circuit is triggered by stress, and later overturned during reactivation, remains to be explored. In the proposed studies, I will uncover the molecular basis of BFD2 regulation through three complimentary aims. Aim 1 will investigate the mechanism of translational activation by BFD2, and how this is influenced by protein-protein interactions. Aim 2 will assess the role of post-translational modification in licensing stress-responsive BFD2 activity. Aim 3 will employ a novel gain-of-function screening approach to identify the factors responsible for antagonizing the BFD1-BFD2 circuit during chronic-stage reactivation. Together, these studies will integrate the genetic mechanisms underlying entry and exit from the T. gondii chronic stage with the signals that trigger stage conversion, which may inform the design of therapies to combat this important human pathogen.