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

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

Toxoplasma gondii‐infected natural killer cells display a hypermotility phenotype in vivo

Toxoplasma gondii is a highly prevalent intracellular protozoan parasite that causes severe disease in congenitally infected or immunocompromised hosts. T. gondii is capable of invading immune cells and it has been suggested that the parasite harnesses the migratory pathways of these cells to spread through the body. Although in vitro evidence suggests that the parasite further enhances its spread by inducing a hypermotility phenotype in parasitized immune cells, in vivo evidence for this phenomenon is scarce. Here we use a physiologically relevant oral model of T. gondii infection, in conjunction with two‐photon laser scanning microscopy, to address this issue. We found that a small proportion of natural killer (NK) cells in mesenteric lymph nodes contained parasites. Compared with uninfected ‘bystander’ NK cells, these infected NK cells showed faster, more directed and more persistent migratory behavior. Consistent with this, infected NK cells showed impaired spreading and clustering of the integrin, LFA‐1, when exposed to plated ligands. Our results provide the first evidence for a hypermigratory phenotype in T. gondii‐infected NK cells in vivo, providing an anatomical context for understanding how the parasite manipulates immune cell motility to spread through the host

From the blood to the brain: avenues of eukaryotic pathogen dissemination to the central nervous system.

Infection of the central nervous system (CNS) is a significant cause of morbidity and mortality, and treatments available to combat the highly debilitating symptoms of CNS infection are limited. The mechanisms by which pathogens in the circulation overcome host immunity and breach the blood-brain barrier are active areas of investigation. In this review, we discuss recent work that has significantly advanced our understanding of the avenues of pathogen dissemination to the CNS for four eukaryotic pathogens of global health importance: Toxoplasma gondii, Plasmodium falciparum, Trypanosoma brucei, and Cryptococcus neoformans. These studies highlight the remarkable diversity of pathogen strategies for trafficking to the brain and will ultimately contribute to an improved ability to combat life-threatening CNS disease

Toxoplasma gondii‐infected natural killer cells display a hypermotility phenotype in vivo

Toxoplasma gondii is a highly prevalent intracellular protozoan parasite that causes severe disease in congenitally infected or immunocompromised hosts. T. gondii is capable of invading immune cells and it has been suggested that the parasite harnesses the migratory pathways of these cells to spread through the body. Although in vitro evidence suggests that the parasite further enhances its spread by inducing a hypermotility phenotype in parasitized immune cells, in vivo evidence for this phenomenon is scarce. Here we use a physiologically relevant oral model of T. gondii infection, in conjunction with two‐photon laser scanning microscopy, to address this issue. We found that a small proportion of natural killer (NK) cells in mesenteric lymph nodes contained parasites. Compared with uninfected ‘bystander’ NK cells, these infected NK cells showed faster, more directed and more persistent migratory behavior. Consistent with this, infected NK cells showed impaired spreading and clustering of the integrin, LFA‐1, when exposed to plated ligands. Our results provide the first evidence for a hypermigratory phenotype in T. gondii‐infected NK cells in vivo, providing an anatomical context for understanding how the parasite manipulates immune cell motility to spread through the host

Transendothelial migration of Toxoplasma‐infected monocytes

Peripheral blood monocytes are actively infected by Toxoplasma gondii and can function as 'Trojan horses' for parasite spread in the bloodstream. Using dynamic live-cell imaging, we visualized the transendothelial migration (TEM) of T. gondii-infected primary human monocytes during the initial minutes following contact with human endothelium. On average, infected and uninfected monocytes required only 9.8 and 4.1 min, respectively, to complete TEM. Infection increased monocyte crawling distances and velocities on endothelium, but overall TEM frequencies were comparable between infected and uninfected cells. In the vasculature, monocytes adhere to endothelium under the conditions of shear stress found in rapidly flowing blood. Remarkably, the addition of fluidic shear stress increased the TEM frequency of infected monocytes 4.5-fold compared to static conditions (to 45.2% from 10.3%). Infection led to a modest increase in expression of the high-affinityconformation of the monocyte integrin Mac-1 (CD11b/CD18), and Mac-1 accumulated near endothelial junctions during TEM. Blocking Mac-1 inhibited the crawling and TEM of infected monocytes to a greater degree than uninfected monocytes, and blocking the Mac-1 ligand, ICAM-1, dramatically reduced crawling and TEM for both populations. These findings contribute to a greater understanding of parasite dissemination from the vasculature into tissues

Real-time imaging of Toxoplasma -infected human monocytes under fluidic shear stress reveals rapid translocation of intracellular parasites across endothelial barriers

Peripheral blood monocytes are actively infected by Toxoplasma gondii and can function as “Trojan horses” for parasite spread in the bloodstream. Using dynamic live-cell imaging, we visualized the transendothelial migration (TEM) of T. gondii-infected primary human monocytes during the initial minutes following contact with human endothelium. On average, infected and uninfected monocytes required only 9.8 and 4.1 minutes, respectively, to complete TEM. Infection increased monocyte crawling distances and velocities on endothelium, but overall TEM frequencies were comparable between infected and uninfected cells. In the vasculature, monocytes adhere to endothelium under the conditions of shear stress found in rapidly flowing blood. Remarkably, the addition of fluidic shear stress increased the TEM frequency of infected monocytes 4.5-fold compared to static conditions (to 45.2% from 10.3%). Infection led to a modest increase in expression of the high affinity conformation of the monocyte integrin Mac-1, and Mac-1 accumulated near endothelial junctions during TEM. Blocking Mac-1 inhibited the crawling and TEM of infected monocytes to a greater degree than uninfected monocytes, and blocking the Mac-1 ligand, ICAM-1, dramatically reduced crawling and TEM for both populations. These findings contribute to a greater understanding of parasite dissemination from the vasculature into tissues