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. 2016 Mar 17;11(3):e0151824. doi: 10.1371/journal.pone.0151824

Fig 3. SpaCBA pili are glycosylated with mannose and fucose.

Fig 3

(A) SpaCBA pili are glycosylated on L. rhamnosus GG cells—Cell wall-associated proteins of L. rhamnosus GG wild type (1), the pilus-deficient ΔspaCBA::TcR mutant (CMPG5357, 2) and the ΔwelE::TcR mutant on which the pili are overexposed (CMPG5351, 3), were probed with mannose- and fucose-specific lectins (HHA and AAL resp.). Pili content was visualized by probing with SpaC antiserum (SpaC, black arrow and HMW: high molecular weight pili). Interference of the Msp1 glycoprotein was ruled out (open arrow). Blots and gels were performed in triplicate. (LK = Precision Plus Protein™ Kaleidoscope™ Standard, Bio-Rad) (B) Purified pili are glycosylated—SDS-PAGE separated pili (pool A) were stained with PAS glycostain and Sypro® to visualize their protein content. Pili content was shown by probing of Western blotted samples with SpaC antiserum (HMW: high molecular weight pili). Purified Msp1 (open arrow) was used as a positive control. Representative gels are shown, experiment was carried out in triplicate. (LK = Precision Plus Protein™ Kaleidoscope™ Standard, Bio-Rad) (C) SpaCBA pili bind mannose-specific lectins—Purified pili fractions (PRM and pili pool B) were subjected to PAS glycostaining and both Sypro® and Silver stain to visualize their protein content. Absence of 75 kDa signals on PAS and lectin blots rule out the interference Msp1 (cf. open arrow). Western blotted samples were probed with SpaC antiserum (HMW: high molecular weight pili) and the mannose-specific lectins HHA and GNA, visualizing the pili content of the samples and the presence of mannose, respectively. Representative gels and blots of in triplicate-repeated experiment. (LK = Precision Plus Protein™ Kaleidoscope™ Standard, Bio-Rad) (D&E) Mannose- and fucose-specific lectins bind SpaCBA pili—Binding of lectins to plate-coated pili was measured to ELISA. Wells coated with coating buffer served as a negative control. Mannan and Lewis X were coated as a positive control for the mannose-specific HHA (Hippeastrum hybrid, D) and fucose-specific AAL (Aleuria aurantia, E) lectins, respectively. Error bars represent standard deviations of three independent experiments (paired t-test, p < 0.05)