Part:BBa_K2632003

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 Gasdermin D (pore-forming domain) acts as a 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 pore causes cell swelling, rupture of the membrane and massive leakage of cytosolic contents¹.
The N-terminal of GSDMD execute the function of pyroptosis in cells.
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 test 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. 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.

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). (Click here to see method)
The N-terminal of GSDMD lyses bacteria
Expression of the N terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) is under the control of P_tet 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 exhibit cytotoxicity in bacteria.

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 express TetR under the control of P_tet. Bacterial colony-forming units (CFU) for vector and eGFP-GSDMD-N275 are shown in the logarithmic form (log10) (n=3). (Click here to see the method)
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 cell line were infected with ΔsifA SL1344. Inducer ATc (16μg/mL) were added after 3 h infetion. Microscopy shows that eGFP-GSDMD-N275 locate 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 cause by bacterial infection without inducer. Morphology of this process is similar to pyroptosis2. Thus, these population of ruptured cells were counted. There are two fold 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 capture after 5 min of induction.

Figure 5. Ruptured cells in a field of view were counted. (Click here to see the mothod)

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:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]