This article describes a luciferase-based assay to quantify the intracellular growth of Toxoplasma gondii, a single-celled protozoan parasite. The method involves measuring luminescence emitted by luciferase after the parasite infects host cells.
Toxoplasma gondii - a single-celled protozoan - is an obligate anaerobic parasite, reproducing only inside host cells.
To quantify the parasite's intracellular growth using a luciferase-based assay, take a suspension of parasite cells expressing luciferase - an enzyme that produces luminescence upon reaction with its substrate. Add the suspension to a culture of human foreskin fibroblasts, and incubate for an adequate duration.
The parasite binds to host cell receptors and gets internalized via an endocytic process, forming a parasitophorous vacuole. The structure provides a niche for the parasite to reproduce.
Post-infection, aspirate the medium, removing the non-internalized parasites. Add a lysis buffer containing a detergent and luciferin - a substrate for luciferase. Detergent in the buffer creates pores in the cell membrane and completely lyses the cells, resulting in the release of luciferase.
The released enzyme oxidizes luciferin molecules, emitting light. Measure the luminescence intensity to determine luciferase activity. Using replicate wells containing infected host cells, measure luciferase activity at predetermined intervals post-infection.
Plot the normalized luminescence intensity versus the time intervals to obtain the fold changes in parasite growth over time. A temporal rise in intensity indicates successful parasite multiplication.
Plot the values in a logarithmic scale to calculate the curve's slope, representing the doubling time of the parasite.
To perform a luciferase-based Toxoplasma growth assay, begin by carefully aspirating the medium from pre-seeded 96-well microplate cultures of human foreskin fibroblasts, and inoculate 150 microliters of parasite-free suspension into the wells in a three-column, five-row format.
After a four-hour incubation in the cell culture incubator, carefully aspirate the medium from each well to remove the non-invaded parasites, and fill the wells in each but the first row with room temperature phenol red-free medium. Then, add 100 microliters of a 12.5 micromolar luciferase substrate solution in PBS into each well of the top row, and incubate the microplates for 10 minutes at room temperature to allow full cell lysis.
At the end of the incubation, measure the luciferase activity on a microplate reader. Each reading represents the initial number of invaded parasites at four hours post-infection. Repeat the analysis every 24 hours for the next four days without changing the medium.
To calculate the normalized fold change of parasite growth, each reading can be divided by the initial reading at four hours post-infection. The log2 of the normalized fold change of parasite growth can then be plotted against each timepoint, and subjected to a linear regression function to obtain the slope, which represents the doubling time.
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