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Signaling determinants in Trojan horse-mediated dissemination of Toxoplasma gondii
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.ORCID iD: 0000-0002-3388-061X
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2019. , p. 23
Keywords [en]
Apicomplexa, leukocyte motility, dendritic cell, amoeboid migration, host-parasite interactions, podosome, integrin, ITGB1, ECM, MAT, TIMP, MMP, MAPK, Erk, RTK, HGFR, Ras, calcium, VDCC, VGCC, calmodulin, CaMkII, CD63, FAK, SRC, Pi3k, hypermigration
National Category
Cell and Molecular Biology
Research subject
Molecular Bioscience
Identifiers
URN: urn:nbn:se:su:diva-175483ISBN: 978-91-7797-917-3 (print)ISBN: 978-91-7797-918-0 (electronic)OAI: oai:DiVA.org:su-175483DiVA, id: diva2:1366563
Public defence
2019-12-12, Vivi Täckholmsalen (Q-salen) NPQ-huset, Svante Arrhenius väg 20, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2019-11-19 Created: 2019-10-29 Last updated: 2019-11-12Bibliographically approved
List of papers
1. Toxoplasma gondii infection shifts dendritic cells into an amoeboid rapid migration mode encompassing podosome dissolution, secretion of TIMP-1, and reduced proteolysis of extracellular matrix
Open this publication in new window or tab >>Toxoplasma gondii infection shifts dendritic cells into an amoeboid rapid migration mode encompassing podosome dissolution, secretion of TIMP-1, and reduced proteolysis of extracellular matrix
2018 (English)In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 20, no 3, article id e12808Article in journal (Refereed) Published
Abstract [en]

Dendritic cells (DCs) infected by Toxoplasma gondii rapidly acquire a hypermigratory phenotype that promotes systemic parasite dissemination by a Trojan horse mechanism in mice. Recent paradigms of leukocyte migration have identified the amoeboid migration mode of DCs as particularly suited for rapid locomotion in extracellular matrix and tissues. Here, we have developed a microscopy-based high-throughput approach to assess motility and matrix degradation by Toxoplasma-challenged murine and human DCs. DCs challenged with T.gondii exhibited dependency on metalloproteinase activity for hypermotility and transmigration but, strikingly, also dramatically reduced pericellular proteolysis. Toxoplasma-challenged DCs up-regulated expression and secretion of tissue inhibitor of metalloproteinases-1 (TIMP-1) and their supernatants impaired matrix degradation by naive DCs and by-stander DCs dose dependently. Gene silencing of TIMP-1 by short hairpin RNA restored matrix degradation activity in Toxoplasma-infected DCs. Additionally, dissolution of podosome structures in parasitised DCs coincided with abrogated matrix degradation. Toxoplasma lysates inhibited pericellular proteolysis in a MyD88-dependent fashion whereas abrogated proteolysis persevered in Toxoplasma-infected MyD88-deficient DCs. This indicated that both TLR/MyD88-dependent and TLR/MyD88-independent signalling pathways mediated podosome dissolution and the abrogated matrix degradation. We report that increased TIMP-1 secretion and cytoskeletal rearrangements encompassing podosome dissolution are features of Toxoplasma-induced hypermigration of DCs with an impact on matrix degradation. Jointly, the data highlight how an obligate intracellular parasite orchestrates key regulatory cellular processes consistent with non-proteolytic amoeboid migration of the vehicle cells that facilitate its dissemination.

Keywords
apicomplexa, coccidia, host-pathogen, leukocyte migration, matrix metalloproteinase, tissue inhibitor of metalloproteinases
National Category
Biological Sciences
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-153605 (URN)10.1111/cmi.12808 (DOI)000424714300003 ()
Available from: 2018-03-14 Created: 2018-03-14 Last updated: 2019-12-09Bibliographically approved
2. Voltage-dependent calcium channel signaling mediates GABA(A) receptor-induced migratory activation of dendritic cells infected by Toxoplasma gondii
Open this publication in new window or tab >>Voltage-dependent calcium channel signaling mediates GABA(A) receptor-induced migratory activation of dendritic cells infected by Toxoplasma gondii
Show others...
2017 (English)In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 13, no 12, article id e1006739Article in journal (Refereed) Published
Abstract [en]

