Structural studies towards the understanding of biosynthesis of the lipopolysaccharide of Haemophilus influenzae

The thesis Structural studies towards the understanding of biosynthesis of the lipopolysaccharide of Haemophilus influenzae involves structural investigation of LPS of four different H. influenzae strains, RM.118-28, RM.118, RM118-26 and RM.7004 AHl-2, paper I-IV.

H. influenzae strains are associated with diseases such as meningitidis, pneumonia, otitis media and respiratory tract infections. LPS is a characteristic surface component of the bacteria and is implicated as a virulence factor. LPS expression of H. influenzae is known to be subject to phase variation resulting in a mixture of different oligosaccharide epitopes. The structural diversity of H. influenzae LPS arising from phase variation has complicated the structural elucidation.

Phase variation is controlled by genes in the lic loci (lic1, lic2 and lic3). A novel substituent, phosphocholine (PCho), was demonstrated in LPS of H. influenzae RM. 118-28, RM. 118-26 and RM. 118. It has been shown that the lic1 locus is required for phase variable expression of PCho. In the strain RM.7004 AHl-2, the lic1 was deleted, resulting in expression of LPS with no PCho. In addition, a globotetraose unit was identified in the LPS of RM.118. It was the first time the epitope was demonstrated in LPS of H. influenzae. The tetrasaccharide is expressed in the mammalian globo series of glycolipids.

To be able to perform detailed structural analysis sofisticated purification methods such as high performance anion exchange chromatography was important. The obtained oligosaccharide fractions were analysed chemically in combination with GLC-MS. Nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS) were the mainly techniques used in order to determine the carbohydrate structure. A method to analyse intact LPS was developed by ESI-MS.