Toxoplasma gondii infection inhibits invasion and migration of human extravillous trophoblasts through dysregulation of FOXO1- and FOXO3a-dependent and -independent mechanisms

The protozoan parasite <i>Toxoplasma gondii</i> causes severe pathologies in the infected fetus following vertical transmission during pregnancy. Primary <i>T. gondii</i> infection increases the risk of miscarriage during the first trimester of gestation; however, the cellular and molecular mechanisms are not fully understood. Extravillous trophoblasts (EVTs) are fetal cells that migrate and invade the maternal decidua to allow placenta formation and embryo implantation. The transcription factors Forkhead box O3a (FOXO3a) and FOXO1 play a central role in the regulation of EVT migration and invasion. Hence, impairment of EVT functions is associated with FOXO3a/FOXO1 dysregulation and poor pregnancy outcomes. Interestingly, <i>T. gondii</i>-driven inactivation of FOXO3a and FOXO1 was reported in fibroblasts and macrophages. Using a combination of cell imaging, reverse genetics and biochemical approaches in the human trophoblast cell line HTR-8/SVneo, herein we provide evidence that infection with type I RH <i>T. gondii</i> strain inhibits invasiveness and migratory activities in EVTs by repressing FOXO3a-and FOXO1-dependent and independent gene expression. Indeed, either <i>T. gondii</i> infection or single knockdown of FOXO3a and FOXO1 reduced invasion and migration properties in EVTs. Selective chemical blockade of parasite motility and host cell entry indicates that active infection is indispensable for reduced EVT migration but is only partially required for <i>T. gondii</i>-driven repression of EVT invasiveness. Mechanistically, <i>T. gondii</i> infection led to AKT-sensitive phosphorylation and nuclear exclusion of FOXO3a and FOXO1 in EVTs. An RT-qPCR-based screening identified a subset of invasion-and migration-associated genes downregulated in <i>T. gondii</i>-infected EVTs (MMP2, MMP3, MMP14, TIMP2, MUC1, and ITGB3). Transcription of genes encoding MMP3 and integrin β3 decreased in FOXO3a KD and FOXO1 KD EVT cell lines, respectively. These data along with pharmacological inhibition of AKT in infected cells provide evidence that <i>T. gondii</i> downregulates MMP3 and ITGB3 gene expression in EVTs in an AKT-FOXO3a/FOXO1-dependent fashion. In all, we have uncovered a novel regulatory mechanism involved in the repression of EVT migration and invasion properties during <i>T. gondii</i> infection. Further investigation using <i>in vivo</i> and <i>ex vivo</i> models of placental infection is required to determine whether <i>T. gondii</i>-driven dysregulation of EVT functions contributes to pregnancy complications associated with congenital toxoplasmosis.