Dissertation - Emily Ross
Dissertation
Date: Thursday 9 June 2022
Time: 09.30 – 12.30
Location: Vivi Täckholmsalen
By:
Emily Ross, MBW, Stockholm University
Supervisor:
Antonio Barragan, MBW, Stockholm University
Opponent:
Ildiko Rita Dunay, Institut für Inflammation und Neurodegeneration, Otto-von-Guericke-Universität
Committee:
Anna Färnert, Department of Medicine Solna, Karolinska Institutet
Staffan Strömblad, Department of Biosciences and Nutrition, Karolinska Institutet
Kristina Jonas, SciLifeLab, MBW, SU
Transmigration of Toxoplasma gondii across biological barriers
Abstract
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.
Keywords: Apicomplexa, blood-brain barrier, leukocyte, immune cell, transendothelial migration, cell adhesion molecule, host-pathogen, FAK, integrin.
Last updated: May 19, 2022
Source: MBW