Signaling determinants in Trojan horse-mediated dissemination of Toxoplasma gondii

Sammanfattning: Toxoplasma gondii is an obligate intracellular parasite that infects all warm-blooded vertebrates including one third of the global human population. While infection is typically asymptomatic in healthy human hosts, reactivated and acute infection in immunosuppressed or immunecompromised individuals can lead to lethal toxoplasmic encephalitis After ingestion, the parasite crosses the intestinal epithelium and rapidly achieves systemic dissemination, ultimately establishing chronic infection in the brain. Shortly after crossing the intestinal epithelium T. gondii encounters dendritic cells (DCs). Paradoxically, T. gondii tachyzoites exploit the inherent migratory ability of DCs for dissemination via a “Trojan horse” mechanism. Within minutes of active invasion by T. gondii tachyzoites, DCs adopt a hypermigratory phenotype that mediates rapid systemic dissemination of T. gondii in mice. Previous studies have demonstrated that the hypermigratory phenotype involves cytoskeletal rearrangement, redistribution of integrins and high-velocity in vitro cell migration (termed hypermotility), which is initiated by GABAergic signaling. However, the downstream effectors of GABAergic signaling in parasitized DCs remain enigmatic. Leukocyte migration often relies on adhesion and proteolysis of extracellular matrix (ECM). However, the role of ECM proteolysis in hypermigration has not been addressed. In this thesis, the migratory activation of T. gondii-infected DCs is characterized in terms of cell signaling and ECM proteolysis.In paper I we demonstrate that MMP-mediated proteolytic activity of DCs is abolished upon T. gondii infection. To investigate DC pericellular proteolysis at the single cell level, we developed a high-content imaging and automated image analysis method. With pharmacological inhibitors and gene silencing, we show that T. gondii-infected DCs lose the ability to degrade ECM through the upregulation of TIMP1 and the loss of podosome structures.In paper II we show that the hypermigratory phenotype induced by GABAergic signaling in T. gondii-infected DCs is dependent on L-type voltage dependent Ca2+ channel (L-VDCC) activation, predominantly CaV1.3. Pharmacological antagonism of CaV1.3 and gene silencing of cav1.3 blocked hypermotility. Further, inhibition of L-VDCCs with benidipine significantly reduced T. gondii dissemination in a mouse model.In paper III we address the impact of TIMP1 on the migratory activation of T. gondii-infected DCs. Using pharmacological antagonism and shRNA-mediated gene silencing, we demonstrate that secreted TIMP1 induces motility and migration in T. gondii-infected DCs by activating ITGB1-FAK signaling through interactions with CD63.In paper IV we report that the GTPase Ras functions as a molecular switch in the migratory activation of T. gondii-infected DCs. We identify that VDCC-CaM-CaMkII and Met signaling converge on Ras-mediated Erk phosphorylation leading to migratory activation of T. gondii-infected DCs.In summary, my thesis details novel host signaling pathways hijacked by the protozoan parasite T. gondii in Trojan horse DCs for dissemination. Through the investigation of host-parasite interactions, we shed new light on mechanisms that govern leukocyte migration and strategies employed by T. gondii to achieve pervasive dissemination. Gaining further insights into the biology that underlies T. gondii pathogenesis and persistence will help ameliorate toxoplasmosis in at-risk groups.