Determining the mechanisms of Toxoplasma gondii colonization and crossing of the placental barrier - PROJECT SUMMARY Our understanding of Toxoplasma gondii (T. gondii) and other pathogens transmitted from mother to fetus is limited. However, they significantly contribute to fetal illness and death worldwide. This proposal aims to address this knowledge gap by identifying and characterizing T. gondii proteins that play a role in crossing the placenta and causing congenital infection. The research team will use innovative methods and tools, such as the similarity of the guinea pig placenta to humans and a new in vitro placental barrier derived from human trophoblast stem cells, to study the role of a particular T. gondii protein, TgMIF, in the parasite's ability to cross the placental barrier. They also aim to identify new parasitic proteins that might contribute to vertical transmission. Preliminary data suggests that only a fraction of extracellular parasites can cross the trophoblast barrier in a manner dependent on a host intercellular adhesion protein called ICAM-1. Furthermore, it has been found that recombinant TgMIF alone can increase ICAM-1 levels on trophoblasts, which might enhance the parasites' ability to cross the placental barrier and infect the fetus. The study's first Aim is to test the hypothesis that TgMIF mediates extracellular parasites to cross the trophoblast barrier and/or T. gondii replication in these cells, using in vitro placental barrier and pregnant guinea pig models. The researchers expect to determine whether both phenomena are involved in fetal infection and pinpoint the role of TgMIF. In the second Aim, the researchers will test the hypothesis that unknown parasitic plasma membrane proteins interact with the host cell protein ICAM-1 and enhance the ability of extracellular parasites to cross the trophoblast barrier. They plan to use a CRISPR/Cas9 loss-of-function screen with the trophoblast barrier to identify these new parasitic proteins. In conclusion, the research team will use stem cells, animal models, genetic tools, and high- throughput screens to achieve the goals of this R21 application. The results of this proposal are expected to identify and characterize crucial proteins that mediate T. gondii vertical transmission and establish a scientific basis for developing therapies to prevent fetal infection.
