Shigella IpaD and IpaB Surface Localizations   

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: 0 210026291 )
   
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-IpaD^131-332 (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.