Quantitative analysis (K(d) and IC(50)) of glycoconjugates interactions with a bacterial lectin on a carbohydrate microarray with DNA Direct Immobilization (DDI)

Abstract

Nowadays, there is a great interest for understanding the structure/function relationship governing recognition of carbohydrates by their receptors for the design of new treatments. Indeed, carbohydrates and glycoconjugates play a major role in key biological events such as cell-cell recognition, pathogenesis inflammation, and host pathogen interactions. Pseudomonas aeruginosa (PA) is one of the predominant bacterium encountered in nosocomial infections. PA infections often lead to chronic inflammation and eventually to death despite aggressive antibiotic therapy: the emergence of resistant strains and biofilm formation seems to give a selective advantage to the bacterium. A promising approach is to inhibit the virulence factors of PA such as PA-IL which is a galactose specific lectin. Herein, we develop a microarray to probe the binding of six galacto-conjugates to PA-IL differing by their spatial configuration and geometry. This microsystem is made of 40 independent microwells in which 64 spots of glycoconjugates probes are arrayed by using DNA Directed Immobilization (DDI). This microsystem allows, in a multiplex fashion, qualitative information on the binding by direct fluorescence readout as well as quantitative information by the determination of IC(50) values in a competition assay and surface dissociation constants (K(d)). According to our data, direct fluorescent signals (FI(635)), IC(50) and K(d) values provided similar ranking for glycoconjugates with respect to PA-IL binding thus affording a general tool for the selection of galacto-conjugates displaying the best affinities toward PA-IL.