Difference between revisions of "Part:BBa K2632003"
 
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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 contents<sup>1</sup>.
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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 contents<sup>1</sup>.
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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 (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).  
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<img src="https://static.igem.org/mediawiki/parts/0/05/T--HZAU-China--Part-GSDMD-N-Figure1.png" width="900px"/>
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<h2>The N-terminal of GSDMD performs the function of pyroptosis in cells</h2>
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We fused eGFP with GSDMD-N275 and GSDMD FL (full length), respectively. Then the corresponding plasmids were transfected into Hela GSDMD KO (knockout) cell. Cell microscopy showed that the cells transfected with GSDMD-N275 underwent pyroptosis while the cells with GSDMD FL did not (<b>Figure 1</b>). We also tested the cell viability though an ATP assay (CellTiter-Glo<sup>®</sup> Luminescent Cell Viability Assay) and demonstrated that GSDMD-N275 and mutants of GSDMD FL have different ability to induce pyroptosis (<b>Figure 2</b>).
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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.  
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<b>Figure 1.</b> 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.
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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)
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<img src="https://static.igem.org/mediawiki/2018/0/0f/T--HZAU-China--GSDMD-mutation-viability.png" width="700px"/>
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<b>Figure 2.</b> 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).</p>
 
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<h2>The N-terminal of GSDMD lyses bacteria</h2>
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Expression of the N terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) in <i>Salmonella enterica</i> serovar Typhimurium str. SL1344 <i>ΔsifA</i>
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                    is under the control of Ptet. The colony-forming unit (CFU) was measured for counting the number of viable bacteria (<b>Figure 3</b>).
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                    The result shows that eGFP-GSDMD-N275 exhibits cytotoxicity to bacteria.
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<b>Figure 3.</b> CFU comparison between the SL1344 <i>ΔsifA</i> cells with eGFP-GSDMD-N275 plasmid and with the empty vector.
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                    In each group, ATc (15μg/ml) was added into medium when bacteria grew to logarithmic phase (OD = 0.6~0.8).
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                    Vector refers to bacteria containing a high copy number plasmid which only expresses TetR under the control of P<sub>tet</sub> .
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                    Bacterial colony-forming units (CFU) for vector and eGFP-GSDMD-N275 are shown in the logarithmic form (log10) (n = 3).
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<h2>The N-terminal of GSDMD from lytic bacteria induces cell pyroptosis</h2>
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Expression of the N-terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) is under the control of tet promoter in <i>ΔsifA</i> SL1344.
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                    Hela GSDMD KO cells were infected with <i>ΔsifA</i> SL1344. Inducer ATc (16μg/mL) was added 3h after infection.
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                    Microscopy shows that eGFP-GSDMD-N275 locates in cytoplasm after 5 min of induction and triggers pyroptosis after 30 min of induction (<b>Figure 4</b>).
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                      By counting the population of ruptured cells, there is a 1.96 fold-change between the induced group and the control group (<b>Figure 5</b>).
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                      So the pyroptosis of host cell in the induced group was triggered by eGFP-GSDMD-N275.
  
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Expression of the N terminal of GSDMD fused with eGFP (eGFP-GSDMD-N275) is under the control of tet promoter in <i>ΔsifA</i> SL1344. Hela GSDMD KO cell line were infect with <i>ΔsifA</i> 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 (Figure 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 pyroptosis<sup>2</sup>. Thus, these population of ruptured cells were counted. There are two fold change between control group and induced group (Figure 4). So  ruptured cells in induced group were triggered pyroptosis by eGFP-GSDMD-N275 but not by bacterial infection.
 
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Figure 3. Hela GSDMD KO cell line were infected with <i>ΔsifA</i> SL1344 containing high copy number plasmids which express eGFP-GSDMD-N275 under the control of ATc. Photos were capture after 5 min of induction.  
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<b>Figure 4.</b> Hela GSDMD KO cells were infected with <i>ΔsifA</i> SL1344 containing high copy number plasmids which express eGFP-GSDMD-N275 under the control of ATc.  
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                    Photos were captured 5 min, 30min, 1.5h after induction, respectively.
 
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<img src="https://static.igem.org/mediawiki/parts/6/62/T--HZAU-China--Part-GSDMD-N-Figure4.png" width="700px"/>
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Figure 4. Ruptured cells in a field of view were counted.
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<b>Figure 5.</b> Numbers of pyroptotic cells before and after ATc induction. Ruptured cells in a field of view were counted.
 
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<h3>Reference</h3>
 
<h3>Reference</h3>
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1. Ding, J. et al. Pore-forming activity and structural autoinhibition of the gasdermin family. Nature 535, 111-116, doi:10.1038/nature18590 (2016).
 
1. Ding, J. et al. Pore-forming activity and structural autoinhibition of the gasdermin family. Nature 535, 111-116, doi:10.1038/nature18590 (2016).
 
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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).
 
 
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Latest revision as of 23:04, 17 October 2018


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 performs 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 (knockout) 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 Ptet. The colony-forming unit (CFU) was measured for counting the number of viable bacteria (Figure 3). The result shows that eGFP-GSDMD-N275 exhibits cytotoxicity to 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 grew 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).

The N-terminal of GSDMD from lytic bacteria induces 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) was added 3h after infection. Microscopy shows that eGFP-GSDMD-N275 locates in cytoplasm after 5 min of induction and triggers pyroptosis after 30 min of induction (Figure 4). By counting the population of ruptured cells, there is a 1.96 fold-change between the induced group and the control group (Figure 5). So the pyroptosis of host cell in the induced group was triggered by eGFP-GSDMD-N275.

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).

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]