Structural diversity of the lipid A and core oligosaccharide moieties of the lipopolysaccharides from nontypeable and serotype f Haemophilus influenzae

This thesis describes structural studies of the oligosaccharide and lipid A moieties of lipopolysaccharides (LPSs) isolated from disease-causing Haemophilus influenzae strains. The nontypeable strains were clinical isolates from the middle ear of children suffering from otitis media and the serotype f strains had been collected from three adults with respiratory tract infections.

The LPS molecules are situated on the cell wall of H. influenzae strains and they play a very important role in colonization, infection, evasion of host immune system and inflammatory response. Previous studies have implicated the heterogeneous repertoire of LPS structures within a strain and mimicry of human cell wall structures to be involved in the diseasecausing behavior of this organism.

Structural analysis of the oligosaccharide moieties with advanced applications of nuclear magnetic resonance (NMR) and various electrospray ionization mass spectrometry (ESI-MS) techniques revealed novel structural features in each of the investigated strains. All of the strains displayed a very complex mixture of LPS structures that differed between and within the pathogens. Moreover, all of the strains had the capacity to express mimics of human glycolipids.

The genetic basis for LPS biosynthesis for H. influenzae is established for the strain of which the complete genome has been determined. In this thesis the function of the genes involved in the biosynthesis of LPS was investigated in a nontypeable strain by using the combination of genetic engineering and structural analysis. The synergy of genomics and analytical carbohydrate chemistry led to the identification of novel structural epitopes, and furthermore, enabled us to identify a new function for one of these genes.

The most recent structural study of lipid A from H. influenzae was conducted in 1988 on a mutant strain. The results of that study established the presence of only one lipid A structure. in this thesis we investigated lipid A from both nontypeable and serotype wild type strains by performing tandem ESI-MS and the results confirmed earlier findings but also evidenced other lipid A structures previously not associated with H. influenzae. Moreover, all of the strains exhibited a heterogeneous population of lipid A molecules.

List of scientific papers

I. Yildirim HH, Hood DW, Moxon ER, Schweda EK (2003). Structural analysis of lipopolysaccharides from Haemophilus influenzae serotype f. Structural diversity observed in three strains. Eur J Biochem. 270(15): 3153-67.
https://pubmed.ncbi.nlm.nih.gov/12869190

II. Mikhail I, Yildirim HH, Lindahl EC, Schweda EK (2005). Structural characterization of lipid A from nontypeable and type f Haemophilus influenzae: variability of fatty acid substitution. Anal Biochem. 340(2): 303-16.
https://pubmed.ncbi.nlm.nih.gov/15840504

III. Yildirim HH, Li J, Richards JC, Hood DW, Moxon ER, Schweda EK (2005). An alternate pattern for globoside oligosaccharide expression in Haemophilus influenzae lipopolysaccharide: structural diversity in nontypeable strain 1124. Biochemistry. 44(13): 5207-24.
https://pubmed.ncbi.nlm.nih.gov/15794658

IV. Yildirim HH, Li J, Richards JC, Hood DW, Moxon ER, Schweda EK (2005). Complex O-acetylation in non-typeable Haemophilus influenzae lipopolysaccharide: evidence for a novel site of O-acetylation. Carbohydr Res. Sep 28: Epub ahead of print.
https://pubmed.ncbi.nlm.nih.gov/16199021