Detailed structural studies towards the understanding of lipopolysaccharide glycan expression in non-typeable Haemophilus influenzae

University dissertation from Stockholm : Karolinska Institutet, Department of Laboratory Medicine

Abstract: Haemophilus influenzae (Hi) is a host-adapted Gram-negative bacterium that regularly colonizes the respiratory tract of humans. Hi is an important cause of disease worldwide and exists in encapsulated and unencapsulated (non-typeable, NT) forms. Lipopolysaccharide (LPS) is a characteristic surface component of the bacteria and has been shown to be an important virulence factor. A great variety of both inter- and intrastrain LPS glycoform structures have been detected and structurally elucidated as well as the genes that are involved in LPS biosynthesis. The knowledge of LPS biosynthetic genes and their related structures has facilitated in vivo studies of LPS in virulence. An ultimate goal is to use this knowledge in the developments of LPS-based vaccines. In this thesis, LPS from three NTHi strains taken from patients with otitis media has been structurally elucidated. The inter-strain differences between the closely related strains 1268 and 1200 compared to the sequenced strain R2846 were very apparent. All three strains indicated great intra-strain heterogeneity regarding both glycose extensions and non-carbohydrate substituents. Furthermore, the strains showed structural outer-core features that had previously not been detected in other Hi strains. In addition to the structural elucidation of LPS from the wild-type strains, the biosynthesis of the outer-core LPS region was investigated using combined genetics and structural studies. Two heptosyltransferase gene candidates, losB1 and losB2 were shown to direct the expression of outer-core heptose in strain R2846. Furthermore, LPS from several lpsA mutant strains were structurally elucidated in order to identify which part of the gene sequence of lpsA is responsible for directing the addition of glucose and galactose to the distal inner-core heptose via alternative linkages. LPS was also analyzed to compare changes in glycoforms between in vivo and in vitro grown bacteria and also importantly, in order to study the expression patterns of LPS during different stages of chinchilla middle-ear infection. It was found that as disease progressed LPS glycoforms became more truncated and less complex. Furthermore, glycoforms containing sialic acid were absent after 9 days post-infection. In order to obtain a complete detailed structural LPS analysis several different methods and techniques were used. Briefly, LPS was isolated by extraction from lyophilized bacteria. LPS was then either subjected to O-deacylation to remove ester linked fatty acids of lipid A or subjected to mild acid hydrolysis in order to release the entire lipid A moiety. The three products, LPS-OH (obtained by O-deacylation) and OS and lipid A (obtained by mild hydrolysis) were further chemically degraded and derivatized or analysed directly by different mass spectrometric (MS) and nuclear magnetic resonance (NMR) techniques.

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