PROJECT SUMMARY/ABSTRACT: Toxoplasma gondii can have devastating consequences in the developing fetus if it is acquired during pregnancy. A significant number of congenital T. gondii infections occur yearly (~4000 in the U.S. and over 200,000 worldwide), making T. gondii among the most important teratogenic pathogens. To date it is not possible to predict whether infection of a T. gondii-naïve pregnant woman will ultimately transmit to the fetus. Congenital toxoplasmosis occurs only after T. gondii breaches the placental barrier, yet very little is known about the cellular and molecular events that occur during this host-pathogen interaction, nor how these events affect infection outcome. In this proposal we outline a series of complementary experiments aimed at understanding what happens when Toxoplasma gondii encounters cells of the placenta and how this might impact infection outcome in the developing fetus. A major focus of our work is on placental trophoblasts which form the primary interface between the developing fetus and maternal blood. Our extensive preliminary and published data firmly establish that placental syncytiotrophoblasts (SYNs) but not cytotrophoblasts (CYTs) resist Toxoplasma infection, and that Toxoplasma infection of trophoblasts induces a transcriptional response that is unique compared to most cell types studied to date. In Aim 1 we exploit multiple genetically tractable models of SYN function and development to identify SYN resistance mechanisms and validate them at the molecular level. Significance of this aim derives from the fact that SYNs are unique in being the only cell type studied to date that is intrinsically resistant to T. gondii infection. In Aim 2 we address the importance of innate immune signaling at the maternofetal interface during congenital transmission. In Subaim 2.2. we will do this by quantifying multiple immunomodulatory cytokines in a unique set of human serum samples that longitudinally cover gestational seroconversion events during pregnancy, and then use multivariate analyses to link immune signaling profiles to different infection outcomes. In Subaim 2.2. we will genetically ablate multiple host-targeting effectors in T. gondii (including one that alters the immunoregulatory landscape specifically in placental cells), and link congenital transmission dynamics to the immunomodulatory landscape in a well-established mouse model of congenital transmission. Impact of the proposed studies derives from (1) the use of primary placental tissues and tractable cell line models of CYTs and SYNs to decode critical molecular mechanisms of resistance and susceptibility, (2) the use of serum samples before, during and after T. gondii infection to robustly quantify changes in the host response during congenital exposure events and (3) the synergistic and well-established collaboration of the co-PI team (Coyne and Boyle) which leverages their expertise in the molecular biology of Toxoplasma host-pathogen inte...