Detection of galectin-3 interaction with commensal bacteria

Abstract

A panel of commensal bacteria was screened for the ability to interact with galectin-3. Two strains of Bifidobacterium longum subsp. infantis interacted to a greater extent than did the pathogenic positive control, Escherichia coli NCTC 12900. Further validation of the interaction was achieved by using agglutination and solid-phase binding assays.

Fig 1

Experimental overview of SPR screening study. (A) Gal-3 interacts with ASF on the chip surface. (B) Preincubation of gal-3 with bacteria decreases the gal-3 interaction with ASF, as indicated by a reduced resonance response.

Fig 2

Inhibition of gal-3 interaction with immobilized ASF by a panel of commensal bacteria by an SPR biosensor approach. E. coli NCTC 12900 was included as a positive control. Inhibition is defined as the ability of bacteria to interact with gal-3, thereby reducing its ability to generate a surface plasmon response through interaction with the ASF chip surface. Gal-3 injected over the ASF surface in the absence of bacteria was used to define the 0% inhibition SPR response. Experiments were carried out in triplicate (n = 3), and the results are presented as mean inhibition ± the standard deviation. ***, P < 0.0001.

Fig 3

SPR analysis of bacterium-free injections of gal-3 over an immobilized ASF surface. Inhibition is defined as the ability of bacteria to interact with gal-3, thereby reducing its ability to generate a surface plasmon response through interaction with the ASF chip surface. Gal-3 injected over the ASF surface in the absence of bacteria was used to define the 0% inhibition SPR response. The effects of injections with and without bacteria are compared, and data are presented as mean inhibition ± the standard deviation. Experiments were carried out in triplicate (n = 3).

Fig 4

Agglutination assay for direct visualization of bacterium–gal-3 interaction. Gal-3 or PBS alone (negative control) was incubated with B. longum subsp. infantis ATCC 15702. Gal-3 exposure resulted in agglutination of the bacteria, as evidence by the absence of a dense pellet of bacteria in the bottom of the well (A). PBS alone did not agglutinate the bacteria, resulting in pellet formation at the bottom of the well (B).

Fig 5

(A) Binding of TAMRA-labeled bacteria to recombinant gal-3 in a solid-phase binding assay. B. longum subsp. infantis ATCC 15702 and ATCC 15697 and E. coli O157:H7 NCTC 12900 were incubated in gal-3-coated wells for 1 h. (B) Binding of TAMRA-labeled B. longum subsp. infantis ATCC 15702 to recombinant gal-3 in a solid-phase binding assay. TAMRA-labeled bacteria were incubated in gal-3-coated wells (white bars) or in gal-3-coated wells in the presence of lactose (gray bars). (C) Binding of TAMRA-labeled B. longum subsp. infantis ATCC 15702 to recombinant gal-3 in a solid-phase binding assay. Bacteria were incubated in gal-3-coated wells (white) or in gal-3-coated wells in the presence of the soluble gal-3 CRD (gal-3C; gray) or full-length soluble gal-3 (black). All experiments were carried out in triplicate (n = 3). The data are presented as means ± the standard deviations. *, P < 0.05; **, P < 0.01; AU, arbitrary units.