Conserved glycolipid termini in capsular polysaccharides synthesized by ATP-binding cassette transporter-dependent pathways in Gram-negative pathogens

Abstract

Bacterial capsules are surface layers made of long-chain polysaccharides. They are anchored to the outer membrane of many Gram-negative bacteria, including pathogens such as Escherichia coli, Neisseria meningitidis, Haemophilus influenzae, and Pasteurella multocida. Capsules protect pathogens from host defenses including complement-mediated killing and phagocytosis and therefore represent a major virulence factor. Capsular polysaccharides are synthesized by enzymes located in the inner (cytoplasmic) membrane and are then translocated to the cell surface. Whereas the enzymes that synthesize the polysaccharides have been studied in detail, the structure and biosynthesis of the anchoring elements have not been definitively resolved. Here we determine the structure of the glycolipid attached to the reducing terminus of the polysialic acid capsular polysaccharides from E. coli K1 and N. meningitidis group B and the heparosan-like capsular polysaccharide from E. coli K5. All possess the same unique glycolipid terminus consisting of a lyso-phosphatidylglycerol moiety with a β-linked poly-(3-deoxy-d-manno-oct-2-ulosonic acid) (poly-Kdo) linker attached to the reducing terminus of the capsular polysaccharide.

Keywords: capsule assembly; polysaccharide export.

Fig. 1.

Identification of the terminal glycolipid structure isolated from E. coli K1 CPS by mass spectrometry. (A) LC-MS data for E. coli K1 CPS terminus. All indicated ions correspond to lyso-phosphatidylglycerol attached to a poly-Kdo linker and one or more NeuAc residues. The composition of the acyl chain is in parentheses and the number of Kdo and NeuAc residues in each ion is indicated. The charge of the ion is shown next to the letter identifying it. (B) LC-MS/MS data for the m/z 868.5 ion. Glycerol and phosphate are designated Gro and P, respectively.

Fig. 2.

Determination of the lipidation state of E. coli K1 CPS in ∆kpsC and ∆kpsS mutants. (A) Immunoblots of whole-cell lysates on PVDF or positively charged nylon membranes. These were probed with the monoclonal α-PSA mAb 2-2B. Whereas CPS from the parent (K1) binds to both membranes, CPSs from the mutants (∆C and ∆S) do not bind to PVDF. (B) PLA treatment of purified K1 CPS is sufficient to eliminate binding to PVDF.