Combined Bacteria Microarray and Quartz Crystal Microbalance Approach for Exploring Glycosignatures of Nontypeable Haemophilus influenzae and Recognition by Host Lectins

Abstract

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.

Siglecs at the Host–Pathogen Interface

“…In addition to the well-studied microorganisms mentioned above, a growing list of bacterial species have been discovered to display sialoglycoconjugates on their surfaces, such as Pseudomonas aeruginosa (Khatua et al 2010(Khatua et al , 2012, Klebsiella pneumoniae (Lee et al 2014), and nontypeable Haemophilus influenzae (NTHi) (Kalograiaki et al 2016). P. aeruginosa recruits host sialoglycoproteins and displays them on the bacterial surface, and these absorbed Sias can enhance bacterial survival by reducing complement deposition and engaging inhibitory Siglec-9 to suppress neutrophil bactericidal machinery (Khatua et al 2010(Khatua et al , 2012.…”

Uncovering the Binding Specificities of Lectins with Cells for Precision Colorectal Cancer Diagnosis Based on Multimodal Imaging

“…These characteristics make lectins very useful for glycoanalysis and medicine development 16, 17, 18, 19, 20, 21. Due to their minimization of sample consumption and high throughput format for analyzing multiple targets simultaneously, lectin microarrays have been employed in the glycomics study 22, 23, 24, 25, 26. However, the interactions of lectins with glycans (dissociation constant, K d  = 10 −4 –10 −7 m ) are much weaker than the interactions of antigens with antibodies ( K d  = 10 −8 –10 −12 m ).…”

Microarray Strategies for Exploring Bacterial Surface Glycans and Their Interactions With Glycan-Binding Proteins

“…More recently, bacteria microarrays have proved to be useful for exploring the presence of carbohydrate structures on bacterial surfaces (Campanero-Rhodes et al, 2015;Kalograiaki et al, 2016Kalograiaki et al, , 2018b. K. pneumoniae O1:K2 strain 52145, a clinically relevant serotype, was first used as model bacterium (Campanero-Rhodes et al, 2015).…”

“…Therefore, other glycan structures different from the CPS were apparently recognized by ConA. The efficiency of bacteria microarrays for exploring the presence of surface glycans of bacteria not presenting CPS and O-antigen-containing LPS was next demonstrated using nontypeable H. influenzae (NTHi) as model (Kalograiaki et al, 2016). Binding assays to microarray-printed NTHi strain 375 (hereafter referred to as NTHi375) with a panel of 19 lectins revealed positive and hapten-inhibitable signals for Gal-, Glc-, and sialic acid-specific lectins, indicating the presence on the bacterial surface of carbohydrate structures specifically recognized by the lectins.…”