Klebsiella pneumoniae is a Gram-negative, ubiquitous bacterium capable of causing severe nosocomial infections in individuals with impaired immune system. Emerging multi-drug resistant strains of this species and particularly carbapenem-resistant strains pose an urgent threat to public health. The lipopolysaccharide (LPS) O-antigen is the main surface antigen. It contributes to the virulence of this species and determines the O-serotype of K. pneumoniae isolates. Among the nine main O-serotypes of K. pneumoniae, O1-and O2-type pathogens are causative agents of over 50% of all infections. Serotype O1, the most common O-serotype, expresses complex LPS consisting of d-galactan-I (a polymer built of →3)-β-d-Galf-(1→3)-α-d-Galp-(1→ repeating units) capped by d-galactan-II (built of [→3)-α-d-Galp-(1→3)-β-d-Galp-(1→] repeating units). Galactan-I is present as the sole polymer in O2 serotype. Recently, in case of serotype O2, conversion of galactan-I to galactan-III (→3)-β-d-Galf-(1→3)-[α-d-Galp-(1→4)]-α-d-Galp-(1→) was reported. Substitution of →3)-α-d-Galp by a branching terminal α-d-Galp was dependent on the presence of the gmlABC operon and had a major impact on the antigenicity of the galactan polymer. Genetic analysis indicated that 40% of the O1 clinical isolates also carry the gmlABC locus; therefore we aimed to characterize the corresponding phenotype of LPS O-antigens. The presence of galactan-III among O1 strains was proven using galactan-III-specific monoclonal antibodies and confirmed by structural analyses performed using sugar and methylation analysis as well as classical and high-resolution magic angle spinning NMR spectroscopy. By using an isogenic mutant pair, we demonstrated that galactan-III expression was dependent on the presence of glycosyltransferases encoded by gmlABC, as was shown previously for the O2 serotype. Furthermore, the galactan-II structures in O1gml+ strains remained unaffected corroborating no functional interactions between the biosynthesis of galactan-III and galactan-II polymers.

Lipopolysaccharide, O-antigen, Klebsiella pneumoniae, Galactan, HR-MAS NMR, d-galactan-I, d-galactan-III, serotype O1

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3	bDGalf	110.2	80.5	85.2	82.6	71.6	63.5
	aDGalp	100.2	68.0	77.8	70.1	72.0	61.7


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3	bDGalf	5.21	4.42	4.07	4.25	3.87	3.68
	aDGalp	5.08	3.95	3.95	4.14	4.14	3.73


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3	bDGalf	110.2/5.21	80.5/4.42	85.2/4.07	82.6/4.25	71.6/3.87	63.5/3.68
	aDGalp	100.2/5.08	68.0/3.95	77.8/3.95	70.1/4.14	72.0/4.14	61.7/3.73

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1[C@H]([C@H](O)CO)O[C@@H](O[C@@H]2[C@@H](O)[C@@H](*)O[C@H](CO)[C@@H]2O)[C@@H]1O

324.282 g/mol (C₁₂H₂₀R₂O₁₀, R = next and previous repeats, not counted in mol weight)