Fig 1.
Binding of wtRS-IIL (A), RS-IIL_A22S (B) and CV-IIL (C) on the sugar chip CM4 with immobilized sugars. The response on the channel with immobilized α-d-mannopyranoside is shown as a solid-line curve, while the response on the channel with immobilized α-l-fucopyranoside is shown as a dotted-line curve. The differential curves were obtained after subtraction of the α-d-galactopyranoside channel.
Fig 2.
Isothermal titration calorimetry data.
Titration of l-fucose (A) and d-mannose (B) to CV-IIL, respectively, and l-fucose (C) and d-mannose (D) to RS-IIL_A22S. Upper panels: Data obtained from 20 automatic injections (2 μl) of the sugar into the protein-containing cell. Lower panels: Plot of total heat released as a function of ligand/protein molar ratio for titrations shown in upper panels. The solid line represents the best fit for the obtained data.
Fig 3.
Hemagglutination inhibition assays with CV-IIL at final concentrations of 400 nM, 200 nM, 100 nM, and 50 nM.
Each lectin sample was mixed with the monosaccharide in a 1: 1 ratio. The mixture was incubated for 1 minute at room temperature and 10 μl of 5% RBC_0+ was added immediately to the mixture. The mixture was incubated for an additional 5 minutes at room temperature, mixed again, applied to a glass slide and observed under a Levenhuk microscope. Pictures were taken with a DEM135 camera (Levenhuk). All negative control experiments did not exhibit any visible agglutination.
Fig 4.
Yeast agglutination inhibition assays with CV-IIL at final concentrations of 25 μM, 12.5 μM, 6.25 μM, and 3.13 μM.
Each lectin sample was mixed with the monosaccharide in a 1: 1 ratio. The mixture was incubated for 1 minute at room temperature, and 10 μl of 5% yeast cell suspension was added immediately to the mixture. The mixture was incubated for 10 additional minutes at room temperature, mixed again, applied to a glass slide and observed under a Levenhuk microscope. Pictures were taken with a DEM135 camera (Levenhuk). All negative control experiments did not exhibit any visible agglutination.
Fig 5.
Hemagglutination (upper panel) and yeast agglutination inhibition assays (lower panel) with RS-IIL_A22S at final concentrations of 200 nM and 6.25 μM.
Each lectin sample was mixed with the monosaccharide in a 1: 1 ratio. The mixture was incubated for 1 minute at room temperature, and then 10 μl of 5% RBC_0+ or yeast suspension was added to the mixture. The mixture was incubated at room temperature for an additional 5 minutes or 10 minutes, respectively, mixed again, applied to a glass slide and observed under a Levenhuk microscope. Pictures were taken with a DEM135 camera (Levenhuk). All negative control experiments did not exhibit any visible agglutination.
Fig 6.
Determination of cytoplasmic extract dilution for hemagglutination and yeast agglutination inhibition assays.
Cytosolic extract containing CV-IIL or RS-IIL_A22S was serially diluted in working buffer, and each sample from the dilution line was mixed with 5% RBC_0+ or yeast cell suspension in a 1: 1 ratio. The mixture was incubated at room temperature for 5 or 10 minutes, respectively, mixed again, applied to a glass slide and observed under a Levenhuk microscope. Pictures were taken with a DEM135 camera (Levenhuk). 128× diluted and 2× diluted cytoplasmic extract was chosen for the CV-IIL hemagglutination and yeast agglutination inhibition assay, respectively. 32× diluted and undiluted cytoplasmic extract were chosen for the RS-IIL_A22S hemagglutination and yeast agglutination inhibition assay, respectively. All negative control experiments did not exhibit any visible agglutination. The positive control was done using purified CV-IIL.
Fig 7.
Agglutination inhibition assay to test specificity of CV-IIL directly in E. coli cytosol.
Hemagglutination (upper panel) and yeast agglutination inhibition assays (lower panel) with 128× and 2× diluted cytoplasmic extract of E. coli expressing cv-2l gene, respectively. Diluted cytosol was mixed with the monosaccharide in a 1: 1 ratio and incubated for 1 minute at room temperature. 10 μl of the solution was mixed with 10 μl of 5% RBC_0+ or 5% yeast suspension and incubated at room temperature for an additional 5 (HI) or 10 (YAI) minutes, mixed again, applied to a glass slide and observed under a Levenhuk microscope. Pictures were taken with a DEM135 camera (Levenhuk).
Fig 8.
Agglutination inhibition assay to test specificity of RS-IIL_A22S directly in E. coli cytosol.
Hemagglutination (upper panel) and yeast agglutination inhibition assays (lower panel) with 32× and undiluted cytoplasmic extract of E. coli expressing rs-2l_A22S gene, respectively. Diluted cytosol was mixed with the monosaccharide in a 1: 1 ratio and incubated for 1 minute at room temperature. 10 μl of the solution was mixed with 10 μl of 5% RBC_0+ or 5% yeast suspension and incubated at room temperature for an additional 5 (HI) or 10 (YAI) minutes, mixed again, applied to a glass slide and observed under a Levenhuk microscope. Pictures were taken with a DEM135 camera (Levenhuk).
Fig 9.
Determination of cytoplasmic extract titer for hemagglutination inhibition assay on microtiter plate.
Bacterial cytoplasmic extracts (undiluted in the first well) dilution increases from left to right in two rows by a ratio of 0.5 between two neighboring wells. Agglutinated red blood cells form a diffuse mat, whereas non-agglutinated red blood cells sediment and form a clear dot in the bottom of the well. The titer was determined for extract containing CV-IIL (well 10 in the first row), wtRS-IIL (well 5 in the first row) and RS-IIL_A22S (well 7 in the first row). E. coli cytoplasmic extract without any plasmid served as negative control. Last wells represent control experiments in absence of lectin.
Fig 10.
Hemagglutination inhibition assay on microtiter plate of cytoplasmic extract containing lectins.
Cytoplasmic extracts containing lectins in dilution corresponding to titer 1 were used. The first well in the first row contains 40 mM d-mannose (upper panel) or l-fucose (lower panel). 8 mM d-mannose or l-fucose were used for cytoplasmic extract containing RS-IIL_A22S. Carbohydrate concentration decreases from left to right in two rows by a ratio of 0.5 between two neighboring wells. Inhibited agglutination results in a clear dot in the bottom of the well, whereas non-inhibited agglutination results in a diffuse mat. Last wells represent control experiments. In positive controls (P) buffer was used instead of monosaccharide. In a negative controls (N) buffer was used instead of lectin.