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	110.6	81.4	85.7	80.5	71.0	63.7
4	aDGalp	101.2	69.9	70.0	69.5	71.4	60.9
	aDGalp	100.9	68.6	77.7	79.1	73.1	61.1


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3	bDGalf	5.22	4.34	4.08	4.30	3.86	3.69
4	aDGalp	5.01	3.83	3.92	4.07	4.24	3.79
	aDGalp	5.10	4.10	3.95	4.18	4.17	3.91


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3	bDGalf	110.6/5.22	81.4/4.34	85.7/4.08	80.5/4.30	71.0/3.86	63.7/3.69
4	aDGalp	101.2/5.01	69.9/3.83	70.0/3.92	69.5/4.07	71.4/4.24	60.9/3.79
	aDGalp	100.9/5.10	68.6/4.10	77.7/3.95	79.1/4.18	73.1/4.17	61.1/3.91

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

486.423 g/mol (C₁₈H₃₀R₂O₁₅, R = next and previous repeats, not counted in mol weight)