Yeast agglutination assay

The yeast agglutination assay for detecting type 1 mannose-sensitive fimbria in adherent-invasive Escherichia coli (AIEC).

The interaction of AIEC with intestinal epithelial cells requires a variant of type 1 pili that provide their adherence and invasion into epithelial cells [Boudeau et al., 2001]. The mannose-sensitive nature of type 1 fimbriae enables their detection in mannan-containing yeast cells’ agglutination reaction [Korhonen, 1979]. In our study [Sobieszczanska et al., 2021], the impact of catecholate hormone norepinephrine on the ability of AIEC strains isolated from children with Crohn’s disease to produce type 1 pili in conditions corresponding to testing of bacterial adherence to epithelial cells. The idea of our study was to reflect the infection of epithelial cells with AIEC strains and the role of type 1 pili in this process.

First, AIEC strains were cultured for 24 h in a nutrient-poor medium SAPI supplemented with 30% of non-inactivated bovine serum (SAPI-serum), rich in complement restricting bacterial growth, and so the synthesis of virulence factors. To test the ability of AIEC to produce type 1 fimbriae in a nutrient-rich media, AIEC following the culture in SAPI-serum medium were sub-cultured for 3 h in MEM (Minimal Essential Medium) cell culture medium enriched with 10% inactivated fetal serum, non-essential amino acids and supplemented or not with 50 mM norepinephrine (NE) at 37° in an atmosphere with 5% CO2. The technique thus mirrors the conditions under which bacterial adhesion to epithelial cells is analyzed, allowing us to examine how these conditions affect the synthesis and expression of type 1 fimbriae.

Materials and Reagents

1. SAPI medium (recipe is given below)

2. Bovine serum (ThermoFisher Scientific, cat. 16030074)

3. MEM (Minimal Essential Medium) with GlutaMaxTM (ThermoFisher Scientific, cat. 32561037)

supplemented with inactivated 10% fetal bovine serum, non-essential amino acids (Lonza, cat. 13

114E), 1% sodium pyruvate (BioWest, cat. L0642)

4. L-norepinephrine bitartrate monohydrate (Sigma-Merc, cat. A9512) was added to the medium at an

appropriate concentration just before AIEC inoculation. The medium cannot be stored.

5. Luria broth base, Miller′s modified (Sigma-Merc, cat. L1900) prepared according to producer

instruction.

6. Phosphate buffered saline (pH 7.2) (Cytogen, cat. 04-3550)

7. Methyl-α-mannopyranoside (corresponding to D-mannose) (Sigma-Merc, catalog number 67770)

8. Fresh bakery yeast was purchased from a local baker’s shop. The baker’s yeast can be substituted

with human erythrocytes group 0 or guinea pig erythrocytes. 

Equipment

1. Bacteriological incubator providing 37°C 

2. Spectrophotometer for optical density measurements (V1600 Meter Toledo)

3. Centrifuge for Eppendorf tubes (Eppendorf, M141R High-Speed Benchtop Refrigerated Centrifuge)

4. Microscopic glass slides (Menzel Gläser; Avantor catalog number 631-0701)

5. Non-tissue culture–treated 96-well round-bottom polystyrene plate (NUNC catalog number: 268200

6. Orbital shaker (DPC Diagnostic Products Corporation)

7. Inverted microscope (Olympus IX71 with camera XC30)

Protocol to detect type 1 fimbria production

1. Disperse 30 µL of the overnight AIEC culture conducted at 37°C in Luria broth in 3 mL of an appropriate medium (SAPI, MEM, MEM with 50 mM NE) and incubate statically for 24 h at 37°C. For some AIEC strains, a ring of bacterial growth can be observed at the border of the medium with air, which contains fimbriated AIEC cells. Hence, before centrifugation, this ring should be rinsed with a pipette into the broth to recover as much fimbriated bacteria as possible. To obtain an adequate pellet of AIEC growing in the growth-restricting SAPI-serum medium, the minimum volume of the liquid medium centrifuged should be 5 mL (inoculated with 50 µL of AIEC culture) due to poor bacterial growth in this medium. In contrast, AIEC grows well in MEM medium, so 3 mL is enough to obtain an adequate AIEC pellet. 

