Toxoplasma gondii interactions with vascular endothelium under fluidic shear stress

Toxoplasma gondii is a highly successful parasite that infects approximately one-third of the human population and can cause fatal disease in immunocompromised individuals. Systemic parasite dissemination to organs such as the brain and eye is critical to disease pathogenesis. T. gondii can disseminate via the circulation, and both intracellular and extracellular modes of transport have been proposed. To examine the dynamics of both of these dissemination mechanisms we have developed a fluidic system combined with time-lapse fluorescence microscopy. Using this approach, we showed that T. gondii-infected primary human monocytes and THP-1 cells exhibited altered adhesion dynamics compared to uninfected monocytes: infected cells rolled at significantly higher velocities (2.5 to 4.6-fold) and over greater distances (2.6 to 4.8-fold) than uninfected monocytes before firmly adhering. Since infected monocytes appeared delayed in their transition to firm adhesion, we examined the effects of infection on integrin expression and function. T. gondii did not affect the expression of LFA-1, VLA-4, or MAC-1 or the ability of Mn2+ to activate these integrins. However, T. gondii infection impaired LFA-1 and VLA-4 clustering and pseudopod extension in response to integrin ligands.;We then applied this same fluidic system to questions of extracellular parasite adhesion to endothelium and showed that shear force influenced parasite adhesion and motility dynamics and the outcome of parasite interactions with endothelium. Extracellular parasites were capable of adhesion to primary human endothelium in shear stress conditions, and interestingly, shear stress enhanced T. gondii helical gliding, resulting in a significantly greater displacement. In addition, shear stress increased the percentage of tachyzoites that invaded or migrated across the endothelium. By examining T. gondii deficient in the adhesion protein MIC2, we found that MIC2 contributed to initial adhesion but was not required for adhesion strengthening. These data suggest that in fluidic conditions, T. gondii adhesion to endothelium may be mediated by a multistep cascade of interactions that is governed by unique combinations of adhesion molecules.;This dissertation work has led to a better understanding of the mechanisms by which T. gondii interacts with and migrates across endothelium into tissues, where the parasites ultimately cause disease.