Team:HZAU-China/Demonstrate
Our project aims is to induce pyroptosis in tumor cells through translocating a pyroptosis associate protein GSDMD by Salmonella. In fact, pyroptosis also can be triggered by the infection of Salmonella, but Hela GSDMD KO cell line undergoes apoptosis after infection. In morphology, apoptosis is characterized by the shrinkage of the cell, which is different from pyroptosis. In order to demonstrate pyroptosis is caused by the N-terminal of GSDMD (GSDMD-N275) rather than the infection of Salmonella itself, Hela GSDMD KO cell line was used in our experiment. Expression of the N-terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) is under the control of the promoter Ptet in ΔsifA Salmonella. Hela GSDMD KO cell line were infected with ΔsifA Salmonella. Inducer anhydrotetracycline (ATc) (16μg/mL) were added 3 h after infection. Microscopy shows that eGFP-GSDMD-N275 located in cytoplasm after 5 min of induction and triggering pyroptosis after 30 min of induction (Figure 1). After 1.5 h of induction, Hela GSDMD KO cells underwent the secondary necrosis caused by bacterial infection without inducer. Morphology of this process is similar to pyroptosis1. Thus, the population of ruptured cells was counted. There are 1.96 fold changes between control group and induced group (Figure 2). So, the pyroptosis of host cell in the induced group was triggered by eGFP-GSDMD-N275 not by bacterial infection. These results demonstrate that we successfully implement our goal.
Figure 1. Hela GSDMD KO cells were infected by Salmonella ΔsifA SL1344 containing high copy number plasmids which express eGFP-GSDMD-N275 under the control of ATc. Photographs were captured 5 min, 30 min, and 90 min after induction, respectively.
Figure 2. Ruptured cells in a field of view were counted (n=8).
1. Grow Hela GSDMD KO cells in a humidified 37℃, 5% CO2 tissue-culture incubator.
2. Count the cells using a hemocytometer. Seed in 24-well (5×10^4 per well) and grow overnight.
Preparation of Bacteria
1. Grow Salmonella bacterial cells overnight (16 h) in 2 mL LB in a 15-mL tube. Incubate at 37℃ in a shaking incubator (200 rpm).
2. Subculture Salmonella bacterial cells by transferring 300 μL of the overnight culture into 5 mL of LB in a loosely capped 50-mL tube. Incubate at 37℃ in a shaking incubator (200 rpm) to late log phase.
3. Pellet 1 mL of the Salmonella bacterial cells subculture by centrifugation at 1,000×g in a microfuge for 2 min at room temperature.
4. Remove 900 μL of supernatant and gently resuspend the pellet in 900 μL PBS.
Infection
1. Aspirate media and rinse the monolayer twice with PBS.
2. Inoculate cells with bacteria cells (MOI = 100) by adding bacteria cells directly to the cell-culture supernatant.
3. Incubate for 2 h at 37℃ in 5% CO2.
4. Aspirate media and wash
5. Add fresh Grow Meduim (GM) containing 100 μg/mL gentamicin and 16 μg/mL incubate at 37℃ in 5% CO2 for 2 h.
6. Replace GM with fresh GM containing 20 μg/mL gentamicin for 1 h.
7.Add 16 μg/mL ATc for remainder of experiment.
Observation is taken after 5 min, 30 min, 90 min.
1 He, W. T. et al. Gasdermin D is an executor of pyroptosis and required for interleukin-1beta secretion. Cell research 25, 1285-1298, doi:10.1038/cr.2015.139 (2015).
