Team:HZAU-China/Public Engagement
Pyroptosis is a form of lytic programmed cell death with inflammation. Recent studies reported that the N-terminal of Gasdermin D (pore-forming domain) acts as an effector of pyroptosis. Full length Gasdermin D is cleaved by Caspase 1 then release the PFD (pore-forming domain) which can oligomerize on the cell membrane. Formation of pores causes cell swelling, rupturing of the membrane and the massive leakage of cytosolic contents1.
We respectively fused eGFP with GSDMD-N275 and GSDMD FL (full length). Then these plasmids were transfected into Hela GSDMD KO cell. Microscopy of cells transfecting GSDMD-N275 undergoing pyroptosis, but GSDMD full length did not induce pyroptosis (Figure 1). We also tested the cell viability through ATP assay (CellTiter-Glo® Luminescent Cell Viability Assay) and demonstrated that GSDMD-N275 and mutants of GSDMD FL had different ability to induce pyroptosis (Figure 2).
Figure 1. pCS2-eGFP-GSDMD FL（left）, pCS2-eGFP-GSDMD-N275（right） were transfected respectively into Hela G¬SDMD KO cells. Pyroptotic cells are pointed by red arrow.
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.
Transfection
1. Dilute 0.5 μg DNA into 50 μl jetPRIME® buffer (supplied). Mix by vortexing.
2. Add 1 μl jetPRIME®, vortex for 10 s, spin down briefly.
3. Incubate for 10 min at RT.
4. Add 50μl of transfection mix per well drop wise onto the cells in serum containing medium, and distribute evenly.
5. Gently rock the plates back and forth and from side to side.
6. If needed, replace transfection medium after 4 h by cell growth medium and return the plates to the incubator.
Observation is taken after 1.5 h
Figure 2. pCS2-Flag-GSDMD FL, pCS2-Flag-GSDMD-N275, pCS2-Flag-GSDMD L290D, pCS2-Flag-GSDMD Y373, pCS2-Flag-GSDMD A377D were transfected respectively into 293T cells. ATP-based cell viability was measured (n=6).
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 (9× 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 3 h at 37 °C in 5% CO2.
4. Aspirate media and rinse the monolayer twice with PBS.
5. Add fresh GM containing 100 μg/mL gentamicin and incubate at 37 °C in 5% CO2.
Observation is taken after 2 h.
Expression of the N terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) was under the control of Ptet in ΔsifA SL1344. The colony-forming unit (CFU) was measured for counting the number of viable bacteria (Figure 3). This result shows that eGFP-GSDMD-N275 exhibits cytotoxicity in bacteria.
Figure 3. In each group, ATc (16μg/ml) was added into medium when bacteria grown to logarithmic phase (OD = 0.6~0.8). Vector refers to bacteria containing a high copy number plasmid which only expresses TetR under the control of Ptet. Bacterial colony-forming units (CFU) for vector and eGFP-GSDMD-N275 are shown in the logarithmic form (log10) (n=3).
2. When OD reaching to 0.6-0.8, add anhydrotetracycline with final concentration of μg/ml to induce the expression of EGFP-GSDMD-N275.
3. Take 100 μl diluted culture to plate on LB agar plates containing appropriate concentration of antibody after 1.5 hours of induce.
Observation is taken overnight.
Expression of the N-terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) was under the control of Tet promoter in ΔsifA SL1344. Hela GSDMD KO cell line were infected with ΔsifA SL1344. Inducer ATc (16μg/mL) were added after 3 h infection. Microscopy showed that eGFP-GSDMD-N275 located in cytoplasm after 5 min of induction and trigger pyroptosis after 30 min of induction (Figure 4). 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, these population of ruptured cells were counted. There are two folds change between control group and induced group (Figure 5). So ruptured cells in induced group were triggered pyroptosis by eGFP-GSDMD-N275 but not by bacterial infection.
Figure 4. Hela GSDMD KO cell line were infected with ΔsifA SL1344 containing high copy number plasmids which express eGFP-GSDMD-N275 under the control of ATc. Photos were captured after 5 min of induction.
Figure 5. 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.
Observation is taken after 5 min, 30 min, 1.5 h.
| Name | Type | Description | Designer | Length(bp) |
|---|---|---|---|---|
| BBa_K2632002 | Promoter | Promoter sifA | Mo Qiqin | 331 |
| BBa_K2632003 | Coding | N-terminal of GasderminD (1-275aa) | Zhujun Xia | 825 |
| BBa_K2632004 | Coding | Full length Gasdermin D | Zhujun Xia | 1455 |
| BBa_K2632005 | Coding | L290D mutant of full length Gasdermin D | Zhujun Xia | 1455 |
| BBa_K2632007 | Coding | A377D mutant of full length Gasdermin D. | Zhujun Xia | 1455 |
1 Ding, J. et al. Pore-forming activity and structural autoinhibition of the gasdermin family. Nature 535, 111-116, doi:10.1038/nature18590 (2016).
2 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).
