Shigella entry unveils a calcium/calpain-dependent mechanism for inhibiting sumoylation
- Institut Pasteur, France
- INSERM, U993, France
- INSERM, U786, France
Figures
Figure 1 with 2 supplements
Shigella infection induces a massive loss in SUMO conjugates in vitro and in vivo.
(A) SUMO1-conjugated protein patterns from uninfected HeLa cells or cells infected with the wild-type Shigella strain M90T for the indicated times. Immunoblot analysis was performed on whole-cell …
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Figure 1—source data 1
Quantification of the immunoblot signals relative to Figure 1D–G.
- https://doi.org/10.7554/eLife.27444.006
Figure 1—figure supplement 1
Shigella infection induces a massive loss in SUMO conjugates in vitro and in vivo.
(A) SUMO2/3-conjugated protein patterns from uninfected HeLa cells or cells infected with the wild-type Shigella strain M90T for the indicated times. Immunoblot analysis was performed on whole-cell …
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Figure 1—figure supplement 1—source data 1
Source data file relative to Figure 1—figure supplement 1B and E.
- https://doi.org/10.7554/eLife.27444.004
Figure 1—figure supplement 2
Study of a panel of Shigella mutant strains for their ability to induce a loss in SUMO conjugates.
(A) SUMO1 patterns of HeLa cells stably expressing TAP-SUMO1, uninfected or infected for 120 min with the wild-type Shigella strain M90T or with mxiD, mxiE or ospG mutants. Immunoblot analysis was …
Figure 2
Shigella inhibits sumoylation by a calpain-dependent mechanism.
(A) HeLa cells were pretreated by vehicle (DMSO) or 100 μM MDL28170 for 1 hr and then left uninfected or infected with the M90T strain or the mxiD mutant for 2 hr. Immunoblot analysis were performed …
Figure 3
Intracellular calcium levels regulate global sumoylation.
(A) HeLa cells were untreated or pretreated with the calcium-chelating agent BAPTA-AM (10 μM) for 1 hr and then left uninfected or infected with the M90T strain for 2 hr. Immunoblot analysis were …
Figure 4 with 1 supplement
SAE2 is a direct calpain substrate.
(A) HeLa cells were pretreated by vehicle (DMSO) or 100 μM MDL28170 for 1 hr and then left uninfected or infected with the M90T strain or the mxiD mutant for 2 hr. Immunoblot analysis were performed …
Figure 4—figure supplement 1
Assessment of desumoylating activities in Shigella lysates.
Reaction buffer alone (control), Shigella M90T non-denatured lysates, or recombinant SENP2 (40 nM) used here as a positive control were incubated with SUMO1-AMC (left) or SUMO2-AMC (right) and their …
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Figure 4—figure supplement 1—source data 1
Source data file relative to Figure 4—figure supplement 1.
- https://doi.org/10.7554/eLife.27444.011
Figure 5 with 1 supplement
Impact of sumoylation on Shigella infection.
(A) HeLa cells were treated with siRNAs for UBC9 and SAE2 or a control siRNA (siScr). Immunoblot analysis were performed using anti-SUMO1, -UBC9, -SAE2 and -tubulin antibodies. (B) Percentage of Shig…
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Figure 5—source data 1
Percentage of Shigella internalization and quantification of the average number of actin foci in cells impaired for sumoylation (Figure 5B, (C,F and G).
- https://doi.org/10.7554/eLife.27444.014
Figure 5—figure supplement 1
SUMO loss does not lead to marked alteration of the actin cytoskeleton.
(A) HT1080 cells, stably expressing an auxin-inducible degron to destroy UBC9 (manuscript in preparation), were left untreated or treated for 24 hr with 200 µM auxin to inhibit sumoylation. …
Figure 6 with 1 supplement
Sumoylation of RhoGDIα regulates Shigella internalization in epithelial cells.
(A) HeLa cells were treated with siRNA for RhoGDIα or a control siRNA (siScr). Immunoblot analysis was performed using anti-RhoGDIα and -tubulin antibodies. (B) Percentage of Shigella …
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Figure 6—source data 1
Source data file relative to Figure 6B, E and H.
- https://doi.org/10.7554/eLife.27444.017
Figure 6—figure supplement 1
Conservation of the sumoylation site within RhoGDIα amino acid sequence in various species.
Alignment has been done using the CLUSTALW2 online software (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Sequences used are from the following organisms: Bos taurus (GDIR1_BOVIN, P19803), Equus …
Figure 7 with 1 supplement
Endogenous RhoGDIα and SUMO are localized at Shigella-induced actin foci.
(A) SUMO1 accumulates at Shigella (M90T) entry sites. Representative ApoTome-generated micrographs of Shigella-infected Ubc9 WT or Ubc9 KO MEFs after 10 min infection. Samples were fixed and …
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Figure 7—source data 1
Source data files relative to Figure 7B, D and F.
- https://doi.org/10.7554/eLife.27444.020
Figure 7—figure supplement 1
SUMO1 and SUMO2/3 accumulate at Shigella (M90T) entry site in Hela cells.
Cells were infected with Shigella strain M90T for 10 min at 37°C. Samples were fixed and processed for immunostaining using anti-SUMO1 (green, upper panel) or anti-SUMO2/3 antibody (green, lower …
Figure 8
Shigella infection leads to loss in SUMO- RhoGDIα.
Endogenous RhoGDIα is modified by SUMO1 and rapidly desumoylated upon Shigella infection. Whole-cell extracts harvested from uninfected or Shigella (M90T)-infected Hela cells (from 0 to 120 min …
Author response image 1
Sumoylation of RhoGDIα is affected by SAE2 impairment.
