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 ␤-Glcp(1-6)-␣-Glcp(1, while in core type 1 the GlcpN residue is substituted at the O-6 position by either the disaccharide ␣-Hep(1-4)-␣-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.

To determine the function of the wabG gene in the biosynthesis of the core lipopolysaccharide (LPS) of Klebsiella pneumoniae, we constructed wabG nonpolar mutants. Data obtained from the comparative chemical and structural analysis of LPS samples obtained from the wild type, the mutant strain, and the complemented mutant demonstrated that the wabG gene is involved in attachment to ␣-L-glycero-D-manno-heptopyranose II (L,D-HeppII) at the O-3 position of an ␣-D-galactopyranosyluronic acid (␣-D-GalAp) residue. K. pneumoniae nonpolar wabG mutants were devoid of the cell-attached capsular polysaccharide but were still able to produce capsular polysaccharide. Similar results were obtained with K. pneumoniae nonpolar waaC and waaF mutants, which produce shorter LPS core molecules than do wabG mutants. Other outer core K. pneumoniae nonpolar mutants in the waa gene cluster were encapsulated. K. pneumoniae waaC, waaF, and wabG mutants were avirulent when tested in different animal models. Furthermore, these mutants were more sensitive to some hydrophobic compounds than the wild-type strains. All these characteristics were rescued by reintroduction of the waaC, waaF, and wabG genes from K. pneumoniae.In gram-negative bacteria the lipopolysaccharide (LPS) is one of the major structural and immunodominant molecules of the outer membrane. LPS consists of three domains: lipid A, core oligosaccharide, and O-specific antigen or O side chain. In smooth LPS, the core region is conceptually divided into two regions: a lipid A proximal inner core and an outer core that provides the attachment site for the O antigen (21). Comparison of the known core LPS structures from Enterobacteriaceae organisms reveals that the first outer core residue might be either glucose (Glc) or a galacturonic acid (GalA) residue. In the four known Escherichia coli core types and in Salmonella enterica, a substitution of the L-glycero-D-manno-heptopyranose II (L,D-HeppII) at the O-3 position for a Glcp residue was found (12). For Klebsiella pneumoniae, Proteus mirabilis, and Yersinia enterocolitica, a substitution of the L,D-HeppII at the O-3 position for an ␣-D-galacturonic acid residue (␣-DGalpA) residue has been described (20,29,30). On the other hand, in most of the Enterobacteriaceae studied, the core LPS contains inner core phosphoryl modifications (21), but K. pneumoniae core LPS is devoid of such modifications (29) (Fig. 1).Important roles in outer membrane permeability and in pathogenesis have been shown for the outer core and for the negative charges contributed by phosphoryl inner core modification in E. coli and/or S. enterica serovar Typhimurium (32,33,34). In view of the peculiarities of the K. pneumoniae core LPS, we sought in this work to determine the importance of the outer core LPS in K. pneumoniae outer membrane permeability and in pathogenesis. The previous knowledge of the K. pneumoniae waa gene cluster (the nomenclature proposed by Reeves et al. [23] for proteins and genes involved in core LPS biosynthesis is used in this work) and t...

Klebsiella pneumoniae strains typically express both smooth lipopolysaccharide (LPS) with O antigen molecules and capsule polysaccharide (K antigen) on the surface. A single mutation in a gene that codes for a UDP galacturonate 4-epimerase (uge) renders a strain with the O ؊ :K ؊ phenotype (lack of capsule and LPS without O antigen molecules and outer core oligosaccharide). The uge gene was present in all the K. pneumoniae strains tested. The K. pneumoniae uge mutants were unable to produce experimental urinary tract infections in rats and were completely avirulent in two different animal models (septicemia and pneumonia). Reintroduction of the single uge wild-type gene in the corresponding mutants completely restored the wild-type phenotype (presence of capsule and smooth LPS) independently of the O or K serotype of the wild type. Furthermore, complemented uge mutants recovered the ability to produce experimental urinary tract infections in rats and virulence in the septicemia and pneumonia animal models.

The core lipopolysaccharide (LPS) of Klebsiella pneumoniae is characterized by the presence of disaccharide ␣GlcN-(1,4)-␣GalA attached by an ␣1,3 linkage to L-glycero-D-manno-heptopyranose II (LD-HeppII). Previously it has been shown that the WabH enzyme catalyzes the incorporation of GlcNAc from UDP-GlcNAc to outer core LPS. The presence of GlcNAc instead of GlcN and the lack of UDP-GlcN in bacteria indicate that an additional enzymatic step is required. In this work we identified a new gene (wabN) in the K. pneumoniae core LPS biosynthetic cluster. Chemical and structural analysis of K. pneumoniae non-polar wabN mutants showed truncated core LPS with GlcNAc instead of GlcN. In vitro assays using LPS truncated at the level of D-galacturonic acid (GalA) and cell-free extract containing WabH and WabN together led to the incorporation of GlcN, whereas none of them alone were able to do it. This result suggests that the later enzyme (WabN) catalyzes the deacetylation of the core LPS containing the GlcNAc residue. Thus, the incorporation of the GlcN residue to core LPS in K. pneumoniae requires two distinct enzymatic steps. WabN homologues are found in Serratia marcescens and some Proteus strains that show the same disaccharide ␣GlcN-(1,4)-␣GalA attached by an ␣1,3 linkage to LD-HeppII.

The core lipopolysaccharide (LPS) of

The complete structures of LPS core types 1 and 2 from Klebsiella pneumoniae have been described by other authors. They are characterized by a lack of phosphoryl residues, but they contain galacturonic acid (GalA) residues, which contribute to the necessary negative charges. The presence of a capsule was determined in core-LPS non-polar mutants from strains 52145 (O1 : K2), DL1 (O1 : K1) and C3 (O8 : K66). O-antigen ligase (waaL) mutants produced a capsule. Core mutants containing the GalA residues were capsulated, while those lacking the residues were non capsulated. Since the proteins involved in the transfer of GalA (WabG) and glucosamine residues (WabH) are known, the chemical basis of the capsular-K2–cell-surface association was studied. Phenol/water extracts from K. pneumoniae 52145ΔwabH waaL and 52145ΔwaaL mutants, but not those from from K. pneumoniae 52145ΔwabG waaL mutant, contained both LPS and capsular polysaccharide, even after hydrophobic chromatography. The two polysaccharides were dissociated by gel-filtration chromatography, eluting with detergent and metal-ion chelators. From these results, it is concluded that the K2 capsular polysaccharide is associated by an ionic interaction to the LPS through the negative charge provided by the carboxyl groups of the GalA residues.

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