Team:HZAU-China/Demonstrate
Our project aims to induce pyroptosis to tumor cells through Salmonella translocate a pyroptosis associate protein GSDMD. In fact, pyroptosis also can be trigger by infection of Salmonella, but Hela GSDMD KO cell line undergoing apoptosis after infection. In morphology, apoptosis is characterised by shrinkage of the cell which is different from pyroptosis. In order to demonstrate the generation of pyroptosis is cased by the N-terminal of GSDMD (GSDMD-N275) rather than infection of Salmonella, 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 Ptet in ΔsifA Salmonella. Hela GSDMD KO cell line were infected with ΔsifA SL1344. Inducer ATc (16μg/mL) were added 3h after infection. Microscopy shows that eGFP-GSDMD-N275 locates in cytoplasm after 5 min of induction and trigger pyroptosis after 30 min of induction (Figure 1). After 1.5 h of induction, Hela GSDMD KO cells undergo second necrosis caused by bacterial infection without inducer. Morphology of this process is similar to pyroptosis2. Thus, the population of ruptured cells was counted. There are 2-fold change between control group and induced group (Figure 5). 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 with ΔsifA SL1344 containing high copy number plasmids which express eGFP-GSDMD-N275 under the control of ATc. Photos were captured 5 min, 30min, 1.5h after induction, respectively.
Figure 2. Numbers of pyroptotic cells before and after ATc induction. Ruptured cells in a field of view were counted.
1. Grow Hela GSDMD KO cells in a humidified 37 °C, 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 bacteria overnight 16 h in 2 mL LB in a 15-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm).
2. Subculture bacteria by transferring 300 μL of the overnight culture into 5 mL of LB in a loosely capped 50-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm) to late log phase.
3. Pellet 1 mL of the Salmonella subculture by centrifugation at 1000 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 (MOI = 100) by adding bacteria directly to the cell-culture supernatant.
3. Incubate for 2 h at 37 °C in 5% CO2.
4. Aspirate media and wash
5. Add fresh GM containing 100 μg/mL gentamicin and 16 μg/mL incubate at 37 °C 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, 1.5 h.
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).
