Bacterial pathogens expose on the cell surface a variety of complex carbohydrate molecules. Gram-negative bacteria produce lipopolysaccharides, which are the main components of the outer membrane of bacterial envelopes and play a major role in host–pathogen interactions. B. pertussis, B. parapertussis, B. bronchiseptica, and B. holmesii, are mammalian respiratory pathogens, having substantial economic impact on human health and agriculture. B. pertussis is responsible for whooping cough (pertussis) and B. holmesii is the second pertussis etiological factor, but the current anti-pertussis vaccines do not provide cross-protection. The structural data on any given hypothetical carbohydrate antigen is a prerequisite for further analysis of structure-related activities and their interaction with hosts. ¹H NMR spectra constitute fingerprints of the analyzed glycans and provide unique identity information. The concept of structure-reporter groups has now been augmented by ¹H,¹³C-correlation spectra of the Bordetella oligosaccharides. The comparative analysis of Bordetellae oligosaccharides (OS) revealed that the hexasaccharide, comprising the α-GlcpN, α-GlcpA, 4,6-disubstituted-β-Glcp, 2,7-disubstituted-l-α-d-Hepp, 3,4-disubstituted-l-α-d-Hepp, and Kdo, constitute the least variable OS segment. This minimal common element in the structure of lipopolysaccharides of Bordetellae could be used to devise a universal cross-protective vaccine component against infections with various bacteria from the genus Bordetella.

Hyaluronan (HA), a member of the GAG family of glycans, has many diverse biological functions that vary a lot depending on the length of the HA chain and its concentration. A better understanding of the structure of different-sized HA at the atomic level is therefore crucial to decipher these biological functions. NMR is a method of choice for conformational studies of biomolecules, but there are limitations due to the low natural abundance of the NMR active nuclei ¹³C and ¹⁵N. We describe here the metabolic labeling of HA using the bacterium Streptococcus equi subsp. Zooepidemicus and the subsequent analysis by NMR and mass spectrometry. The level of ¹³C and ¹⁵N isotope enrichment at each position was determined quantitatively by NMR spectroscopy and was further confirmed by high-resolution mass spectrometry analysis. This study provides a valid methodological approach that can be applied to the quantitative assessment of isotopically labeled glycans and will help improve detection capabilities and facilitate future structure-function relationship analysis of complex glycans.

Glycoproteins are processed endosomally prior to presentation to T cells and subsequent induction of specific antibodies. The sugar part of glycoconjugate may be degraded while the type of the process depends on the features of the particular structure. The generated carbohydrate epitopes may differ from native structures and influence immunogenicity of the antigens. We have devised a model of endosomal-like pre-processing of Bordetella pertussis 186 oligosaccharides (OSs) to verify how it affects the immunogenicity of their conjugates. The glycoconjugates of structurally defined forms of the dodecasaccharide OS were synthesized and their immunogenicity was assessed using immunochemical methods. The structural features of the oligosaccharides and their sensitivity to deamination were analyzed by NMR spectroscopy. The distal trisaccharide-comprising pentasaccharide conjugated to a protein was the most effective in inducing immune response against the B. pertussis 186 LOS and the immune response to the complete OS conjugates was significantly lower. This could be explained by the loss of the distal trisaccharide during the in-cell deamination process suggesting that the native structure is not optimal for a vaccine antigen. Consequently, our research has shown that designing of new glycoconjugate vaccines requires the antigen structures to be verified in context of possible endosomal reactions beforehand.

The hypoxic microenvironment is crucial for tumour cell growth and invasiveness. Tumour tissue results from adaptation to reduced oxygen availability. Hypoxia first activates pro‐angiogenic signals for alleviation. Pathologic, tumour angiogenesis maintains hypoxia, impairing treatment outcomes. Vessel normalisation requires physioxia. Oxygen delivery by red blood cell (RBC) carrying haemoglobin (Hb) is enhanced by myo‐inositol trispyrophosphate (ITPP), an effector of oxygen transport by RBCs. Altering glycolytic activity, it lowers intracellular pH and increases oxygen release from Hb. ³¹P NMR tracking of 2,3‐diphosphoglycerate (2,3‐DPG), allosteric effector of Hb and non‐penetrating anion in RBCs, reports on erythrocytes internal environment. ³¹P resonances of 2,3‐DPG are pH‐sensitive, their positions indicate the oxygenation state of RBCs and interactions with effectors such as ITPP. Here we show in vitro and in vivo, that modifying Hb activity through band‐3 anion transporter, ITPP enhances oxygen release and controls RBC internal pH. Its blood availability validates applicability of ITPP‐based strategies.

Pertussis pathogenesis is the result of multiple virulence factors. In addition to the secretory proteins of Bordetella pertussis , surface molecules such as adhesins and endotoxin play a role in the pathogenesis of this disease. There are conflicting reports on the existence and nature of the Bordetella capsular polysaccharides or exoglycans. The data concerning the glycome of Bordetellae is incomplete. This conclusion is primarily derived from genomic data, with only limited indications regarding the actual structures. In this study, we present novel data on the exoglycan produced by all strains and species of the investigated bacteria from the genus Bordetella , including Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, and Bordetella holmesii . This is the first time this type of data has been provided. The exoglycan was consistently recovered from the chemically defined culture media of various Bordetellae species and strains. The compound was identified by nuclear magnetic resonance (NMR) as a free hexasaccharide released into the medium and thus received its name, Bordetella oligosaccharide (BOS). The biosynthetic origin of the BOS was confirmed by NMR combined with metabolic labeling in culture, using ¹³ C, ¹⁵ N-L-glutamate as a primary carbon source. The identification of BOS has the potential to enhance our comprehension of the complete array of virulence factors contributing to the pathogenesis of Bordetella pertussis , particularly in regard to their relations with other Bordetella species. In the field of vaccine design, glycan structures are typically of utmost importance; however, they were hardly ever considered in the case of pertussis.