Difference between revisions of "Team:HZAU-China/Basic Part"

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.

We fused eGFP with GSDMD-N275 (N-terminal 275 amino acids) and GSDMD FL (full length), respectively. Then the corresponding plasmids were transfected into Hela GSDMD knockout (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 through an ATP assay (CellTiter-Glo^® Luminescent Cell Viability Assay) and demonstrated that GSDMD-N275 and mutants of GSDMD FL have different abilities to induce pyroptosis (Figure 2).

Figure 1. Microscopy of the Hela GSDMD KO cells transfected with pCS2-eGFP-GSDMD FL (above) and pCS2-eGFP-GSDMD-N275 (below), 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).

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 bacterial cells (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 bacterium grew to logarithmic phase (OD = 0.6~0.8). Vector refers to bacterium containing a high copy number plasmid which only expresses TetR under the control of P_tet. CFU for vector and eGFP-GSDMD-N275 are shown in the logarithmic form (log10) (n=3).

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 located in cytoplasm after 5 min of induction and triggered pyroptosis after 30 min of induction (Figure 4). After 1.5 h of induction, Hela GSDMD KO cells underwent second necrosis caused by bacterial infection without inducer. Morphology of this process is similar to pyroptosis⁴. Thus, the population of ruptured cells was counted. There is 1.96 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. Photographs were captured 5 min, 30 min, 90 min after induction, respectively.

Figure 5.Ruptured cells in a field of view were counted.