Whooping cough is a highly contagious disease caused predominantly by Bordetella pertussis, but it also comprises of a pertussis-like illness caused by B. holmesii. The virulence factors of B. holmesii and their role in the pathogenesis remain unknown. Lipopolysaccharide is the main surface antigen of all Bordetellae. Data on the structural features of the lipopolysaccharide (LPS) of B. holmesii are scarce. The poly- and oligosaccharide components released by mild acidic hydrolysis of the LPS were separated and investigated by ¹H and ¹³C NMR spectroscopy, mass spectrometry, and chemical methods. The structures of the O-specific polysaccharide and the core oligosaccharide of B. holmesii ATCC 51541 have been identified for the first time. The novel pentasaccharide repeating unit of the B. holmesii O-specific polysaccharide has the following structure: {→2)-α-l-Rhap-(1→6)-α-d-Glcp-(1→4)-[β-d-GlcpNAc-(1→3]-α-d-Galp-(1→3)-α-d-GlcpNAc-(1→}_n. The SDS-PAGE and serological cross-reactivities of the B. holmesii LPS suggested the similarity between the core oligosaccharides of B. holmesii ATCC 51541 and B. pertussis strain 606. The main oligosaccharide fraction contained a nonasaccharide. The comparative analysis of the NMR spectra of B. holmesii core oligosaccharide fraction with this of the B. pertussis strain 606 indicated that the investigated core oligosaccharides were identical.

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.

The high-resolution magic angle spinning nuclear magnetic resonance spectroscopy (HR-MAS NMR) analysis of Plesiomonas shigelloides 78/89 lipopolysaccharide directly on bacteria revealed the characteristic structural features of the O-acetylated polysaccharide in the NMR spectra. The O-antigen profiles were unique, yet the pattern of signals in the, spectra along with their ¹H,¹³C chemical shift values, resembled these of d-galactan I of Klebsiella pneumoniae. The isolated O-specific polysaccharide (O-PS) of P. shigelloides strain CNCTC 78/89 was investigated by ¹H and ¹³C NMR spectroscopy, mass spectrometry and chemical methods. The analyses demonstrated that the P. shigelloides 78/89 O-PS is composed of →3)-α-d-Galp-(1→3)-β-d-Galf2OAc-(1→ disaccharide repeating units. The O-acetylation was incomplete and resulted in a microheterogeneity of the O-antigen. This O-acetylation generates additional antigenic determinants within the O-antigen, forms a new chemotype, and contributes to the epitopes recognized by the O-serotype specific antibodies. The serological cross-reactivities further confirmed the inter-specific structural similarity of these O-antigens.

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.

Streszczenie: Krztusiec jest zakaźną chorobą, typową dla wieku dziecięcego. Znamiennym objawem jest napadowy, silny i bezproduktywny kaszel, pogarszający się w nocy, który może prowadzić do wymiotów, bezdechu oraz sinicy. Pod względem klinicznym diagnostyka krztuśca jest problematyczna, ponieważ u zaszczepionych dzieci, nastolatków oraz dorosłych objawy mogą być znacznie łagodniejsze i mniej charakterystyczne, a więc trudniejsze do zdiagnozowania. Wprowadzenie szczepionki pełnokomórkowej (DTwP) oraz obowiązku szczepień skutecznie zredukowało liczbę przypadków. W wielu krajach, szczepionki DTwP zawierające pełne komórki bakteryjne, ze względu na swoją reaktogenność, zostały całkowicie lub częściowo, zastąpione szczepionkami bezkomórkowymi (DTaP) zawierającymi kilka wybranych i oczyszczonych białkowych antygenów bakteryjnych. Pomimo powszechności szczepień ochronnych notuje się wzrost zachorowań na krztusiec, nie tylko wśród najbardziej wrażliwej grupy – noworodków, ale również zaszczepionych nastolatków i dorosłych. Główną przyczyną wzrostu zachorowań jest niepełna ochrona oraz słaba skuteczność szczepionki bezkomórkowej w zapobieganiu transmisji Bordetella pertussis , która jest głównym czynnikiem etiologicznym krztuśca. Nowoczesne metody badawcze pozwalają także na identyfikację innych gatunków bakterii z rodzaju Bordetella , tj. B. parapertussis oraz B. holmesii wywołujących parakrztusiec o podobnych objawach. Obecnie stosowane szczepionki nie chronią przed szczepami gatunku B. holmesii , który staje się drugim, pod względem częstości zachorowań, czynnikiem etiologicznym krztuśca. Niedoskonałość dostępnych szczepionek jest przyczyną intensywnych poszukiwań nowych antygenów szczepionkowych i adiuwantów, powtórnego zainteresowania szczepionkami pełnokomórkowymi oraz podejścia hybrydowego przy projektowaniu szczepionki bezkomórkowej opartej o pęcherzyki zewnątrzbłonowe. Największe szanse na wprowadzenie ma szczepionka BPZE1, która jest osłabionym szczepem bakterii B. pertussis i jest przeznaczona do podawania w postaci żywej szczepionki donosowej.