A second outer-core region in Klebsiella pneumoniae lipopolysaccharide

Abstract

Up to now only one major type of core oligosaccharide has been found in the lipopolysaccharide of all Klebsiella pneumoniae strains analyzed. Applying a different screening approach, we identified a novel Klebsiella pneumoniae core (type 2). Both Klebsiella core types share the same inner core and the outer-core-proximal disaccharide, GlcN-(1,4)-GalA, but they differ in the GlcN substituents. In core type 2, the GlcpN residue is substituted at the O-4 position by the disaccharide beta-Glcp(1-6)-alpha-Glcp(1, while in core type 1 the GlcpN residue is substituted at the O-6 position by either the disaccharide alpha-Hep(1-4)-alpha-Kdo(2 or a Kdo residue (Kdo is 3-deoxy-D-manno-octulosonic acid). This difference correlates with the presence of a three-gene region in the corresponding core biosynthetic clusters. Engineering of both core types by interchanging this specific region allowed studying the effect on virulence. The replacement of Klebsiella core type 1 in a highly type 2 virulent strain (52145) induces lower virulence than core type 2 in a murine infection model.

FIG. 1.

K. pneumoniae oligosaccharide (OS) core LPS structures. (A) Type 1 core (40, 42). Depending on the K. pneumoniae strain, residues J and K could be H or GalA, and residue P could be H or Hep. Broken lines denote the truncation level for the different core biosynthetic gene mutations (12, 18-20, 32). (B) Type 2 core found in K. pneumoniae 52145 (O1:K2). Oligosaccharides OS1 and OS2 from K. pneumoniae 52145 waaL mutant LPS obtained by complete O and N deacylation under alkaline conditions and mild acid hydrolysis, respectively. Letters denoting OS1 and OS2 residues are the same used in Fig. 4 and 5 (also Tables S2 and S3 in the supplemental material). Kdop, l,d-Hepp, Glcp, GlcNp, and GalAp.

FIG. 2.

Diagram of the K. pneumoniae 52145 waa gene cluster and comparison of this cluster to those of K. pneumoniae C3 (32) and S. marcescens N28b (6). White boxes denote PCR-amplified DNA fragments. Common inner core genes (black arrows), common outer-core genes (gray arrows). Common outer-core genes between K. pneumoniae 52145 and S. marcescens N28b (striped arrows). Cluster-specific genes (white arrows). K. pneumoniae 52145 waa gene cluster, GenBank accession no. AY823268.

FIG. 3.

SDS-Tricine-PAGE analysis of LPS samples from K. pneumoniae 52145- and 52145-derived nonpolar core biosynthetic mutants.

FIG. 4.

Anomeric region of the ¹H-NMR spectra of OS1 obtained from K. pneumoniae 52145 waaL mutant LPS by complete O and N deacylation. Letters denote residues as in Fig. 1B OS1.

FIG. 5.

(A) Anomeric region of the ¹H-NMR spectra and (B) ROESY spectra of OS2 obtained from K. pneumoniae 52145 waaL mutant LPS by mild acid hydrolysis. Letters denote residues as in Fig. 1B OS2.

FIG. 6.

Positive ion MALDI-TOF spectrum of acid-released core LPS oligosaccharides from K. pneumoniae 52145Δ waaL.

FIG. 7.

Positive ion MALDI-TOF/TOF spectrum of m/z 1814.55 signal of acid-released core LPS oligosaccharide isolated from K. pneumoniae 52145ΔwaaL, in the positive ion mode. Insert shows the fragmentation pattern.

FIG. 8.

Positive ion MALDI-TOF/TOF spectrum of m/z 1652.07 signal of acid-released core LPS oligosaccharide isolated from K. pneumoniae 52145ΔwaaL, in the positive ion mode. Insert shows the fragmentation pattern.

FIG. 9.

SDS-PAGE (A) and SDS-Tricine-PAGE (B) analysis of LPS samples from K. pneumoniae type 2 core strains 52145 (O1:K2) and B5055 (O1:K2), 52145-derived triple core biosynthesis mutants, homologous and heterologous complemented mutants, and type 1 core strain C3 (O8:K66).