Methyl α d mannopyranoside

A yeast agglutination assay was performed using the protocol described previously (Pretzer et al.12 (link)) but with minor modifications. Briefly, overnight-grown cultures of Lactobacillus strains were washed, suspended in phosphate buffered saline (PBS, pH 7.2) to a final concentration of 1 × 10¹⁰ CFUs/ml. Similarly, overnight cultures of S. cerevisiae or C. albicans cells were washed and suspended in PBS so as to a make a 1% w/v cell suspension. For agglutination, 25 μl of bacterial suspension was added to 25 μl of PBS followed by 50 μl of yeast cells suspension in a 96-well U-bottom well sterile plate (Greiner bio-one). The mixtures were incubated for 10 min at RT with gentle shaking. To study inhibition, 25 μl of methyl-α-D-mannopyranoside (50 mM; Sigma-Aldrich) was added to the bacterial suspension instead of PBS. To examine agglutination, the mixtures were spotted on a slide and viewed under a phase-contrast microscope (400-fold magnification, Zeiss Axio Imager Z1 microscope equipped with an AxioCam MRm Rev.3 monochrome digital camera). Alternatively C. albicans and S. cerevisiae were incubated with 200 μg/ml of fluorescein isothiocyanate (FITC)-labeled lectin domain of Cmpg5300.05_29 alone and in combination with 25 μl of methyl-α-D-mannopyranoside (50 mM; Sigma-Aldrich). As a control, a lectin L-type domain of lectin-like protein 2 (Llp2) from L. rhamnosus GG was used.

The glycosylation profile of UDA15a and UDA15b cell lines was studied by lectin blotting using ConA and TL. Both, sVSG (2 μg) and cell pellets (1 x 10⁶ cell equivalents/sample) coming from hypotonic lysis were denatured in SDS-sample buffer containing 8 M urea and 50 mM DTT, subjected to electrophoresis on a NuPAGE Bis-Tris 4–12% gradient gel (Invitrogen) in MOPS buffer and transferred to a nitrocellulose membrane. Proteins were stained with Ponceau S (Sigma) as loading control and blocked with 3% BSA in PBS. Membranes were probed with biotinylated TL (0.33 μg/mL, Vector Laboratories, Inc.) in a solution containing 50 mM Tris-HCl pH 7.4, 0.5 M NaCl, 0.05% IGEPAL and 0.25% BSA, or biotinylated ConA (0.05 μg/mL, Sigma) in PBS containing 1 mM MgCl₂, 1 mM CaCl₂, 1 mM MnCl₂, 0.05% IGEPAL and 0.25% BSA. Specific inhibitors of lectin binding such as chitin hydrolysate (1:10 dilution, Vector Laboratories, Inc.) for TL and methyl α-D- mannopyranoside (0.5 M, Sigma) for ConA were also used as carbohydrate-specific binding controls. Finally, glycoproteins were detected with Extravidin-peroxidase conjugated (Sigma) by chemiluminescent detection ECL Western Blotting Detection Reagents (GE Healthcare).

Mannose lectin FRIL was purified by affinity chromatography on D-mannose agarose and eluted competitively with methyl α-D-mannopyranoside (Sigma–Aldrich, Oakville, ON, Canada) as described by Colucci et al. (1999) (link). The identity of protein bands was confirmed by LC-MS after tryptic digestion as described in (Marsolais et al., 2010 (link)). The peak list was searched against NCBInr/Other green plants using Mascot¹.