Structural and genetic characterization of lipopolysaccharides extracted from disease causing non-typeable Haemophilus influenzae strains

This thesis deals with the structure and genetic blueprint of lipopolysaccharides (LPS) expressed by the Gram-negative bacterium Haemophilus influenzae (H.influenzae). H. influenzae is an opportunistic pathogen that regularly colonizes the upper respiratory tract and exists in encapsulated (typeable) or nonencapsulated (non-typeable) forms (NTHi). Prior research has indicated that the surface expressed lipopolysaccharides (LPS) is a major virulence factor of H. influenzae. Pathogenic behavior can for example result in respiratory tract infections, otitis media (OM) or invasive disease such as meningitis. The thesis contains detailed studies of the phase-variable glycosyltransferase lex2. This transferase was shown to act either as a glucosyltransferase or galactosyltransferase depending on one single key amino acid. This was established by elucidation of LPS expressed by genetically defined lex2 mutant strains or transformant strains in which the transferase activity was removed or altered. Moreover, two structural characterizations of non-typeable clinical isolates, strains 2019 and R2866 are investigated. NTHi R2866 is an atypical non-typeable strain as it was isolated from a child with meningitis. Our data indicate that R2866 produces an extremely heterogeneous population of glycoforms with expression of L-glycero-Dmanno-heptose (L,D-Hep) in its outer core. This residue was evidenced to carry a phoshocholine (PCho) residue in O-7 position, a substitution which is novel. Moreover, this study includes data obtained for genetically defined mutant strains R2866lpsA and R2866losB2 as these demonstrated detailed LPS structures not seen in wild-type. The characterization of LPS expressed by 2019 wild-type strain and mutant strains 2019lex2, 2019lpt3 and 2019pgmB add to the previously published structure of 2019. In 1992 it was established that lactose is linked to the proximal heptose (Hep I) of the conserved triheptosyl inner-core moiety, no other structures were reported. We show that the middle heptose (Hep II) can express a β-D-Galp-(1→4)-β-D-Glcp-(1→4)-α-DGlcp-(1→3 epitope. Interestingly, the lex2 mutant was indicated to be substituted at O2 at Hep III by β-D-Glcp which, in turn, can be further extended. Such elongations have never been reported for NTHi 2019 before. NTHi 2019 belongs to a small subset of strains that express one additonal phosphoethanol amine (PEtn) residue in its outer core. Here, we establish that PEtn substitutes O-3 of the distal heptose (Hep III). This PEtn substituent was absent in the lpt3 mutant indicating Lpt3 to be the transferase required to add PEtn to Hep III. LIST OF PUBLICATIONS This thesis is based on the following papers, which in the text will be referred to by their roman numerals (I-IV). I. Engskog MKR, Yildirim HH, Li J, Richards JC, Deadman M, Hood DW and Schweda EKH. A dual role for the lex2 locus: identification of galactosyltransferase activity in non-typeable Haemophilus influenzae strains 1124 and 2019. Carbohydrate Research. 344(5): 632-641, (2009) II. Deadman ME, Hermant P, Engskog MKR, Makepeace K, Moxon ER, Schweda EKH and Hood DW. Lex2B, a phase-variable glycosyltransferase, adds either a glucose or a galactose to Haemophilus influenzae lipopolysaccharide. Infection and Immunity. 77(6): 2376-2387, (2009) III. Engskog MKR, Deadman ME, Hood DW and Schweda EKH. Detailed structural features of lipopolysaccharide glycoforms in NTHi strain 2019