Differential recognition of Haemophilus influenzae whole bacterial cells and isolated lipooligosaccharides by galactose-specific lectins

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.

Exploration of Galectin Ligands Displayed on Gram-Negative Respiratory Bacterial Pathogens with Different Cell Surface Architectures

“…Each Hep of the inner core can be a point for addition of strain-specific and phase-variable short carbohydrate extensions that constitute the outer core, which may also mimic host glycan epitopes and could potentially serve as galectin ligand. Other carbohydrate structures on the bacterial surface might also be recognized, as was inferred in a previous study on the binding of two model galactose-specific lectins to NTHi entire cells and isolated LOSs [ 18 ].…”

“…Recognition of the high molecular weight adhesin HMW1 by the galactose-specific agglutinin VAA was inferred in a previous study in which the binding of this lectin to NTHi strains expressing HMW1 or not was comparatively examined [ 18 ]. HMW1 is glycosylated at multiple sites with N-linked Gal/Gal-Glc units [ 39 ], and it is expressed by the particular NTHi strain studied here.…”

“…LPSs were further treated with DNase, RNase and proteinase K before precipitation with 0.375 M magnesium chloride in 95% ethanol. NTHi wild type, Δ lgtF , and Δ lpsA LOSs were isolated from the aqueous layer of phenol-treated bacteria suspensions by precipitation with methanol containing 1% sodium acetate-saturated methanol [ 18 ]. For practical reasons, the wild type LOS was isolated from the Δ ompP5 mutant.…”

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

“…In particular, the absence of the Galα(1-4)Galβ epitope from the chain extension linked to the distal manno-heptose of the Hep trisaccharide inner core ( Figure 5) resulted in decreased binding of VAA but had no significant effect on the binding of RCA, suggesting that RCA might not bind this LOS. Indeed, a follow up study using microarrays containing the purified LOS showed only marginal binding of RCA as opposed to strong binding of VAA, which was drastically reduced for a truncated LOS lacking Galα(1-4)Galβ (Kalograiaki et al, 2018c). In striking contrast, RCA bound strongly to the microarray-printed LOS from the capsule-deficient H. influenzae laboratory strain RdKW20, whose major glycoform displays terminal Galβ(1-4)Glc at the distal Hep extension, while, although ∼19% of this LOS bears terminal Galα(1-4)Galβ, VAA bound only weakly.…”

“…In-depth analysis of the LOS epitopes recognized by RCA and VAA in each case, using STD-NMR experiments assisted by molecular dynamics simulations, revealed that RCA bound the RdKW20 LOS glycoform displaying terminal Galβ(1-4)Glcβ, whereas VAA recognized the Galα(1-4)Galβ(1-4)Glcβ epitope ( Figure 5) in NTHi375 LOS but not in RdKW20 LOS, what could be due to different conformational preferences of the branch and ensuing presentation of the epitope. Binding assays to wild type and selected mutant/transformed whole bacterial cells ran in parallel 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, stressing the importance of examining binding to entire bacterial cells (Kalograiaki et al, 2018c).…”