Plate-Based Quantification of Stimulated Toxoplasma Egress

Abstract

Apicomplexans are obligate parasites that replicate inside host cells, within a subcellular compartment called the parasitophorous vacuole. Egress is the process by which apicomplexan parasites like Toxoplasma gondii exit from host cells, rupturing the parasitophorous vacuole and host-cell plasma membranes in the process. T. gondii retains the ability to egress throughout most of its intracellular replicative cycle, and this process has been associated with parasite signaling pathways that include the modulation of intracellular calcium, cyclic nucleotides, phosphatidic acid, and pH, which can be manipulated genetically or pharmacologically. Here we describe two methods of assessing stimulated parasite egress from host cells by measuring the permeabilization of host-cell membranes that occurs during this process. The first method measures the release of lactate dehydrogenase (LDH) from host cells, which is quantified in a colorimetric assay that detects LDH by the enzymatic generation of red formazan. The second method measures entry of the cell-impermeant 4′,6-diamidino-2-phenylindole (DAPI) DNA dye, which stains host-cell nuclei (HCN) as parasites egress. Both described methods complement, with higher throughput, video-microscopy approaches that are well suited to examine the dissociation of parasite vacuoles that follows host-cell permeabilization.