Bacteriophage-mediated Glucosylation Can Modify Lipopolysaccharide O-Antigens Synthesized by an ATP-binding Cassette (ABC) Transporter-dependent Assembly Mechanism

Abstract

Lysogenic bacteriophages may encode enzymes that modify the structures of lipopolysaccharide O-antigen glycans, altering the structure of the bacteriophage receptor and resulting in serotype conversion. This can enhance virulence and has implications for antigenic diversity and vaccine development. Side chain glucosylation is a common modification strategy found in a number of bacterial species. To date, glucosylation has only been observed in O-antigens synthesized by Wzy-dependent pathways, one of the two most prevalent O-antigen synthesis systems. Here we exploited a heterologous system to study the glucosylation potential of a model O-antigen produced in an ATP-binding cassette (ABC) transporter-dependent system. Although O-antigen production is cryptic in Escherichia coli K-12, because of a mutation in the synthesis genes, it possesses a prophage glucosylation cluster, which modifies the GlcNAc residue in an α-l-Rha-(1→3)-d-GlcNAc motif found in the original O16 antigen. Raoultella terrigena ATCC 33257 produces an O-antigen possessing the same disaccharide motif, but its assembly uses an ABC transporter-dependent system. E. coli harboring the R. terrigena O-antigen biosynthesis genes produced an O-antigen displaying reduced reactivity toward antisera raised against the native R. terrigena repeat structure, indicative of an altered chemical structure. Structural determination using NMR revealed the addition of glucose side chains to the repeat units. O-antigen modification was dependent on a functional ABC transporter, consistent with modification in the periplasm, and was eliminated by deletion of the glucosylation genes from the E. coli chromosome, restoring native level antisera sensitivity and structure. There are therefore no intrinsic mechanistic barriers for bacteriophage-mediated O-antigen glucosylation in ABC transporter-dependent pathways.

Keywords: ABC transporter; O-antigen; bacteriophage; biosynthesis; glycosylation; lipopolysaccharide (LPS); nuclear magnetic resonance (NMR); outer membrane; polysaccharide.

FIGURE 1.

Structures of the O-polysaccharide repeat units. The component sugars are glucose (Glc), 2-acetylamino-2-deoxyglucose (N-acetylglucosamine; GlcNAc), and 6-deoxymannose (rhamnose; Rha).

FIGURE 2.

Organization of the O-antigen biosynthesis genes in E. coli. Shown are the wb* O-antigen biosynthesis gene clusters from E. coli K-12 (GenBank^TM accession number NC_010473) and R. terrigena ATCC 33257 (GenBank^TM accession number AY376146), as well as the KplE1 prophage (also named CPS-53) in the E. coli K-12 genome, encoding the three Gtr proteins required for glucosylation of O-antigens.

FIGURE 3.

Reaction of LPS from R. terrigena ATCC 33257 and E. coli recombinant strains with antibodies raised against the K. pneumoniae O12 antigen. The figure shows the silver-stained SDS-PAGE LPS profile (upper panel) and the corresponding immunoblot (lower panel). The samples were proteinase K-treated whole cell lysates. The markers indicated on the left are protein standards to allow comparison of LPS migrations in different backgrounds.

FIGURE 4.

¹³C NMR spectra of the O-polysaccharides. The spectra represent samples from E. coli CWG1217 (Δwzx-wbbK) (top panel) and CWG1218 (Δwzx-wbbK ΔgtrA) (bottom panel) harboring pKM114. The numbers refer to carbons in sugar residues designated as shown in Table 1. The displacement of the A6 and A5 signals (C-6 and C-5 of GlcNAc) is due to positive α-effect on the linkage carbon and negative β-effect on the neighboring carbon and indicates the position of glucosylation.

FIGURE 5.

Partial ¹H,¹³C heteronuclear single-quantum coherence spectrum of the O-polysaccharide of E. coli CWG1217 Δwzx-wbbK harboring the R. terrigena O-antigen gene cluster. The corresponding parts of the ¹H and ¹³C NMR spectra are shown along the axes. The numbers refer to H/C pairs in sugar residues, designated as shown in Table 1.

FIGURE 6.

O-antigen modification is dependent on a functional ABC transporter. The samples were proteinase K-treated whole cell lysates of E. coli CWG1217 (Δwzx-wbbK) and CWG1219 (Δwzx-wbbK ΔgtrA) transformed with pWQ677 (carrying wbbL-wzm-wbbB) and pWQ114 (encoding FLAG-Wzt). Gene expression was induced with 2.5 ng ml⁻¹ anhydrotetracycline to give a constant level of the biosynthesis enzymes. The amount of FLAG-Wzt (NBD) was varied by repression with glucose or induction with different concentrations of l-arabinose. The presence of the O12 antigen in LPS and Und-PP-linked glycans was assessed using silver stain (top panel) and immunoblotting (middle panel) of proteinase K-treated whole cell lysates. The silver stain profile detects only lipid-A linked O-antigen, whereas the immunoblot detects O-antigen linked to both lipid-A and UndPP. The Western immunoblot of FLAG-tagged Wzt confirmed the presence of the NBD (bottom panel). The markers indicated on the left are protein standards to allow comparison of LPS migrations in different backgrounds.