Symptomatic toxoplasmosis in the context of immune suppression is typically associated with the reactivation of the chronic form of the infection mediated by bradyzoites that reside within tissue cysts. Despite their central role in the pathogenesis, very little is known about the basic biology of bradyzoites. In fact, our work (Watts et al. mBio 2015) was the first to definitively demonstrate that bradyzoites within tissue cysts in vivo are dynamic replication competent entities. This view directly challenged the prevailing dogma of bradyzoites and the tissue cysts that house them were metabolically inert. This view has been proffered as the reason for why bradyzoites are resistant to drugs that effectively kill the actively growing tachyzoites. Understanding the underlying basis for the inherent resistance to drug-mediated clearance needs to be addressed on a mechanistic basis which is now possible given our recent identification of quantifiable markers for the physiological status of individual bradyzoites within tissue cysts. This can further be extended to developing a framework for the evaluation of potential new drugs in vivo that rely on quantifiable physiological readouts that will greatly enhance sensitivity by addressing the behavior of populations of bradyzoites within tissue cysts as opposed to the currently used tissue cyst burden. This crude metric limits the sensitivity of efficacy to 2 orders of magnitude as percentage decreases in cyst burden and little else are measured. Our development of BradyCount1.0, an imaging based application, to quantify the actual bradyzoite levels within tissue cysts, revealed a previously underappreciated level of heterogeneity in bradyzoite numbers relative to tissue cyst size. We exploited what was known about the dynamics of the scaffold protein TgIMC3 and nuclear morphology in the context of replication, to capture not only replicating bradyzoites but also information of their relative ?age?. Additional studies revealed that bradyzoite mitochondrial activity and the accumulation amylopectin granules (AG), a measure of stored energy, were also heterogeneous. This provides three distinct and likely interrelated physiological metrics that can be assessed at the level of individual bradyzoites within in vivo derived tissue cysts. With this study, we aim to develop a new standard to assess the progression of the chronic Toxoplasma infections, and to translate this novel approach to assess the effectiveness of existing and future drugs. Toward this end we propose develop BradyCount 2.0 to morphologically quantify nuclear profiles and bradyzoite scaffolds (as a markers of active and past replication), mitochondrial profiles and the distribution of AG as reporters of energy and intermediary metabolism. By recording multiple parameters individually and collectively on bradyzoites within tissue cysts, we will establish a framework to define the chronic infection and the impact of drug intervention employing quantifiable physiological metric. These inputs will be used to refine a Markov Chain-based data-driven computational model to understand this enigmatic phase, for which effective treatments are lacking. These studies will greatly improve the sensitivity of testing of both existing and future drugs while providing crucial mechanistic insights.
A significant barrier to the identification of potential drug treatments targeted at the Toxoplasma gondii chronic infection is the lack of suitable quantifiable readouts for the effect of drugs on the encysted bradyzoites. In this study we leverage our findings regarding bradyzoite replication and energy metabolism to establish imaging based approaches and develop a computational model framework to assess drug efficacy in the murine model.
