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

Joshua H Cook,Norikiyo Ueno,Melissa B Lodoen

doi:10.1074/jbc.m117.793281

Joshua H Cook, Norikiyo Ueno + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m117.793281

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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 investigated the molecular basis for the hypermotility of primary human peripheral blood monocytes and THP-1 cells infected with T. gondii Compared with 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 lipopolysaccharide (LPS) or Escherichia coli Furthermore, T. gondii infection disrupted the phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 (Tyr-397) and Tyr-925 and of the related protein proline-rich tyrosine kinase (Pyk2) at Tyr-402. 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. gondii-infected monocytes. Using time-lapse confocal microscopy of THP-1 cells expressing enhanced GFP (eGFP)-FAK during settling on fibronectin, we found that T. gondii-induced monocyte hypermotility was characterized by a reduced number of enhanced GFP-FAK-containing clusters over time compared with 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. gondii-induced monocyte hypermotility.

Highlights

Monocytes are migratory cells that circulate in the blood and home to sites of infection or injury by extravasating from the
Clustering of activated ␤1 integrins is reduced in T. gondii–infected monocytes settled on fibronectin
We investigated the effect of T. gondii infection on the ability of activated ␤1 integrins to cluster, the initial step in focal adhesion formation

Summary

Results

Clustering of activated ␤1 integrins is reduced in T. gondii–infected monocytes settled on fibronectin. There was a statistically detectable reduction in the area of adhesions marked by either paxillin or vinculin (Fig. 5C), the magnitude of these changes was not large These data suggest that the decrease in p-FAK Tyr-397 and p-Pyk Tyr-402 in infected cells resulted from fewer ␤1 integrin clusters and was associated with an impairment of paxillin, FAK, and vinculin localization to focal adhesions. Uninfected monocytes spread over the fibronectin surface and formed structures resembling focal adhesions, which were visible as small regions of high eGFP-FAK density (Fig. 6A) These structures increased in mock-infected cells over time, during the first 15 min of settling, after which point, they appeared to stabilize (Fig. 6B). The T. gondii–infected cells changed shape rapidly as they roamed over the ECM (Fig. 6, A and C and Video S2) and moved at significantly higher speeds (Fig. 6D) than uninfected cells, consistent with the increased motility of T. gondii–infected monocytes [28] and the microscopy analyses of cells fixed at specific time points after settling on fibronectin (Fig. 4A)

Discussion

Mammalian and parasite cell culture and infections

Flow cytometry

Western blotting and densitometry