2. Centrifuge (10 000 rpm) a 3 – 5 mL of AIEC broth culture in an Eppendorf tube. Discharge the supernatant carefully. Do not wash the bacterial pellet. 

3. Resuspend the pellet thoroughly in 500 µL of phosphate-buffered saline (PBS; pH 7.2), transfer it to the cuvette, and establish the suspension’s optical density spectrophotometrically to 9 x 108 colony forming units/mL (CFU per mL). 

Qualitative agglutination assay

A 25 µL yeast suspension was dropped onto a clean glass slide twice. To one of the drops, an equal volume of D-mannose was added to inhibit mannans on the surface of yeast cells, and to another drop, a 25 µL of PBS was added. Next, a 25 µL of E. coli suspension of established density was dropped into both yeast suspensions and mixed on an orbital shaker for 1 min. The reaction of yeast agglutination was read visually.

Quantitative agglutination assay

To establish the yeast agglutination titer, E. coli suspension was first diluted exponentially with PBS in the tubes, and consecutive dilutions at 25 µL were added to wells of a 96-well plate with yeast suspension or yeast mixed with D-mannose. After mixing on an orbital shaker for 5 min, the reaction was read visually and under an inverted microscope. The agglutination titer is the highest dilution of the E. coli suspension at which yeast agglutination is visible.

Recipes

1. SAPI-serum medium supplemented with 30% bovine serum according to Lyte et al. 1993.

Medium composition: 

6.25 mM NH₄NO₃

1.84 mM KH₂PO₄

3.35 mM KCl

1.01 mM MgSO₄

2.77 mM glucose

Dilute in 0.8 L of deionized water 

Adjust pH to 7.5 with 10% HCl and top up to 1 L.

Sterilize the medium by autoclaving for 30 min at 250°F (121°C) in saturated steam under pressure of approximately 15 pounds per square inch. The medium should be safely stored at 4°C for a month. To culture E. coli, warm the medium to room temperature and then add a non-inactivated sterile bovine serum (FBS) to obtain a 30% (v/v) concentration. 

2.         Baker’s yeast (Saccharomyces cerevisiae) suspension

Weigh 0.1 g of yeast mass and resuspended it carefully in 10 mL of PBS to obtain a uniform 1% solution.

3.         1% D-mannose 

Weigh 0.1 g of methyl-α-mannopyranoside (corresponding to D-mannose) and resuspend in 10 mL of PBS to obtain 1% solution.

References

1. Boudeau J, Barnich N, Darfeuille-Michaud A. Type 1 pili-mediated adherence of Escherichia coli strain LF82 isolated from Crohn’s disease is involved in bacterial invasion of intestinal epithelial cells. Mol Microbiol. 2001;39:1272–1284. https://doi.org.10.1111/j.1365-2958.2001.02315.x
2. Korhonen T.K. Yeast agglutination by purified Enterobacterial pili. FEMS Microbiol Letters 1979; 6: 421–425. https://doi.org/10.1111/j.1574-6968.1979.tb03756.x
3. Sobieszczańska B., Turniak M., Olbromski M., Walczuk U., Choroszy M., Tukiendorf A, Dzięgiel P. Norepinephrine affects the interaction of adherent-invasive Escherichia coli with intestinal epithelial cells. Virulence 2021;12:630-637. https://doi.org/10.1080/21505594.2021.1882780
4. Lyte M, Ernst S. Alpha and beta-adrenergic receptor involvement in catecholate-induced growth of Gram-negative bacteria. Biochem Biophys Res Comm. 1993;190:447–452. https://doi.org.10.1006/bbrc.1993.1068