Molecular basis for the structural diversity in serogroup O2-antigen polysaccharides in Klebsiella pneumoniae

Abstract

Klebsiella pneumoniae is a major health threat. Vaccination and passive immunization are considered as alternative therapeutic strategies for managing Klebsiella infections. Lipopolysaccharide O antigens are attractive candidates because of the relatively small range of known O-antigen polysaccharide structures, but immunotherapeutic applications require a complete understanding of the structures found in clinical settings. Currently, the precise number of Klebsiella O antigens is unknown because available serological tests have limited resolution, and their association with defined chemical structures is sometimes uncertain. Molecular serotyping methods can evaluate clinical prevalence of O serotypes but require a full understanding of the genetic determinants for each O-antigen structure. This is problematic with Klebsiella pneumoniae because genes outside the main rfb (O-antigen biosynthesis) locus can have profound effects on the final structure. Here, we report two new loci encoding enzymes that modify a conserved polysaccharide backbone comprising disaccharide repeat units [→3)-α-d-Galp-(1→3)-β-d-Galf-(1→] (O2a antigen). We identified in serotype O2aeh a three-component system that modifies completed O2a glycan in the periplasm by adding 1,2-linked α-Galp side-group residues. In serotype O2ac, a polysaccharide comprising disaccharide repeat units [→5)-β-d-Galf-(1→3)-β-d-GlcpNAc-(1→] (O2c antigen) is attached to the non-reducing termini of O2a-antigen chains. O2c-polysaccharide synthesis is dependent on a locus encoding three glycosyltransferase enzymes. The authentic O2aeh and O2c antigens were recapitulated in recombinant Escherichia coli hosts to establish the essential gene set for their synthesis. These findings now provide a complete understanding of the molecular genetic basis for the known variations in Klebsiella O-antigen carbohydrate structures based on the O2a backbone.

Keywords: Gram-negative bacteria; Klebsiella pneumonia; O antigen; carbohydrate structure; glycosyltransferase; lipopolysaccharide (LPS); nuclear magnetic resonance (NMR); polysaccharide; polysaccharide structure; serotyping.

Figure 1.

The known carbohydrate repeating unit structures of OPSs of K. pneumoniae. The sugars contained within the square brackets are from the repeat units of the OPS. The O1 and O2c antigens are polysaccharides attached to the non-reducing end of the O2a antigen in the serotypes O1/O8 and O2ac, respectively. A subset of O2afg serotypes can also contain the O1 antigen. Not shown here are non-stoichiometric O-acetyl groups that distinguish serotypes O1 and O8, for example.

Figure 2.

Organization of the genetic determinants of O-antigen biosynthesis in K. pneumoniae serotypes derived from O2a. The O serotypes are indicated at the left along with the designations of the strains from which the DNA sequences were obtained. hisI defines the 3′ end of the O2a-antigen biosynthesis cluster (rfb region). The wzm-wbbO genes are necessary and sufficient for biosynthesis of the O2a antigen. The function of orf7 is unknown, and it is not necessary for biosynthesis of OPS in E. coli K-12. In serotype O2afg, the gmlABC cluster is located adjacent to the rfb region, whereas in O2aeh and O2ae, this cluster (gmlABD) is located next to the genomic proA locus. orf8 is present at the 3′ end of the rfb region in CWK53 and encodes a putative acetyltransferase. The O1 (wbbY) and O2c gene clusters (wbmVWX) are not linked to the rfb region, and they are flanked by transposase genes (indicated by tnp and the insertion element (IS) family to which they belong). wbbY can be present in strains expressing either the O2a or the O2afg (gmlABC) antigens, and an example of each is shown.

Figure 3.

SDS-PAGE and immunoblotting of LPS showing gmlABC^2afg- and gmlABD^2aeh-dependent seroconversion of the O2a antigen to the O2afg and O2aeh antigens. A and D, silver-stained SDS-PAGE of LPS in whole-cell lysates. The corresponding immunoblots were probed with antiserum specific for O2a (B and E), the O2afg OPS of CWK55 (C), or the O2aeh OPS of CWK53 (F). The rfb^2a genes were expressed from pWQ288. The gmlABC^2afg and gmlABD^2aeh gene clusters were contained on pWQ393 and pWQ394, respectively. Some cross-reactivity was observed with the anti-O2a serum and unsubstituted lipid A-core molecules in CWK53 and CWK55. The orphan band in the last lane of E reflects an undigested protein antigen, which sometimes occurs in such lysates. In F, the higher reactivity of the wildtype CWK53 LPS (compared with the recombinant) could reflect an epitope(s) contributed by O-acetyl groups present in CWK53 but absent from the recombinant.