The obligate intracellular parasite Toxoplasma gondii exploits cells of the immune system to disseminate. Upon T. gondii-infection,. Upsilon-aminobutyric acid (GABA)/GABAA receptor signaling triggers a hypermigratory phenotype in dendritic cells (DCs) by unknown signal transduction pathways. Here, we demonstrate that calcium (Ca2+) signaling in DCs is indispensable for T. gondii-induced DC hypermotility and transmigration in vitro. We report that activation of GABAA receptors by GABA induces transient Ca2+ entry in DCs. Murine bone marrow-derived DCs preferentially expressed the L-type voltage-dependent Ca2+ channel (VDCC) subtype Cav1.3. Silencing of Cav1.3 by short hairpin RNA or selective pharmacological antagonism of VDCCs abolished the Toxoplasma-induced hypermigratory phenotype. In a mouse model of toxoplasmosis, VDCC inhibition of adoptively transferred Toxoplasma-infected DCs delayed the appearance of cell-associated parasites in the blood circulation and reduced parasite dissemination to target organs. The present data establish that T. gondii-induced hypermigration of DCs requires signaling via VDCCs and that Ca2+ acts as a second messenger to GABAergic signaling via the VDCC Cav1.3. The findings define a novel motility-related signaling axis in DCs and unveil that interneurons and DCs share common GABAergic motogenic pathways. T. gondii employs GABAergic non-canonical pathways to induce host cell migration and facilitate dissemination.

National Category
Cell and Molecular Biology Cell Biology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-152517 (URN)10.1371/journal.ppat.1006739 (DOI)000419019800010 ()
Available from: 2018-02-07 Created: 2018-02-07 Last updated: 2019-12-09Bibliographically approved
3. TIMP-1 promotes hypermigration of Toxoplasma-infected primary dendritic cells via CD63-ITGB1-FAK signaling
Open this publication in new window or tab >>TIMP-1 promotes hypermigration of Toxoplasma-infected primary dendritic cells via CD63-ITGB1-FAK signaling
2019 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 132, no 3, article id UNSP jcs225193Article in journal (Refereed) Published
Abstract [en]

Tissue inhibitor of metalloproteinases-1 (TIMP-1) exerts pleiotropic effects on cells including conferring metastatic properties to cancer cells. As for metastatic cells, recent paradigms of leukocyte migration attribute important roles to the amoeboid migration mode of dendritic cells (DCs) for rapid locomotion in tissues. However, the role of TIMP-1 in immune cell migration and in the context of infection has not been addressed. We report that, upon challenge with the obligate intracellular parasite Toxoplasma gondii, primary DCs secrete TIMP-1 with implications for their migratory properties. Using a short hairpin RNA (shRNA) gene silencing approach, we demonstrate that secreted TIMP-1 and its ligand CD63 are required for the onset of hypermotility in DCs challenged with T. gondii. Further, gene silencing and antibody blockade of the beta 1-integrin CD29 (ITGB1) inhibited DC hypermotility, indicating that signal transduction occurred via ITGB1. Finally, gene silencing of the ITGB1-associated focal adhesion kinase (FAK, also known as PTK2), as well as pharmacological antagonism of FAK and associated kinases SRC and PI3K, abrogated hypermotility. The present study identifies a TIMP-1-CD63-ITGB1-FAK signaling axis in primary DCs, which T. gondii hijacks to drive high-speed amoeboid migration of the vehicle cells that facilitate its systemic dissemination.

Keywords
Apicomplexa, Leukocyte motility, Amoeboid migration, CD29, LAMP-3
National Category
Biological Sciences
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-166566 (URN)10.1242/jcs.225193 (DOI)000458849100011 ()30635444 (PubMedID)
Available from: 2019-03-04 Created: 2019-03-04 Last updated: 2019-11-01Bibliographically approved
4. Convergent Met and Voltage-gated Ca2+ channel signaling drives hy- permigration of Toxoplasma-infected dendritic cells
Open this publication in new window or tab >>Convergent Met and Voltage-gated Ca2+ channel signaling drives hy- permigration of Toxoplasma-infected dendritic cells
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Ras-Erk MAPK signaling controls many of the principal pathways involved in metazoan cell motility, drives metasta- sis of multiple cancer types and is targeted in chemothera- py. Yet, its putative roles in immune cell functions or in infections have remained elusive. Here, using primary den- dritic cells (DCs) in an infection model with the protozoan Toxoplasma gondii, we show that two pathways activated by infection converge on Ras-Erk MAPK signaling to promote migration of parasitized DCs. We identify signaling through the receptor tyrosine kinase Met (also known as HGFR) as a driver of T. gondii-induced DC hypermotility. Further, we show that voltage-gated Ca2+ channel (VGCC, subtype CaV1.3) signaling impacts the migratory activation of DCs via calmodulin-calmodulin kinase II. We report that VGCC and Met signaling converge on Ras GTPase to drive.

The data provide a molecular basis for the hypermigratory mesenchymal-to-amoeboid transition (MAT) of parasitized DCs. The emerging concept suggests that parasitized DCs acquire metastasis-like migratory propertiesto promote infection-related dissemination.

Keywords
Reptor tyrosine kinase, calcium signaling, leukocyte motility, amoeboid migration, Apicomplexa
National Category
Biological Sciences
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-175961 (URN)
Note

Submitted manuscript part of thesis.

Available from: 2019-11-11 Created: 2019-11-11 Last updated: 2019-11-12Bibliographically approved

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