Team:HZAU-China/Results
SifA maintains the integrity of Salmonella-containing vacuole (SCV) where Salmonella survive and replicate1. The existence of SCV limits the releasing of GSDMD-N275 into cytoplasm. In addition, the growth of inhibition of ΔsifA mutant in macrophage is remarkable2. Thus, we knocked out the sifA gene in order to decrease the stability of SCV and reduce the virulence of Salmonella.
ΔsifA mutant was constructed by using gene editing system based on two-step allelic exchange3.
PCR verification indicated that the chromosomal gene sifA was knocked out (Figure 1). Primers named DSIFA F and DSIFA R were used in this PCR (Figure 2).
Figure 1. PCR verification of sifA gene knock out. Lane 1 refers to ΔsifA mutant. Lane 2 refers to WT Salmonella enterica serovar Typhimurium strain SL1344 as a control.
Figure 2. DSIFA F and DSIFA R are the primers in the ORF of sifA. This pair of primers can extend a 436bp product in WT.
SifA is essential for maintaining vacuolar membrane stability. Cytosolic bacterium can be generated by the function loss of SCV. This population of bacterium was easily recognized and cleaned up by macrophage. Hence, ΔsifA mutant decreases the toxicity to the host. Microscopy demonstrated that ΔsifA mutant was defective for replication in macrophage (Figure 3).
Figure 3. Microscopy of immortalized bone-marrow-derived macrophages (iBMDM) infected with the ΔsifA mutant and WT Salmonella SL1344, respectively. These strains contain high copy number plasmids to express RFP constitutively.
1. Grow iBMDMs in a humidified 37 °C, 5% CO2 tissue-culture incubator.
2. Count the cells using a hemocytometer. Seed in 24-well (5×10^4 per well) and grow overnight .
Preparation of the Bacterial Cells
1. Grow bacterial cells overnight 16 h in 2 mL LB in a 15-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm).
2. Subculture bacterial cells by transferring 300 μL of the overnight culture into 5 mL of LB in a loosely capped 50-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm) to late log phase.
3. Pellet 1 mL of the Salmonella bacteria cells subculture by centrifugation at 1,000×g in a microfuge for 2 min at room temperature.
4. Remove 900 μL of supernatant and gently resuspend the pellet in 900 μL PBS.
Infection
1. Aspirate media and rinse the monolayer twice with PBS.
2. Inoculate cells with bacterial cells (MOI = 20) by adding bacterial cells directly to the cell-culture supernatant. Centrifugate at 110 g for 5 min at room temperature.
3. Incubate for 25 min at 37 °C in 5% CO2.
4. Aspirate media and rinse the monolayer twice with PBS, to remove extracellular bacterial cells.
5. Add fresh GM containing 100 μg/mL gentamicin and incubate for 2 h at 37 °C in 5% CO2.
6. Replace GM with fresh GM containing 20 μg/mL gentamicin for remainder of experiment.
Observation is taken after 11 h.
RGD motif can specifically bind to alpha V beta 3 (αvβ3), a well-known biomarker on the surface of tumor cells. We express RGD motif fused with OmpA to surface display it on the outer membrane of bacterial cells. Microscopy shows that bacterial cells expressed Lpp-OmpA-RGD induced by 0.1 mM IPTG can bind to αvβ3-positive MDA-MB-231 cell line (Figure 4),but cannot bind to αvβ3-negative MCF7 cell line (Figure 5). These results demonstrated that the bacterium gain the function of tumor targeting though display RGD motif.
Figure 4. Microscopy of αvβ3-positive MDA-MB-231 cell line were incubated with E. coli which constructively expressed RFP and inductively expressed RGD motif under the control of lac promoter. The locations of bacteria cells are pointed by red arrow.
Figure 5. Microscopy of αvβ3-negative MCF7 cell line were incubated with E. coli which constructively expressed RFP and inductively expressed RGD motif under the control of lac promoter.
1. Grow MDA-MB-231 and MCF7 in a humidified 37 °C, 5% CO2 tissue-culture incubator.
2. Count the cells using a hemocytometer. Seed in 24-well (1.5× 10^5 per well) and grow overnight.
Preparation of Bacterial cells
1. Grow bacterial cells overnight 14 h in 2 mL LB in a 15-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm).
2. Subculture bacterial cells by transferring 300 μL of the overnight culture into 5 mL of LB containing 0.1 mM IPTG in a loosely capped 50-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm) to early stationary phase.
3. Pellet 1 mL of the bacterial cells subculture by centrifugation at 1000 g in a microfuge for 2 min at room temperature.
4. Remove 900 μL of supernatant and gently resuspend the pellet in 900 μL PBS.
Infection 1. Aspirate media and rinse the monolayer twice with PBS.
2. Inoculate cells with bacterial cells (MOI = 100) by adding bacterial cells directly to the cell-culture supernatant.
3. Incubate for 1.5 h at 37 °C in 5% CO2.
4. Aspirate media and rinse the monolayer three times with PBS.
Observation is taken immediately.
We fused eGFP with GSDMD-N275 (N-terminal 275 amino acid) and GSDMD FL (full length), respectively. Then the corresponding plasmids were transfected into Hela GSDMD KO cell. Cell microscopy showed that the cells transfected with GSDMD-N275 underwent pyroptosis while the cells with GSDMD FL did not (Figure 6). 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 7).
Figure 6. 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.
