Toxoplasma gondii disrupts β1 integrin signaling and focal adhesion formation during monocyte hypermotility

Abstract

The motility of blood monocytes is orchestrated by the activity of cell surface integrins, which translate extracellular signals into cytoskeletal changes to mediate adhesion and migration. Toxoplasma gondii is an intracellular parasite that infects migratory cells and enhances their motility, but the mechanisms underlying T. gondii-induced hypermotility are incompletely understood. We have investigated the molecular basis for the hypermotility of primary human peripheral blood monocytes and THP-1 cells infected with T. gondii. Compared to uninfected monocytes, T. gondii infection of monocytes reduced cell spreading and the number of activated β1 integrin clusters in contact with fibronectin during settling, an effect not observed in monocytes treated with LPS or E. coli. Furthermore, T. gondii infection disrupted the phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 (Y397) and Y925 and of the related protein proline-rich tyrosine kinase (Pyk2) at Y402. The localization of paxillin, FAK, and vinculin to focal adhesions and the colocalization of these proteins with activated β1 integrins were also impaired in T. gondiiinfected monocytes. Using time-lapse confocal microscopy of THP-1 cells expressing eGFP-FAK during settling on fibronectin, we found that T. gondii-induced monocyte hypermotility was characterized by a reduced number of eGFP-FAKcontaining clusters over time compared to uninfected cells. This study demonstrates an integrin conformation-independent regulation of the β1 integrin adhesion pathway, providing further insight into the molecular mechanism of T. gondiiinduced monocyte hypermotility.

Dysregulation of focal adhesion kinase upon Toxoplasma gondii infection facilitates parasite translocation across polarised primary brain endothelial cell monolayers

“…Impact of focal adhesion kinase (FAK) inhibition on cellular barrier integrity and transmigration of Toxoplasma gondii(a) Transcellular electrical resistance (TCER) of Caco2 and murine brain endothelial cell (MBEC) monolayers upon treatment with FAK inhibitor 12.5 μM). In contrast, challenge with MOI-equivalent or higher doses of T. gondii lysate (or LPS) did not reduce pFAK levels.This indicated an effect mediated by live intracellular tachyzoites and is well in line with recent reports of FAK dysregulation in toxoplasma-infected DCs and monocytic cells(Cook, Ueno, & Lodoen, 2018;Olafsson, Ross, Varas-Godoy, & Barragan, 2019). (b) Permeability to FITC-dextran (3 kDa) in same setup as in (a).…”

“…Although that study did not address the impact of infection on polarisation or barrier integrity, it is plausible that similar mechanisms explain the dephosphorylation of FAK upon T. gondii infection. Indeed, β1-integrin regulation and motility in toxoplasma-infected DCs and monocytes was recently linked to FAK dysregulation (Cook et al, 2018;Olafsson et al, 2019). Additionally, because redistribution of β1-integrins is observed upon T. gondii infection in DCs (Weidner et al, 2013), it is also possible that FAK dysregulation impacts on β1-integrin signalling and focal adhesion reorganisation in endothelial and epithelial cells and that the resulting increased vascular or epithelial permeability facilitates passage of T. gondii (Izawa et al, 2017).…”

The hitchhiker's guide to parasite dissemination

“…Somewhat perplexingly, these studies reported that infection inhibited integrin‐mediated adherence in vitro yet enhanced endothelial crawling in a manner sensitive to antibody blockade of integrin–ligand pairings (Cook et al, ; Harker et al, ). A separate study reported that T. gondii infection transiently decreases adherence of peritoneal macrophages and J774 macrophages to the integrin ligands fibronectin, laminin, and collagen IV (Ga Dama, Ribeiro‐Gomes, Guimaraes, & Arnholdt, ).…”

“…Alternatively, T. gondii infection might reprogram monocyte motility to promote dissemination. This idea is suggested by a collection of in vitro studies that showed that infection alters the rolling and crawling of monocytes interacting with endothelial monolayers or fibronectin-coated substrates (Cook, Ueno, & Lodoen, 2018;Harker et al, 2013;Ueno et al, 2014).…”

“…Integrin-dependent motility enables transendothelial migration of leukocytes from the blood into tissues, and interstitial migration through tissues. Integrin-independent motility only functions in confined three-dimensional spaces such as tissue interstitium ligand pairings (Cook et al, 2018;Harker et al, 2013). A separate study reported that T. gondii infection transiently decreases adherence of peritoneal macrophages and J774 macrophages to the integrin ligands fibronectin, laminin, and collagen IV (Ga Dama, Ribeiro-Gomes, Guimaraes, & Arnholdt, 2004).…”