Structural studies on lipopolysaccharides from haemophilus species

Abstract: Background: Carbohydrates are indispensable mediators for a variety of cellular interactions. In biological systems carbohydrates are usually linked to a carrier as e.g. proteins or lipids. One example is the molecule lipopolysaccharide (LPS). LPS is one of the major outer membrane constituents found in Gram-negative bacteria and they play key roles in the biology of these organisms. Notably, they have been found to be important virulence factors in pathogenic species. Aims: In this thesis the biosynthesis and the molecular structures of LPS expressed by Haemophilus influenzae and Haemophilus parainfluenzae were investigated. These two bacteria colonize the human nasopharynx. H. influenzae with capsule type b is involved in invasive diseases such as meningitidis and epiglottis while non-encapsulated H. influenzae (non-typeable, NTHi) often cause otitis media, and acute and chronic lower respiratory tract infections in infants. Though, closely related H. parainfluenzae is a commensal. Materials and Methods: Structural elucidation of LPS involves initial de-lipidation to obtain water-soluble material that is suitable for subsequent analyses by chemical, nuclear magnetic resonance (NMR) and mass spectrometric (MS) methods. Results: The function of the gene lic2B in H. influenzae type b strain Eagan was investigated. The LPS expressed by the mutant strain Eaganlic2Blic2C+ was analyzed and found to encode for a glucosyltransferase responsible for the addition of β-D-Glcp to O-4 of α-D-Glcp-(1→ elongating from the middle inner core heptose. Further, the LPS structures of NTHi strains 1247 and 1008 were determined. NTHi strain 1247 expressed globotetraose elongating from the phosphocholine bearing GlcI, [β-DGalpNAc-(1→3)-α-D-Galp-(1→4)-β-D-Galp-(1→4)-β-D-Glcp-(1→4)-[PCho→6]-β-DGlcIp-(1→], or truncated versions thereof. Globotetraose was also found to elongate from the distal heptose in NTHi 1247. The lpsA mutant of strain 1247 allowed the identification of a novel disialyllactose epitope, [α-Neu5Ac-(2→8)-α-Neu5Ac-(2→3)- β-D-Galp-(1→4)-β-D-Glcp-(1→], from the proximal inner core heptose. Alternatively, a globotetraose was found to elongate from GlcI. NTHi strain 1008 showed almost identical glycoforms to NTHi strain 1247 but lacked terminal N-acetyl galactoseamine. All of the three investigated H. parainfluenzae strains were shown to express the same lipid A and inner core as NTHi. H. parainfluenzae genome strain T3T1 and strain 22 were shown to express rough-type LPS having novel outer core structures elongating from GlcI that were [α-Neu5,9Ac2-(2→6)-β-D-GalpNAc-(1→4)-β-D-Galp-(1→3)-β-DFucpNAc4N-(1→] in strain T3T1 and [Neu5Ac-(2→6)-α-D-Galp-(1→6)-β-D-Glcp- (1→3)-β-D-FucpNAc4N-(1→] in strain 22. H. parainfluenzae strain 13 expressed an Orepeating chain with the structure [→6)-[Ac→3]-β-D-Galf-(1→3)-[PEtn→6]-β-DGlcpNAc-(1→]. Conclusion: It was showed that the H. parainfluenzae LPS investigated here lacks all virulence determining LPS attributes expressed by H. influenzae such as phosphocholine and phase variable expression of outer core structures. This may provide further insight into the factors relating to commensal vs. pathogenic behavior inside the host.