Figure 4.

¹³C NMR spectra of the OPSs isolated from recombinant E. coli K-12 strains expressing the rfb^2a locus alone (O2a) and in combination with the gmlABC^2afg genes from K. pneumoniae CWK55 (O2afg) and gmlABD^2aeh from CWK53 (O2aeh). Signals were assigned based on ¹H NMR and 2D COSY, TOCSY, ROESY, ¹H-¹³C HSQC, and HMBC experiments (for HSQC and HMBC spectra, see Figs. S1 and S3). The alphanumeric designations above the NMR peaks refer to carbons in the sugar residues labeled on the repeat-unit structure at the right of each spectrum. The ¹³C NMR glycosylation effects on G C-1 (+8.1 ppm in O2afg and +3.2 ppm in O2aeh) are in good agreement with the reported values of +8.0 ppm and +4.0 ppm for the α-(1→4) and α-(1→2) linkages, respectively (71). Downfield displacement of the signals for P C-4 in the O2afg spectrum and P C-2 in the O2aeh spectrum (by 9.0 and 2.5 ppm, respectively, compared with their position in →3)-substituted P′) confirmed the positions of side-chain galactose. A small portion of the repeating units were unmodified in both the O2afg (∼6%) and the O2aeh (∼15%) polysaccharides. The rfb^2a genes were expressed from pWQ288. The gmlABC^2afg and gmlABD^2aeh gene clusters were contained on pWQ393 and pWQ394, respectively.

Figure 5.

A gene cluster unlinked to the rfb region is involved in thermoregulated biosynthesis of the O2c antigen. A, silver-stained SDS-PAGE of LPS from whole-cell lysates of the wild-type K. pneumoniae 5053 and E. coli DH5α harboring recombinant plasmids containing genes required for the biosynthesis of the O2a and the O2c OPS. B and C, corresponding immunoblots probed with antisera specific for the O2c and O2a OPS, respectively. Cultures were grown at 30 or 37 °C. The rfb^2a genes were expressed from pWQ288. The wbmVWX genes were provided by pWQ395.

Figure 6.

Production of the O2c antigen requires three genes (wbmVWX). A, to determine whether all three genes in the wbmVWX are necessary for the expression of the O2c antigen, a series of constructs were made that each eliminate one of the genes. B, silver-stained SDS-PAGE of LPS from whole-cell lysates of E. coli DH5α harboring recombinant plasmids containing genes required for the biosynthesis of the O2a OPS and derivatives of the O2c wbm gene cluster. C and D, corresponding immunoblots probed with antisera specific for the O2c and O2a OPS, respectively. Cultures were grown at 30 °C. The rfb^2a genes were expressed from pWQ288. Plasmids pWQ395, pWQ895, pWQ896, and pWQ897 expressed the gene combinations wbmVWX, wbmVX, wbmVW, and wbmWX, respectively.

Figure 7.

Biosynthesis of the K. pneumoniae O2c antigen is dependent on a functional rfb^2a gene cluster. E. coli CWG286 was transformed with pWQ395 (wbmVWX) together with a series of plasmids containing single mutations in each gene in the rfb^2a cluster. LPS in whole-cell lysates was separated by SDS-PAGE and visualized by silver-staining (A) and immunoblotting with antisera specific for O2a (B) and the O2c antigen (C). Und-PP–linked OPS accumulates intracellularly in ABC-transporter (wzm wzt) mutants. This material is poorly stained with silver but detectable by immunoblotting (70). Individual rfb^2a mutations were provided by plasmids pWQ517 (ΔwbbM), pWQ549 (wbbN*; frameshift mutation), pWQ516 (ΔwbbO), pWQ289 (Δwzm Δwzt), and pWQ633 (Δglf).