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 (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).  
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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 (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 pyroptosis<sup>2</sup>. 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.
 
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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]