Toxoplasma gondii is an obligate intracellular Apicomplexan parasite that could possibly infect all warm-blooded animals. Acute infections with T. gondii produce generally mild symptoms in healthy individuals, but infections of the fetus during pregnancy and infections in those that are immunocompromised can cause severe and life-threatening pathology. The parasite gains entry to the host by crossing the biological barriers of the intestine or placenta. Mononuclear phagocytes (MPs) and other leukocytes at these barriers become infected by T. gondii. Previous work has established that upon T. gondii infection, dendritic cells (DCs) undergo morphological and phenotypical changes and display enhanced migration. Components of this ‘hypermigratory phenotype’ have been confirmed in other MPs, such as monocytes, macrophages and microglia. The mechanisms underlying the hypermigratory phenotype are the subject of this thesis.

In paper I, we describe a non-canonical extended upregulation of the transcription factor Egr1 in T. gondii-infected DCs. While the rapid and transient canonical induction of Egr1 depends on the ERK1/2-pathway, the extended upregulation was dependent on p38 MAPK and p38-activating parasite-derived effector GRA24. The hypermotility component of the hypermigratory phenotype did not depend on GRA24/p38, but on ERK1/2. We determined that EGR1 acts as an inhibitor of phenotypic maturation and that GRA24 stimulates Il2 and Il12p40 expression in T. gondii-infected DCs.

In paper II, we characterize actors upstream of ERK1/2 in hypermotility of T. gondii-infected DCs. Two axes that output on the ERK1/2 pathway were found to be required for hypermotility. The first involves Ca²⁺ influx through voltage-gated calcium channel Ca_v1.3, resulting in activation of calcium/calmodulin-dependent protein kinase II (CaMKII) via Ca²⁺ sensor calmodulin (CaM). The other axis relies on hepatocyte growth factor (HGF), which is secreted by DCs, and its receptor Met. Both axes converge on the ERK1/2 pathway via the GTPase Ras.

In paper III, we study the migratory behavior of T. gondii-infected DCs on and across endothelial cell monolayers. Particularly infected DCs transmigrated across endothelial cell monolayers, but were, unlike on 2D surfaces, not hypermotile on endothelial cells. We characterize the differential involvement of β1 and β2 integrins, cell adhesion molecules ICAM-1 and PECAM-1 and pan-integrin-cytoskeleton linker talin in transmigration across endothelial cells and in migration on endothelial cells and 2D surfaces.

Finally, we report in paper IV that T. gondii imparts a DC-like transcriptional signature on infected macrophages. Infected macrophages upregulate chemokine receptor CCR7 and display chemotaxis to CCR7-ligand CCL19, like DCs. Concomitantly, these macrophages upregulate the expression of transcription factors associated with DCs and of immune activation-related genes and markers. T. gondii-infected macrophages thus display a remarkable transcriptional and functional plasticity. We identify GRA28 as the primary T. gondii-derived effector protein responsible for these phenotypes, with parasite-derived ROP16 having partially opposing effects.

Altogether, my thesis identifies novel aspects of the hypermigratory phenotype in T. gondii-infected MPs and provides insights into the molecular components and signaling that underlie them.

Toxoplasma gondii is an obligate intracellular parasite that can likely infect all warm-blooded vertebrates, with estimates of up to 30% of the global human population being infected. Although infection with T. gondii is usually asymptomatic or mild, in immunocompromised individuals infection can lead to lethal toxoplasmic encephalitis. Infection acquired during pregnancy can also lead to serious ocular or neurological damage and even death of the foetus. Following ingestion, the parasite is able to cross the first biological barrier it encounters, the gut epithelium and convert to the rapidly replicating tachyzoite stage. It can then disseminate throughout the body of the host, eventually reaching sites such as the brain, after crossing the blood-brain barrier (BBB). Previous findings have shown that T. gondii can use leukocytes, such as dendritic cells (DCs), for dissemination via a “Trojan horse”-type mechanism, but how T. gondii then crosses restrictive barriers such as the BBB is still not fully understood. The overall objective of this work has been to investigate how T. gondii crosses biological barriers and how infection impacts host cell signalling.

In paper I we demonstrate that T. gondii can cross polarised cell monolayers without significantly perturbing barrier integrity. Reduced phosphorylation of focal adhesion kinase (FAK) was observed in cell monolayers upon T. gondii challenge, and inhibition or gene silencing of FAK (Ptk2) facilitated transmigration of T. gondii across polarised cell monolayers. In paper II we found that upon T. gondii infection of DCs, secreted TIMP-1 induces hypermotility by activating β1 integrin-FAK signalling through interactions with CD63. In paper III we show that T. gondii can cross polarised endothelial cell monolayers inside DCs. We also report that parasitised DCs on endothelium do not display a hypermotile phenotype, switching to integrin-dependent motility. Blockade of β1 and β2 integrins or ICAM-1, and gene silencing of β1 (Itgb1) or talin (Tln1) restored infected-DC motility, and reduced the frequency of transmigration of T. gondii-challenged DCs across endothelium. In paper IV we demonstrate that, shortly after T. gondii inoculation in mice, parasites mainly localised to cortical capillaries of the brain. Early invasion to the brain parenchyma occurred in absence of a significant increase in BBB permeability, perivascular leukocyte cuffs or haemorrhage. Further, pharmacological inhibition or endothelial cell-specific knockout of FAK facilitated parasite transmigration to the brain parenchyma.

In paper V we report that DCs challenged with type II T. gondii transmigrate across polarised endothelial cell monolayers at a higher frequency than type I T. gondii, while type I infected DCs exhibited increased migratory velocities on endothelium. We also show that T. gondii-induced upregulation of ICAM-1 in DCs is genotype-dependent, and requires the T. gondii secreted effector GRA15. Finally, gene silencing of leukocyte ICAM-1 (Icam-1) or deletion of T. gondii GRA15 reduced transmigration across endothelial cell monolayers.

In summary, the work in this thesis provides novel insights into how T. gondii can potentially cross biological barriers on its journey to the brain. We find that T. gondii can cross polarised monolayers both as free parasites and using DCs as a “Trojan horse”, and identify new ways in which T. gondii can alter host cell dynamics to benefit its own dissemination.