Part:BBa_K2632003

N-terminal of GasderminD (1-275aa)

Pyroptosis is a form of lytic programmed cell death with inflammation. Recent studies reported that N-terminal of Gasdermin D 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. We respectively fused eGFP with GSDMD-N275, GSDMD FL (full length) and L290D, Y373D, A377D mutants of GSDMD FL. 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 (Fig.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 (Fig.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)

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 infect with ΔsifA SL1344. Inducer ATc (16μg/mL) were added after 3h infetion. Microscopy shows that eGFP-GSDMD-N275 locate in cytoplasm after 5 min of induction and trigger pyroptosis after 30 min of induction (Fig.3).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 (Fig.4). So ruptured cells in induced group were triggered pyroptosis by eGFP-GSDMD-N275 but not by bacterial infection.

Figure 3. 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 4. 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:

• 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]