1. Grow Hela GSDMD KO cells in a humidified 37 °C, 5% CO2 tissue-culture incubator.
2. Count the cells using a hemocytometer. Seed in 24-well (5 × 10^4 per well) and grow.
Transfection
1. Dilute 0.5 μg DNA into 50 μl jetPRIME® buffer (supplied). Mix by vortexing.
2. Add 1 μl jetPRIME®, vortex for 10 s, spin down briefly.
3. Incubate for 10 min at RT.
4. Add 50μl of transfection mix per well drop wise onto the cells in serum containing medium, and distribute evenly.
5. Gently rock the plates back and forth and from side to side.
6. If needed, replace transfection medium after 4 h by cell growth medium and return the plates to the incubator.
Observation is taken after 1.5 h.
Figure 7. 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).
1. Grow HEK293T cells in a humidified 37 °C, 5% CO2 tissue-culture incubator.
2. Count the cells using a hemocytometer. Seed in 96-well (1 × 10^4 per well) and grow overnight.
3. Transfect 0.5 μg DNA per well.
4. Equilibrate the plate and its contents at room temperature for approximately 30 minutes after 20 h.
5. Add a volume of CellTiter-Glo® Reagent equal to the volume of cell culture medium present in each well. (add 100 μl of reagent to 100 μl of medium containing cells for a 96-well plate).
6. Mix contents for 2 minutes on an orbital shaker to induce cell lysis.
7. Allow the plate to incubate at room temperature for 10 minutes to stabilize luminescent signal.
8. Record luminescence.
PsifA is a intracellular environment-dependent promoter. SipD is required for bacterial internalization. ΔsipD mutant cannot enter host cells. Thus, we use this strain as a control. Microscopy suggested that only intracellular bacteria express eGFP which under the control of PsifA (Figure 8).
Figure 8. Microscopy of hela GSDMD KO cells infected with the ΔsipD or ΔsifA mutant, respectively. These mutants contain low copy number plasmids to express eGFP which was regulated by PsifA.
1. Grow Hela GSDMD KO cells in a humidified 37 °C, 5% CO2 tissue-culture incubator.
2. Count the cells using a hemocytometer. Seed in 24-well (9× 10^4 per well) and grow overnight.
Preparation of Bacterial Cells
1. Grow bacterial cells overnight 16 h in 2 mL LB in a 15-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm).
2. Subculture bacterial cells by transferring 300 μL of the overnight culture into 5 mL of LB in a loosely capped 50-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm) to late log phase.
3. Pellet 1 mL of the Salmonella subculture by centrifugation at 1000 g in a microfuge for 2 min at room temperature.
4. Remove 900 μL of supernatant and gently resuspend the pellet in 900 μL PBS.
Infection
1. Aspirate media and rinse the monolayer twice with PBS.
2. Inoculate cells with bacterial cells (MOI = 100) by adding bacterial cells directly to the cell-culture supernatant.
3. Incubate for 3 h at 37 °C in 5% CO2.
4. Aspirate media and rinse the monolayer twice with PBS.
5. Add fresh GM containing 100 μg/mL gentamicin and incubate at 37 °C in 5% CO2. Observation is taken after 2 h.
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 bacterial cells (Figure 9). This result shows that eGFP-GSDMD-N275 exhibits cytotoxicity in bacteria.
Figure 9. 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 grown to logarithmic phase (OD = 0.6~0.8). Vector refers to bacterium containing a high copy number plasmid which only express TetR under the control of Ptet. CFU for vector and eGFP-GSDMD-N275 are shown in the logarithmic form (log10) (n=3).
2. When OD reaching to 0.6-0.8, add anhydrotetracycline with final concentration of μg/ml to induce the expression of EGFP-GSDMD-N275.
3. Take 100 μl diluted culture to plate on LB agar plates containing appropriate concentration of antibody after 1.5 hours of induce.
Observation is taken overnight.
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 10). 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 11). So the pyroptosis of host cell in the induced group was triggered by eGFP-GSDMD-N275 not by bacterial infection.
In these experiment, the choice of MOI and infection time were conducted by Modeling.
Figure 10. 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 11. Ruptured cells in a field of view were counted.
1. Grow Hela GSDMD KO cells in a humidified 37 °C, 5% CO2 tissue-culture incubator.
2. Count the cells using a hemocytometer. Seed in 24-well (5 × 10^4 per well) and grow overnight.
Preparation of Bacteria
1. Grow bacteria overnight 16 h in 2 mL LB in a 15-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm).
2. Subculture bacteria by transferring 300 μL of the overnight culture into 5 mL of LB in a loosely capped 50-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm) to late log phase.
3. Pellet 1 mL of the Salmonella subculture by centrifugation at 1,000×g in a microfuge for 2 min at room temperature.
4. Remove 900 μL of supernatant and gently resuspend the pellet in 900 μL PBS.
Infection
1. Aspirate media and rinse the monolayer twice with PBS.
2. Inoculate cells with bacteria (MOI = 100) by adding bacteria directly to the cell-culture supernatant.
3. Incubate for 2 h at 37 °C in 5% CO2.
4. Aspirate media and wash.
5. Add fresh GM containing 100 μg/mL gentamicin and 16 μg/mL incubate at 37 °C in 5% CO2.
Observation is taken after 5 min, 30 min, 90min.
1 Dumont, A. et al. SKIP, the host target of the Salmonella virulence factor SifA, promotes kinesin-1-dependent vacuolar membrane exchanges. Traffic 11, 899-911, doi:10.1111/j.1600-0854.2010.01069.x (2010).
2 Thurston, T. L. et al. Growth inhibition of cytosolic Salmonella by caspase-1 and caspase-11 precedes host cell death. Nature communications 7, 13292, doi:10.1038/ncomms13292 (2016).
3 Hmelo, L. R. et al. Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange. Nat Protoc 10, 1820-1841, doi:10.1038/nprot.2015.115 (2015).
4 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).
