PMC

(a) 5 μM Gal-8, Gal-8R233H, or Gal-8R69H were added to mid-log phase E. coli O86 (BG B⁺ E. coli). Viable bacteria were quantified by dilution plating, n=3, 1 representative experiment in duplicate over 2 dilutions shown, error bars=SD. (b) Flow cytometric analysis following incubation of E. coli O86 (BG B⁺ E. coli) with Gal-8N or Gal-8C at ~0.1 μM with or without inclusion of 20 mM lactose (Lac) where indicated. (c) 5 μM Gal-8, Gal-8N, or Gal-8C were added to mid-log phase E. coli O86 (BG B⁺ E. coli). Viable bacteria were quantified by dilution plating, n=3, 1 representative experiment in duplicate over 2 dilutions shown, error bars=SD. (d) Flow cytometric analysis following incubation of E. coli O86 (BG B⁺ E. coli) with Gal-4N or Gal-4C at ~0.1 μM with or without inclusion of 20 mM lactose (Lac) where indicated. (e) 5 μM Gal-4, Gal-4N, or Gal-4C were added to mid-log phase E. coli O86 (BG B⁺ E. coli). Viable bacteria were quantified by dilution plating, n=3, 1 representative experiment in duplicate over 2 dilutions shown, error bars=SD.

(a–d) Glycan microarray data obtained following incubation with (a) 0.2 μM Gal-1, (b) 0.2 μM Gal-3, (c) 0.5 μM Gal-4, and (d) 0.02 μM Gal-8. RFU = relative fluorescence units represented on the y-axis. Error bars = +/- 1 SEM. See for complete list of glycans represented on the x-axis. (e) Structure of E. coli O86 O antigen. (f-i) Flow cytometric analysis following incubation of E. coli O86 with (f) Gal-1, (g) Gal-3, (h) Gal-4, and (i) Gal-8 all tested at ~0.1 μM with or without inclusion of 20 mM lactose (Lac) where indicated.

(a) Schematic of O antigen structures on wild type (WT) BG B⁺ E. coli and mutants of BG B⁺ E. coli WaaL⁻ (Ligase-) and Wzy⁻ (Polymerase-) lacking a complete O antigen. (b) Flow cytometric analysis following incubation of BG B⁺ E. coli and mutants WaaL⁻ and Wzy⁻ with ∼0.1 μM Gal-8. (c–d) Incubation of (c) WT and WaaL⁻ mutant BG B⁺ E. coli or (d) WT and Wzy⁻ mutant BG B⁺ E. coli with 5 μM Gal-4 or Gal-8 as indicated. Viable bacteria were quantified by dilution plating, n=3, representative experiment in duplicate over 2 dilutions shown, error bars=SD.

(a) Flow cytometric analysis of galectin binding after incubation of BG B⁺ E. coli and two different BG B⁻ E. coli reference strains obtained from a clinical laboratory with ∼0.1 μM Gal-8. (b–c) Incubation of (b) BG B⁺ E. coli or (c) BG B⁻ E. coli strain 1 with 5 μM Gal-1, Gal-3, Gal-4, or Gal-8 as indicated. Viable bacteria were quantified by dilution plating, n=3, representative experiment in duplicate over 2 dilutions shown, error bars=SD. (d) Flow cytometric analysis following incubation of BG B⁺ E. coli, K. pneumoniae, P. aeruginosa, and S. aureus with ∼0.1 μM Gal-8. (e–g) Incubation of (e) K. pneumoniae, (f) P. aeruginosa, or (g) S. aureus with 5 μM Gal-1, Gal-3, Gal-4, or Gal-8 as indicated. Viable bacteria were quantified by dilution plating, n=3, representative experiment in duplicate over 2 dilutions shown, error bars=SD. (h–i) Incubation with or without 5 μM Gal-8 with (h) GFP⁺ P. aeruginosa alone or (i) GFP⁺ P. aeruginosa mixed with BG B⁺ E. coli followed by determination of percent GFP⁺ P. aeruginosa by flow cytometric analysis in a mixing experiment. Gated values of GFP⁺ bacteria treated with PBS (blue) or Gal-8 (red) are shown. (j) Quantification of percent GFP⁺ bacteria utilizing flow cytometric analysis obtained following incubation of Gal-8 with either GFP⁺ P. aeruginosa alone (P.a.) or GFP⁺ P. aeruginosa mixed with BG B⁺ E. coli (P.a. + BG B⁺ E.c.).

E. coli O86 (BG B⁺ E. coli) were mixed with (a) 5 μM Gal-1, Gal-3, Gal-4, or Gal-8, (b) 5 μM Gal-4 with or without 20 mM lactose (Lact.) or 20 mM sucrose (Sucr.), (c) 5 μM Gal-8 with or without 20 mM lactose (Lact.) or 20 mM sucrose (Sucr.), or (d) the indicated concentrations of Gal-1, Gal-3, Gal-4, or Gal-8. Viable bacteria were quantified by dilution plating, n=3, 1 representative experiment in duplicate over 2 dilutions shown (a–c), error bars=SD. (e) Still-frame images from real-time video microscopy demonstrating bacterial mobility at 10-s intervals before and after addition of 5 μM Gal-8 as indicated (see ). Arrows indicate one group of immobilized bacteria. Scale bars = 100 μm. (f) E. coli O86 (BG B⁺ E. coli) were grown to mid-log phase followed by addition of 5 μM Gal-8. Untreated and Gal-8 treated bacteria were stained with propidium iodide (red) and visualized by fluorescence microscopy. Scale bars = 100 μm. (g) Transmission electron microscopy images of E. coli O86 (BG B⁺ E. coli) following addition of PBS (NT) or 5 μM Gal-8. Lower panels show close up view of single bacterium. Scale bars = 500 nm. (h) Scanning electron microscopy images of E. coli O86 (BG B⁺ E. coli) followed by addition of PBS (NT) or Gal-8. Scale bars = 500 nm.

(a) Flow cytometric analysis following incubation of BG B⁺ E. coli with ∼0.1 μM mGal-4 with or without lactose. (b) Flow cytometric analysis following incubation of BG B⁺ E. coli and mutants WaaL⁻ with ∼0.1 μM mGal-4. (c) mGal-4 binding to the CFG glycan microarray at 20 μg/ml (0.5 μM). BGB= blood group B glycans, BGA= blood group A glycans (See ). (d) Quantification of WT BG B⁺ and ΔwaaL mutant E. coli after incubation with ~5 μM mGal-4. Viable bacteria were quantified by dilution plating; n = 3 experiments; one representative experiment in duplicate over two dilutions is shown; error bars represent means ± s.d. (e) Live antibiotic-treated mice were fed PBS, Wild type (WT), or WaaL⁻ mutant BG B+ E. coli. The number of viable bacteria in the intestine of mice sacrificed 24 h after feeding was quantified by dilution plating. * = p value 0.049. (f) Growth of WT and WaaL⁻ mutant BG B⁺ E. coli in the presence and absence of TDG. * = p value 0.008. (g) Schematic of O antigen structures on α-Gal E. coli. (h) Flow cytometric analysis of α-Gal expressing bacteria after incubation of α-Gal–expressing bacteria with ~0.1 μM human Gal-4 or Gal-8. (i) Bar graph showing the percentage of α-Gal–expressing bacteria and BG B⁻ bacteria remaining after incubation with 5 μM Gal-4 and Gal-8 as compared to PBS-treated control bacteria. Viable bacteria were quantified by dilution plating, n=3, representative experiment in duplicate over 2 dilutions shown, error bars=SD.