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	aDGalp	95.9-96.1	68.1-68.2	79.9-80.0	69.9-70.0	71.3-71.4	61.7-61.9
	bDGalp	105.1	70.4-70.5	77.7	65.5-65.6	75.6-75.7	61.9-63.5


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3	aDGalp	5.18-5.19	4.04	4.15	4.29	4.23-4.24	3.73-3.76
	bDGalp	4.68-4.69	3.74-3.75	3.80	4.19	3.68	3.68-3.76


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3	aDGalp	95.9-96.1/5.18-5.19	68.1-68.2/4.04	79.9-80.0/4.15	69.9-70.0/4.29	71.3-71.4/4.23-4.24	61.7-61.9/3.73-3.76
	bDGalp	105.1/4.68-4.69	70.4-70.5/3.74-3.75	77.7/3.80	65.5-65.6/4.19	75.6-75.7/3.68	61.9-63.5/3.68-3.76

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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