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Shigella uses a type III secretion system to invade host cell and to cause disease. Secretion control and insertion of a translocation pore into cell membrane are critical steps for pathogenesis and are tightly linked to the formation of the needle tip complex formed by the IpaB and IpaD proteins (Veenendaal et al., 2007). Surface localizations of IpaD and IpaB were monitored by FACS analysis according to the localization protocol for Pseudomonas aeruginosa homolog PcrV (Lee et al., 2010).

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Shigella IpaD and IpaB Surface Localizations
志贺氏杆菌IPaD和IPAB表面定位

微生物学 > 微生物生物化学 > 蛋白质 > 免疫检测
作者: Lionel Schiavolin
Lionel SchiavolinAffiliation: Molecular Bacteriology Lab, Université Libre de Bruxelles, Anderlecht, Belgium
For correspondence: lschiavo@ulb.ac.be
Bio-protocol author page: a992
Alaeddine Meghraoui
Alaeddine MeghraouiAffiliation: Molecular Bacteriology Lab, Université Libre de Bruxelles, Anderlecht, Belgium
Bio-protocol author page: a993
 and Abdelmounnaim Allaoui
Abdelmounnaim AllaouiAffiliation: Molecular Bacteriology Lab, Université Libre de Bruxelles, Anderlecht, Belgium
Bio-protocol author page: a994
Vol 3, Iss 22, 11/20/2013, 2592 views, 1 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.975

[Abstract] Shigella uses a type III secretion system to invade host cell and to cause disease. Secretion control and insertion of a translocation pore into cell membrane are critical steps for pathogenesis and are tightly linked to the formation of the needle tip complex formed by the IpaB and IpaD proteins (Veenendaal et al., 2007). Surface localizations of IpaD and IpaB were monitored by FACS analysis according to the localization protocol for Pseudomonas aeruginosa homolog PcrV (Lee et al., 2010).

Keywords: Type 3 secretion system(3型分泌系统), Tip complex(尖端复杂的), Shigella(志贺菌), Flow cytometry(流式细胞仪)

Materials and Reagents

  1. Shigella strains
  2. Tryptic Soy Broth (TSB) (VWR International, catalog number: for Europe 1.00525.5000 and for U.S. EM1.00525.5007)
  3. Agar (MP Biomedicals, catalog number: 0210026291)
  4. Congo Red (VWR International, catalog number: for Europe 34140.184)
  5. Anti-IpaD and -IpaB polyclonal antibodies (house made)
  6. PBS (Fischer Scientific, catalog number: BP399-20)
  7. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148)
  8. Triton X-100 (VWR International, for Europe catalog number: 1.08603.1000)
  9. Trizmabase (Sigma-Aldrich, catalog number: T1503)
  10. Bovine serum albumin (BSA) (VWR International, catalog number: 422361V)
  11. Anti-mouse secondary antibody CF647-conjugated (Sigma-Aldrich, catalog number: SAB4600351)
  12. PBS + PFA 4% stock solution (see Recipes)
  13. Congo Red agar plates (see Recipes)

Equipment

  1. 250 ml Erlenmeyer
  2. Microtubes
  3. CR agar plate
  4. Polystyrene tube for FACS analysis
  5. Centrifuge Heating magnetic stirrer
  6. 37 °C shaker
  7. Rotator mixer
  8. Flow cytometry with a four-colour FACS Calibur cytometer (Becton Dickinson and Company)

Procedure

  1. Preparation of bacterial samples
    1. Launch overnight precultures in TSB (37 °C with shaking) from Congo Red (CR) positives colonies of Shigella on CR agar plates.
    2. Dilute 1: 100 precultures in fresh TSB (for volume see step A3) and incubate at 37 °C with shaking until an OD600 ≈ 1.5 is reached. (Medium must be filtered or autoclaved with stirring to avoid glucose caramelization which interferes with type III secretion.)
    3. Harvest 2 x 108 bacteria per tested conditions at 2,000 x g for 4 min at room temperature (RT) (OD600 of 1 correspond to approximately 5 x 108 bacteria).
    4. Wash twice with 500 μl of ice-cold PBS with 0.1% Triton X-100 (centrifugation conditions as in step A3). Be careful when you take out the liquid supernatant as the pellet can detach gradually all along the procedure.
    5. Resuspend in 500 μl of ice-cold PBS with 0.1% Triton X-100 and add 500 μl of PBS + PFA (4%) to fix bacteria.
    6. Mix by inversion and incubate 20 min at RT.
    7. Add 50 μl Tris HCl 1 M (pH 7.5), mix by inversion and incubate 5 min at RT to quench the cross-linker.
    8. Harvest bacteria at 10,000 x g for 2 min at RT (All further centrifugation steps are performed with these parameters.).
    9. Wash once with 1 ml PBS + 0.1% Triton X-100 and once with 1 ml PBS.

