KpsC and KpsS are retaining 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferases involved in synthesis of bacterial capsules

Abstract

Capsular polysaccharides (CPSs) are high-molecular-mass cell-surface polysaccharides, that act as important virulence factors for many pathogenic bacteria. Several clinically important Gram-negative pathogens share similar systems for CPS biosynthesis and export; examples include Escherichia coli, Campylobacter jejuni, Haemophilus influenzae, Neisseria meningitidis, and Pasteurella multocida. Each CPS contains a serotype-specific repeat-unit structure, but the glycans all possess a lipid moiety at their reducing termini. In E. coli and N. meningitidis, the predominant lipid is a lysophosphatidylglycerol moiety that is attached to the repeat-unit domain of the CPS via multiple residues of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), referred to as a poly-Kdo linker. The Kdo residues are β-linked, suggesting that they are synthesized by retaining glycosyltransferases. To date, the only characterized Kdo transferases are the inverting enzymes that catalyze the α-linkages found in lipopolysaccharide. Here, we identify two conserved proteins from CPS assembly systems, KpsC and KpsS, as the β-Kdo-transferases and demonstrate in vitro reconstitution of poly-Kdo linker assembly on a fluorescent phosphatidylglycerol acceptor. KpsS adds the first Kdo residue, and this reaction product is then extended by KpsC. Cross-complementation experiments demonstrate that the E. coli and N. meningitidis protein homologs are functionally conserved.

Fig. 1.

Heterologous complementation of kpsC and kpsS mutations in E. coli and N. meningitidis. E. coli ΔkpsC and ΔkpsS mutants (A) were transformed with plasmids carrying the genes indicated, and protein expression was induced with 0.1% l-arabinose. N. meningitidis strains were analyzed using chromosomally encoded expression of PSA in a strain constructed by allelic replacement (B). PSA production was examined in immunoblots on PVDF membrane, probed with mAb 2.2B. The inability of nonlipidated CPS produced by ΔkpsC and ΔkpsS mutants was reported previously (14). Small variations in apparent chain length may be due to different levels of expression of plasmid-encoded proteins.

Fig. 2.

Restoration of surface PSA CPS in E. coli ΔkpsC cells transformed with plasmids encoding polypeptides encompassing predicted domains of N. meningitidis KpsC. Full-length KpsC contains 704 amino acids, and the numbers refer to the residues included in each construct. The concentrations of l-arabinose and anhydrotetracycline used to induce gene expression were 0.1% and 1 μg/mL, respectively. The numbers refer to residues from KpsC encompassed by each polypeptide. PSA production was examined on a PVDF immunoblot probed with mAb 2.2B, and surface PSA was detected by sensitivity to bacteriophage K1F. kpsC^1-704 encodes the full-length N. meningitidis KpsC.

Fig. 3.

TLC separation of the reaction mixtures using NBD-PG acceptor. The enzymes included in each reaction are indicated.

Fig. 4.

Proposed model for CPS biosynthesis. The headgroup of (lyso)phosphatidylglycerol is represented by a red rectangle, Kdo residues as green diamonds, and CPS repeat unit sugars as gray hexagons. KpsC is depicted as a single protein with two domains. CS, chondroitin synthase; GT, glycosyltransferase; HAS, hyaluronan synthase; HS, heparosan synthase; PLA, phospholipase A; PST, polysialyltransferase. Candidate initiating GTs and known polymerizing GTs from different capsule-assembly systems are identified.