HeLa cells were treated with control siRNA (siScr) or siSAE2, and then co-transferred with expression vectors for GFP-RhoGDIα and SUMO1. Immunoblot analysis was performed on whole cell extracts …
Author response image 2
The virA defective mutant induces calpain activation and loss of SUMO conjugates and SAE2.
SUMO1 and SUMO2 patterns in HeLa cells, uninfected or infected for 120min with the wild-type Shigella strain M90T or mutants mxiD or virA. Immunoblot analysis was performed on whole-cell lysates …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers |
|---|---|---|---|
| strain, strain background (Shigella flexneri serotype 5a) | M90T strain | PMID: 6279518 | Taxonomy ID: 1086030 |
| strain, strain background (Shigella flexneri serotype 5a | mxiD | PMID: 8437520 | N/A |
| strain, strain background (Shigella flexneri serotype 5a) | mxiE | PMID: 12142411 | N/A |
| strain, strain background (Shigella flexneri serotype 5a) | ospG | PMID: 16162672 | N/A |
| strain, strain background (Shigella flexneri serotype 5a) | ipaB | PMID: 1582426 | N/A |
| strain, strain background (Shigella flexneri serotype 5a) | ipaC | PMID: 19165331 | N/A |
| strain, strain background (Shigella flexneri serotype 5a) | ipaC/pC57 | PMID: 19165331 | N/A |
| strain, strain background (Shigella flexneri serotype 5a) | ipaC/pC351 | PMID: 19165331 | N/A |
| strain, strain background (Shigella flexneri serotype 5a) | virA | PMID: 22423964 | N/A |
| strain, strain background (Shigella flexneri serotype 5a) | ipgD | PMID: 8478058 | N/A |
| cell line (Hela) | CCL-2 | ATCC | ATCC CCL2/CVCL_0030 |
| cell line (HT1080) | CCL-121 | ATCC | ATCC CCL-121/CVCL_0317 |
| cell line (HT1080) | GFP-PML-IV | PMID: 23530056 | N/A |
| cell line (Hela) | TAP-SUMO1 | PMID: 25097252 | N/A |
| cell line (Hela) | TAP-SUMO2 | PMID: 25097252 | N/A |
| cell line (HT1080) | UBC9-auxin degron | this paper | N/A |
| genetic reagent (siRNA) | control | Dharmacon | #D-001810–10 |
| genetic reagent (siRNA) | Capns1 | Dharmacon | #L-009979–00 |
| genetic reagent (siRNA) | Ubc9 | Dharmacon | #L-004910–00 |
| genetic reagent (siRNA) | SAE2 | Dharmacon | #L-005248–01 |
| genetic reagent (siRNA) | RhoGDIα | Dharmacon | #L-016253–00 |
| transfected construct (Plasmid) | pEGFP-RhoGDIα WT | PMID: 22393046 | N/A |
| transfected construct (Plasmid) | pEGFP-RhoGDIα K138R | PMID: 22393046 | N/A |
| antibody | anti-SAE1 | Abcam | #ab97523/AB_10681015 |
| antibody | anti-SAE2 | Abcam | #ab22104/AB_446785 |
| antibody | anti-SUMO1 | Abcam | Y299/AB_778173 |
| antibody | anti-SUMO2/3 | Abcam | 8A2/AB_1658424 |
| antibody | anti-UBC9 | Abcam | EP2938Y/AB_1267373 |
| antibody | anti-SUMO1 | DSHB Iowa | 21C7/AB_2198257 |
| antibody | anti-Calpastatin | Cell Signaling Technology | #4146/AB_2244162 |
| antibody | anti-SAE2 | Cell Signaling Technology | D15C11/AB_10889561 |
| antibody | anti-Cdc42 | Cell Signaling Technology | 11A11/AB_10695738 |
| antibody | anti-RhoA | Cell Signaling Technology | 67B9/AB_10693922 |
| antibody | anti-Tubulin | Cell Signaling Technology | DM1A/AB_1904178 |
| antibody | anti-RanGAP1 | Santa Cruz | C-5/AB_2176987 |
| antibody | anti-GFP | Santa Cruz | C-2 |
| antibody | anti-RhoGDIα | Merck Millipore | #06–730/AB_310229 |
| antibody | anti-Capns1 | Merck Millipore | MAB3083/AB_2070014 |
| antibody | anti-Rac1 | Merck Millipore | 23A8/AB_309712 |
| antibody | anti-LPS | PMID: 25097252 | N/A |
| antibody | anti-SP100 | PMID: 7559785 | N/A |
| chemical compound, drug | Phalloidin–Tetramethylrhodamine B isothiocyanate | Sigma | P1951 / AB_2315148 |
| chemical compound, drug | Dapi | Sigma | D9542 |
| chemical compound, drug | Cytochalasin D | Sigma | C8273 |
| chemical compound, drug | MDL 28170 | Sigma | M6690 |
| chemical compound, drug | Ionomycin | Sigma | I3909 |
| chemical compound, drug | BAPTA-AM | Enzo life sciences | BML-CA411-0025 |
| chemical compound, drug | Indole-3-acetic acid | Sigma | I5148 |
| chemical compound, drug | N-Ethylmaleimide | Sigma | E3876 |
| peptide, recombinant protein | Recombinant SAE2 | Novus biologicals | NBP2-50574-20ug |
| peptide, recombinant protein | Recombinant Calpain-1 | Merck Millipore | 208712 |
| peptide, recombinant protein | GST-SENP2cat | this paper | NP_06760.2 |
| peptide, recombinant protein | SUMO1-AMC | Boston Biochem | UL-551 |
| peptide, recombinant protein | SUMO2-AMC | Boston Biochem | UL-758 |
| software, algorithm | Icy software | Institut Pasteur | PMID: 22743774 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.27444.022