  2. Immunological staining
    1. Harvest bacteria and resuspend in 500 μl of the blocking solution (PBS + 4% BSA).
    2. Incubate bacteria on rotator mixer 1 h at 4 °C.
    3. Harvest bacteria and resuspend in 250 μl PBS + 4% BSA with mouse sera diluted 1: 100.
      Sera were from Swiss mice immunized with GST-IpaD131-332 (Schiavolin et al., 2013) or His-IpgC + IpaB (Page et al., 1999). Negative controls for IpaD and IpaB localizations are respectively an IpaD protein lacking its last residues which do not bind the needle tip (Espina et al., 2006) and an ipaD KO mutant where IpaB is not retained at the tip (Veenendaal et al., 2007). In both cases secreted IpaD or IpaB do not interfere with the assay.
    4. Incubate overnight at 4 °C with agitation.
    5. Add 750 μl PBS to bacteria and then wash twice with 1 ml PBS. All further steps are made in the dark to preserve fluorescent properties of the secondary antibody. Microtubes or rack are covered with aluminum foil.
    6. Resuspend in 250 μl of PBS + 4% BSA with goat CF647-conjugated anti-mouse IgG antibody diluted 1: 500.
    7. Agitate 1 h or longer at 4 °C (incubation time may last overnight).
    8. Add 750 μl PBS to bacteria and then wash twice with 1 ml PBS.
    9. Resuspend bacterial pellet in 500 μl of PBS and dilute 1: 10 in FACS polystyrene tube.
    10. Analyze by flow cytometry with a four-colour FACS Calibur cytometer for instance (you will find examples of results in the supporting information of the third reference (Schiavolin et al., 2013) available for free on Mol. Microbiol. website).
    11. Parameters used (no compensation):
      detectors
      voltage
      mode
      FSC
      E02
      Log
      SSC
      366
      Log
      FL4
      800
      Log

Recipes

  1. PBS + PFA 4% stock solution (100 ml)
    Weigh out 4 g of paraformaldehyde in a 250 ml erlenmeyer
    Add 80 ml of double-distilled water
    Add 50 μl of 1 M NaOH
    Add a magnetic bar and close the erlenmeyer
    Heat at 70 °C until complete solubilization (the solution should become clear)
    Put on ice and allow the solution to cool down to RT
    Adjust volume to 90 ml with double-distilled water
    Add 10 ml of PBS 10x and mix
    Filter solution with a 0.2 μm 25 mm nylon syringe filter and aliquot in 15 ml falcon tube
    Freshly prepared PFA can be stored at -20 °C for further assays (thaw gently at RT)
  2. Congo Red agar plates
    Prepare a 30 g/L TSB solution with bidistilled water and dissolve 15 g/L agar
    Autoclave the medium for 15 min at 120 °C (If you autoclave your medium for 20 min, sugar will caramelize and your plates will be darker, and even darkest after incubation with bacteria at 37 °C.)
    Add the Congo Red at a final concentration of 250 µg/ml (Stock solution is prepared in bidistilled water at a concentration of 10 mg/ml.) when the bottle can be safely handled with a protective glove.

Acknowledgments

This protocol was adapted from Lee et al. (2010). This study was supported by grants from the Belgian Fonds National de la Recherche Scientifique Médicale (FRS-FNRS; Convention 3.4556.11) and from the European Community’s Seventh framework program FP7/2011–2015 under grant agreement No. 261742. L.S. and A.M are recipients of a PhD fellowship from the Belgian Fonds National de Recherches Industrielles et Agronomiques (FRIA). A part of this work was also supported by the Fonds Defay, and by Van Buuren and Héger-Masson foundations. We thank A. Op de Beeck for her help in performing the FACS experiment.

References

  1. Espina, M., Olive, A. J., Kenjale, R., Moore, D. S., Ausar, S. F., Kaminski, R. W., Oaks, E. V., Middaugh, C. R., Picking, W. D. and Picking, W. L. (2006). IpaD localizes to the tip of the type III secretion system needle of Shigella flexneri. Infect Immun 74(8): 4391-4400.
  2. Lee, P. C., Stopford, C. M., Svenson, A. G. and Rietsch, A. (2010). Control of effector export by the Pseudomonas aeruginosa type III secretion proteins PcrG and PcrV. Mol Microbiol 75(4): 924-941. 
  3. Page, A. L., Ohayon, H., Sansonetti, P. J. and Parsot, C. (1999). The secreted IpaB and IpaC invasins and their cytoplasmic chaperone IpgC are required for intercellular dissemination of Shigella flexneri. Cell Microbiol 1(2): 183-193.
  4. Schiavolin, L., Meghraoui, A., Cherradi, Y., Biskri, L., Botteaux, A. and Allaoui, A. (2013). Functional insights into the Shigella type III needle tip IpaD in secretion control and cell contact. Mol Microbiol 88(2): 268-282.
  5. Veenendaal, A. K., Hodgkinson, J. L., Schwarzer, L., Stabat, D., Zenk, S. F. and Blocker, A. J. (2007). The type III secretion system needle tip complex mediates host cell sensing and translocon insertion. Mol Microbiol 63(6): 1719-1730.


How to cite this protocol: Schiavolin, L., Meghraoui, A. and Allaoui, A. (2013). Shigella IpaD and IpaB Surface Localizations. Bio-protocol 3(22): e975. DOI: 10.21769/BioProtoc.975; Full Text



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7/10/2014 11:17:54 AM  

Brooke Napier
Stanford University

The catalog # for the Congo Red from VWR is not correct.

Also, would be helpful to include the recipe for the CR agar plates to supplement this protocol.

7/11/2014 8:51:39 AM  

Lionel Schiavolin (Author)
Molecular Bacteriology Lab, Université Libre de Bruxelles, Belgium

Hi,

you'll find the CR from VWR at this URL:
https://uk.vwr.com/app/catalog/Product;jsessionid=6nvO4UhfRBxKo5Evg2WuAw**.node3?article_number=34140.184
I haven't found the same product from US VWR...

for CR agar plates' preparation :

- prepare a 30 g/l TSB solution with bidistilled water and dissolve 15 g/l agar.
- autoclave the medium for 15 min at 120°C (if you autoclave your medium for 20 min, sugar will caramelize and your plates will be darker, and even darkest after incubation with bacteria at 37°C)
- Add the CR at a final concentration of 250 µg/ml (stock solution is prepared in bidistilled water at a concentration of 10 mg/ml) when the bottle can be safely handled with a protective glove

I hope it'll help you.
Don't hesitate if you have more questions...

Lionel

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