Institutionen för laboratoriemedicin / Department of Laboratory Medicine
Abstract
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 itwas 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
beta-D-Galp-(1--->4)-beta-D-Glcp-(1--->4)-alpha-DGlcp-(1--->3 epitope.
Interestingly, the lex2 mutant was indicated to be substituted at O-2 at
Hep III by beta-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