Recognition of bacterial surface epitopes by host receptors plays an important role in the infectious process and is intimately associated with bacterial virulence. Delineation of bacteria-host interactions commonly relies on the detection of binding events between purified bacteria- and host-target molecules. In this work, we describe a combined microarray and quartz crystal microbalance (QCM) approach for the analysis of carbohydrate-mediated interactions directly on the bacterial surface, thus preserving the native environment of the bacterial targets. Nontypeable Haemophilus influenzae (NTHi) was selected as a model pathogenic species not displaying a polysaccharide capsule or O-antigen-containing lipopolysaccharide, a trait commonly found in several important respiratory pathogens. Here, we demonstrate the usefulness of NTHi microarrays for exploring the presence of carbohydrate structures on the bacterial surface. Furthermore, the microarray approach is shown to be efficient for detecting strain-selective binding of three innate immune lectins, namely, surfactant protein D, human galectin-8, and Siglec-14, to different NTHi clinical isolates. In parallel, QCM bacteria-chips were developed for the analysis of lectin-binding kinetics and affinity. This novel QCM approach involves capture of NTHi on lectin-derivatized chips followed by formaldehyde fixation, rendering the bacteria an integrated part of the sensor chip, and subsequent binding assays with label-free lectins. The binding parameters obtained for selected NTHi-lectin pairs provide further insights into the interactions occurring at the bacterial surface.

No abstract

Bacterial surfaces are decorated with carbohydrate structures that may serve as ligands for host receptors. Based on their ability to recognize specific sugar epitopes, plant lectins are extensively used for bacteria typing. We previously observed that the galactose-specific agglutinins from Ricinus communis (RCA) and Viscum album (VAA) exhibited differential binding to nontypeable Haemophilus influenzae (NTHi) clinical isolates, their binding being distinctly affected by truncation of the lipooligosaccharide (LOS). Here, we examined their binding to the structurally similar LOS molecules isolated from strains NTHi375 and RdKW20, using microarray binding assays, saturation transfer difference NMR, and molecular dynamics simulations. RCA bound the LOSRdKW20 glycoform displaying terminal Galβ(1,4)Glcβ, whereas VAA recognized the Galα(1,4)Galβ(1,4)Glcβ epitope in LOSNTHi375 but not in LOSRdKW20, unveiling a different presentation. Binding assays to whole bacterial cells were consistent with LOSNTHi375 serving as ligand for VAA, and also suggested recognition of the glycoprotein HMW1. Regarding RCA, comparable binding to NTHi375 and RdKW20 cells was observed. Interestingly, an increase in LOSNTHi375 abundance or expression of HMW1 in RdKW20 impaired RCA binding. Overall, the results revealed that, besides the LOS, other carbohydrate structures on the bacterial surface serve as lectin ligands, and highlighted the impact of the specific display of cell surface components on lectin binding.