Coding
GSDMD-N275
Part:BBa_K2632003
Designed by: Zhujun Xia Group: iGEM18_HZAU-China (2018-09-15)
N-terminal of GasderminD (1-275aa)
Pyroptosis is a form of lytic programmed cell death with inflammation. Recent studies reported that the N-terminal of GSDMD acts as an effector of pyroptosis. Full length GSDMD (GSDMD FL) is cleaved by Caspase 1, releasing the pore-forming domain (GSDMD-N275) which can oligomerize and make pores on the cell membrane. Formation of pores causes cell to swell, leading to membrane rupture and massive leakage of cytosolic contents1.
The N-terminal of GSDMD execute the function of pyroptosis in cells.
We fused eGFP with GSDMD-N275 and GSDMD FL (full length), respectively. Then the corresponding plasmids were transfected into Hela GSDMD KO cell. Cell microscopy showed that the cells transfected with GSDMD-N275 underwent pyroptosis while the cells with GSDMD FL did not (Figure 1). We also tested the cell viability though an ATP assay (CellTiter-Glo® Luminescent Cell Viability Assay) and demonstrated that GSDMD-N275 and mutants of GSDMD FL have different ability to induce pyroptosis (Figure 2).
Figure 1. Microscopy of the Hela GSDMD KO cells transfected with pCS2-eGFP-GSDMD FL and pCS2-eGFP-GSDMD-N275, respectively. Pyroptotic cells are pointed by red arrow.
Figure 2. Cell viability of the 293T cells transfected with pCS2-Flag-GSDMD FL, pCS2-Flag-GSDMD-N275, pCS2-Flag-GSDMD L290D, pCS2-Flag-GSDMD Y373D, pCS2-Flag-GSDMD A377D, respectively. Asterisks indicate the statistically significant differences. ATP-based cell viability was measured (n=6).
The N-terminal of GSDMD lyses bacteria
>
Expression of the N terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) in Salmonella enterica serovar Typhimurium str. SL1344 ΔsifA
is under the control of P_tet. 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. CFU comparison between the SL1344 ΔsifA cells with eGFP-GSDMD-N275 plasmid and with the empty vector.
In each group, ATc (15μg/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 express 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).
The N-terminal of GSDMD from lytic bacteria induce cell pyroptosis.
Expression of the N-terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) is under the control of tet promoter in ΔsifA SL1344.
Hela GSDMD KO cells 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 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 pyroptosis2sup>. 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.
Figure 4. 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 5. Numbers of pyroptotic cells before and after ATc induction. Ruptured cells in a field of view were counted.
Reference
We fused eGFP with GSDMD-N275 and GSDMD FL (full length), respectively. Then the corresponding plasmids were transfected into Hela GSDMD KO cell. Cell microscopy showed that the cells transfected with GSDMD-N275 underwent pyroptosis while the cells with GSDMD FL did not (Figure 1). We also tested the cell viability though an ATP assay (CellTiter-Glo® Luminescent Cell Viability Assay) and demonstrated that GSDMD-N275 and mutants of GSDMD FL have different ability to induce pyroptosis (Figure 2).
Figure 1. Microscopy of the Hela GSDMD KO cells transfected with pCS2-eGFP-GSDMD FL and pCS2-eGFP-GSDMD-N275, respectively. Pyroptotic cells are pointed by red arrow.
Figure 2. Cell viability of the 293T cells transfected with pCS2-Flag-GSDMD FL, pCS2-Flag-GSDMD-N275, pCS2-Flag-GSDMD L290D, pCS2-Flag-GSDMD Y373D, pCS2-Flag-GSDMD A377D, respectively. Asterisks indicate the statistically significant differences. ATP-based cell viability was measured (n=6).
The N-terminal of GSDMD lyses bacteria
>Expression of the N terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) in Salmonella enterica serovar Typhimurium str. SL1344 ΔsifA is under the control of P_tet. 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. CFU comparison between the SL1344 ΔsifA cells with eGFP-GSDMD-N275 plasmid and with the empty vector. In each group, ATc (15μg/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 express 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).
The N-terminal of GSDMD from lytic bacteria induce cell pyroptosis.
Expression of the N-terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) is under the control of tet promoter in ΔsifA SL1344. Hela GSDMD KO cells 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 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 pyroptosis2sup>. 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.
Figure 4. 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 5. Numbers of pyroptotic cells before and after ATc induction. Ruptured cells in a field of view were counted.
Reference
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).
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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Categories
Parameters
| None |