Campylobacter jejuni γ-glutamyltranspeptidase Activity Assay    

Materials and Reagents

1. 25 mm culture flasks with vent cap (Corning, catalog number: 430639 )
   
2. 96 well plate flat bottom (Corning, Costar^®, catalog number: 3599 )
3. Campylobacter culture
   
4. UltraPure™ Tris Buffer (powder format) (Thermo Fisher Scientific, Invitrogen™, catalog number: 15504-020 )
   
5. Lysozyme from chicken egg white (Sigma-Aldrich, catalog number: L-6876 )
   
6. L-Glutamic acid γ-(3-carboxy-4-nitroanilide) ammonium salt (Sigma-Aldrich, catalog number: 49525 )
   
7. Glycylglycine (Sigma-Aldrich, catalog number: G3915 )
   
8. Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Thermo Scientific™, catalog number: 23227 )
   
9. 4-nitroaniline (Sigma-Aldrich, catalog number: 185310 )
   
10. Hearth Infusion medium (Thomas Scientific, Oxoid, catalog number: CM1032B )
    
11. Tris/HCl (pH 7.6 and pH 8.2)
    
12. Lysis buffer (see Recipes)
    
13. Reaction buffer (see Recipes)
    
14. pNA stock solution (see Recipes)
    

Equipment

1. Absorbance microplate reader (such as BMG Labtech GmbH, Fluorstar Omega or equivalent)
   
2. Vibra-Cell™ Sonicater (Sonics & Materials, model: VC40 )
   
3. Refrigerated table top centrifuge
   
4. The anoxomat system (MART Microbiology)
   

Procedure

1. Dilute a Campylobacter preculture in 5 ml Heart Infusion (HI) broth to an optical density 600 nm of 0.05. Growth the culture in vertically standing 25 mm culture flasks with vent cap in a microaerobic atmosphere (MART) at 42 °C, 160 rpm in HI broth for until stationary phase ~20 h.
   Note: If other bacterial species are assayed use species specific growth conditions.
   
2. Pellet ~1.10⁹ bacteria of the bacterial culture, remove the supernatant and store the pellet at -80 °C for at least 1 h.
   Note: The -80 °C freezing step will help to disrupt the cells, shorter incubation will lead to less disrupted cells.
   
3. Resuspend the pellet in ice cold 250 μl lysis buffer, and incubated for 30 min on ice.
   
4. Sonicate 6 times (5 sec each, with 15 sec intervals on ice) at 40 mV and the output control set at 80%.
   
5. Centrifuge the cell lysate for 10 min at 12,000 x g at 4 °C.
   
6. Harvest the supernatant and keep on ice before use.
   
7. Measure the protein concentration of 20 μl supernatant (BCA kit with standard) in a 96 wells plate according to the kit’s protocol.
   
8. Make a serial dilution in a range between 0 and 200 µM of the standard pNA [4-nitroaniline (stock solution 1 mM)] in 50 mM Tris/HCl (pH 7.6).
   
9. Mix 20 μl supernatant or standard pNA in a 96 wells plate with 180 μl of reaction buffer.
   
10. Measure the optical density of the samples and standard in the 96 wells plate at a wavelength of 405 nm (OD₄₀₅) every 60 s during an incubation period of 30 min at 37 °C (Figures 3 and 4).
    
11. Calculate the slope of all values in a linear range of the obtained graphs (see below).
    
12. GGT activity is expressed as nmol min^-1 mg protein^-1.
    
    

Figure 3. A typical pNA standard curve


Figure 4. Graph showing in time the pNA increase in the bacterial lysates made of C.jejuni wt (red) and racRS mutant (blue)

To calculate the GGT activity first plot the pNA standard curve. Calculate the Δabsorbance (A time x - A time y) in a linear range. Next, compare the calculated Δabsorbance of each sample to the standard pNA curve to determine the amount of pNA generated between time x and time y (nmol pNA).

The GGT activity is calculated by the formula: (nmol pNA)
T x M
T = the time incubated (in min)
M = mg protein determined by BCA protein kit
GGT activity is reported as nmole/min^-1/mg protein^-1

Example:
Δabsorbance(wt) = 0.58 (15 min) - 0.43 (5 min)=0.15
Δabsorbance 0.15 = 40 nmole pNA
Time x - time y = 10 min
M = 0.02 mg protein
GGT activity is: 40/(10 x 0.02) = 200 nmole/min^-1/mg protein^-1

Recipes

1. Lysis buffer
   50 mM Tris/HCl (pH 7.6)
   1 μg/ml lysozyme
   
2. Reaction buffer
   2.9 mM L-γ-glutamyl-3-carboxy-4-nitroanilide
   100 mM glycylglycine
   100 mM Tris/HCl (pH 8.2)
   
3. pNA stock solution
   1 mM 4-nitroaniline
   50 mM Tris/HCl (pH 7.6)
   Can be stored at -20 °C
   

Acknowledgments

The γ-glutamyltranspeptidase activity protocol described here and in, van der Stel et al. (2015), is modified from a protocol described in Chevalier et al. (1999). This work was supported by the NWO-ECHO grant 711.012.007 to MW.

References

1. Chevalier, C., Thiberge, J. M., Ferrero, R. L. and Labigne, A. (1999). Essential role of Helicobacter pylori γ-glutamyltranspeptidase for the colonization of the gastric mucosa of mice. Mol Microbiol 31(5): 1359-1372.
   
2. Hanigan, M. H. (1998). gamma-Glutamyl transpeptidase, a glutathionase: its expression and function in carcinogenesis. Chem Biol Interact 111-112: 333-342.
   
3. Heisterkamp, N., Groffen, J., Warburton, D. and Sneddon, T. P. (2008). The human gamma-glutamyltransferase gene family. Hum Genet 123(4): 321-332.
   
4. Ong, P. L., Yao, Y. F., Weng, Y. M., Hsu, W. H. and Lin, L. L. (2008). Residues Arg114 and Arg337 are critical for the proper function of Escherichia coli gamma-glutamyltranspeptidase. Biochem Biophys Res Commun 366(2): 294-300.
   
5. van der Stel, A. X., van Mourik, A., Laniewski, P., van Putten, J. P., Jagusztyn-Krynicka, E. K. and Wosten, M. M. (2015). The Campylobacter jejuni RacRS two-component system activates the glutamate synthesis by directly upregulating gamma-glutamyltranspeptidase (GGT). Front Microbiol 6: 567.