TechNotes No. 5-1999

Binding and inhibition P-fimbriated uropathogenic E. coli to b–globotetraoside GlycoWell™ plates.

Introduction
Human P-fimbriated uropathogenic Escherichia coli bacteria express a carbohydrate-specific adhesin protein, which is believed to be an important virulence factor. The endogenous carbohydrate ligand for this adhesin in globotetraose (Gb4, b–D–GalNAc–(1-3)–a–D–Gal–(1-4)–b–D–Gal–(1-4)–b–D–Glc)¹. The submolecular details of the carbohydrate epitope of this adhesin have been investigated by inhibiting the bacteria binding to a Gb4 GlycoWell™ plate with synthetic carbohydrate analogs of Gb4². This TechNote describes the binding of P–fimbriated E. coli to Globotetraose GlycoWell™ plates, as well as competitive inhibition using a soluble derivative of the central disaccharide of Gb4, galabiose (a–D–Gal–(1-4)–b–D–Gal–OCH₂CH₂SiMe₃)³, as inhibitor.

Experimental

1. Bacteria preparation. Grow the bacteria on TS agar with 100 µg/mL Carbenicillin and passage for 3 days. Suspend bacteria in PBS to an A540 of 1.0. Spin down a know volume of the bacterial suspension and resuspend the pellet in PBS containing 2 % BSA (1/20th of the original volume). Use this bacterial suspension in the experiment described below.
2. Blocking for non-specific binding. Block the wells for non-specific binding by incubation overnight at 4ºC with 400 µL of PBS containing 2% BSA and E. coli cells devoid of adhesin suspended to a klett of 50. Wash three times with PBS and leave the last portion of washing for 15 min in the wells.
3. Addition of inhibitor. Add to three wells of Gb4 GlycoWell™ plate 50µL of inhibitor solution and to three wells of Globotetraose GlycoWell™ plate 50µL of PBS. Add to three wells of N–acetyl (blank) GlycoWell plate 50µL of PBS.dilution series. Dissolve the inhibitor to 18 mM in PBS. Dispense 75 µL of inhibitor solution to the first well of Gb4 GlycoWell™ plate. Dispense 50 µL of PBS to all other wells. Make serial three–fold dilutions by transferring 25 µL from the first well to the second well, mixing, transferring 25 µL from the second well to the third, and so on until the eleventh well. Remove and discard 25 µL from the eleventh well. The very last well (twelfth) will contain no inhibitor. Make the inhibitor dilution series in triplicate.
4. Bacteria incubation. Add 50 µL bacteria suspension (HB101/pPAP5) to all wells except control wells. Add 50 µL bacteria (HB101/pPAP24) devoid of papG adhesin to control wells. Shake the plate gently. Incubate for 45 min at room temperature.
5. Washing. Empty the wells and wash three times with CovaBuffer. Leave the last wash in the wells for 15 min.
6. Incubation with 1° antiserum. Add 100 µL anti–pili rabbit antiserum solution. Incubate for 60 min at room temperature.
7. Washing. Empty the wells and wash three times with CovaBuffer. Leave the last wash in the wells for 15 min.
8. Incubation with 2° antiserum Add 100 µL alkaline phosphatase–conjugated anti–rabbit IgG solution. Incubate for 60 min at room temperature.
9. Washing. Empty the wells and wash three times with CovaBuffer. Leave the last wash in the wells for 15 min. Wash once with substrate buffer.
10. Substrate incubation. Add 100 µL of substrate solution to each well. Read OD at 405 nm in a microwell plate reader. Subtract the optical density reading from control wells (incubated with HB101/pPAP24) from the optical density reading from other wells.

• PBS buffer pH 7.2, 0.15 M Na⁺: 24.0 g NaCl, 0.6 g KCl, 3.46 g Na₂HPO₄·2H₂O, 0.6 g KH₂PO₄, 3 L H₂O.
• CovaBuffer: 116.9 g NaCl, 10.0 g MgSO₄·7H₂O, 0.50 mL TWEEN 20, 1 L PBS.
• Gb4 (SW-04-001) plates.
• Inhibitor: (a–D–Gal–(1-4)–b–D–Gal–OCH₂CH₂SiMe₃)³.
• E. coli bacteria⁴ expressing the PapG adhesin (HB101/pPAP5).
• E. coli bacteria⁵ devoid of the PapG adhesin (HB101/pPAP24).
• TS agar.
• Carbenicillin.
• Bovine Serum Albumin (BSA).
• Anti-pili rabbit antiserum (WU93 diluted 1/200 in 2% BSA/PBS).
• Alkaline phosphatase–conjugated anti–rabbit IgG (Sigma A7539, diluted 1/500).
• Substrate buffer: 10% diethanolamine, 0.5 mM MgCl2, pH 9.4.
• Sigma 104 phosphatase substrate (i.e. p-nitrophenyl phosphate).
• Substrate solution: 1 mg/mL Sigma 104 phosphatase substrate in substrate buffer filtered through 0.22 µM filter.
  
  

Figure 1. P–fimbriated uropathogenic E. coli binds to Gb4 GlycoWell™ plates (SW-04-001). A soluble disacchride fragment of Gb4, galabiose [a–D–Gal–(1-4)–b–D–Gal–OCH2CH2SiMe3], inhibits binding of the bacteria to the Gb4 GlycoWell™ plate.