Secondary acylation of Vibrio cholerae lipopolysaccharide requires phosphorylation of Kdo

Abstract

The lipopolysaccharide of Vibrio cholerae has been reported to contain a single 3-deoxy-d-manno-octulosonic acid (Kdo) residue that is phosphorylated. The phosphorylated Kdo sugar further links the hexa-acylated V. cholerae lipid A domain to the core oliogosaccharide and O-antigen. In this report, we confirm that V. cholerae possesses the enzymatic machinery to synthesize a phosphorylated Kdo residue. Further, we have determined that the presence of the phosphate group on the Kdo residue is necessary for secondary acylation in V. cholerae. The requirement for a secondary substituent on the Kdo residue (either an additional Kdo sugar or a phosphate group) was also found to be critical for secondary acylation catalyzed by LpxL proteins from Bordetella pertussis, Escherichia coli, and Haemophilus influenzae. Although three putative late acyltransferase orthologs have been identified in the V. cholerae genome (Vc0212, Vc0213, and Vc1577), only Vc0213 appears to be functional. Vc0213 functions as a myristoyl transferase acylating lipid A at the 2'-position of the glucosamine disaccharide. Generally acyl-ACPs serve as fatty acyl donors for the acyltransferases required for lipopolysaccharide biosynthesis; however, in vitro assays indicate that Vc0213 preferentially utilizes myristoyl-CoA as an acyl donor. This is the first report to biochemically characterize enzymes involved in the biosynthesis of the V. cholerae Kdo-lipid A domain.

FIGURE 1.

Comparison of E. coli K12 lipid A species to V. cholerae O1 and V. cholerae O139 lipid A species. The covalent modifications of lipid A are indicated with dashed bonds, and the lengths of the acyl chains are indicated below each structure. The lipid A of E. coli K12 is a hexa-acylated structure, bearing two secondary acyl chains at the 2′- and 3′-positions. The E. coli lipid A structure is glycosylated at the 6′-position with two Kdo moieties and is phosphorylated at the 1- and 4′-positions of the disaccharide backbone. Similar to E. coli, the lipid A species of V. cholerae serogroup O1 is hexa-acylated, but with a symmetrical acyl chain distribution. The proposed lipid A structure of V. cholerae O139 is the octa-acylated structure. Both V. cholerae serogroups O1 and O139 reported lipid A species have a single Kdo sugar that is phosphorylated (red) and a phosphoethanolamine (magenta) attached to the 1-phosphate.

FIGURE 2.

Enzymatic assay of the V. cholerae Kdo transferase and Kdo kinase. A, E. coli NovaBlue (DE3) membranes containing either pET21 or WaaA plasmids from E. coli, H. influenzae, or V. cholerae were assayed for Kdo transferase activity at 0.001 mg/ml for 1 h at 30 °C with [4′-³²P]lipid IV_A substrate. The small amount Kdo₂-l4′-³²P]lipid IV_A in the vector control lane results from endogenous levels of E. coli WaaA. B, E. coli HMS174 (DE3) membranes expressing Vc0227 (0.1 mg/ml) were assayed for Kdo kinase activity using Kdo-[4′-³²P]lipid IV_A substrate in the presence or absence of ATP.

FIGURE 3.

Assay of the V. cholerae late acyltransferases in the presence of phosphorylated Kdo-lipid IV_A (P-Kdo-lipid IV_A), or Kdo₂-lipid IV_A. E. coli MKV15 (DE3) membranes expressing the V. cholerae late acyltransferases (Vc0212, Vc0213, or Vc1577) were assayed for activity at 0.01 mg/ml for 1 h at 30 °C with a mixture of acyl-ACPs (C12:0, C14:0, C16:0, and C18:0) and either P-Kdo-[4′-³²P]lipid IV_A (A) or Kdo₂-[4′-³²P]lipid IV_A (B).

FIGURE 4.

H. influenzae and B. pertussis LpxL proteins also require phosphorylated-Kdo lipid IV_A for optimal activity. A, E. coli MKV15 (DE3) membranes expressing Vc0213, Bp3073, or Hi1527 were assayed for activity at 0.01 mg/ml for 1 h at 30 °C using a mixture of acyl-ACPs (C12:0, C14:0, C16:0, and C18:0) and either Kdo-[4′-³²P]lipid IV_A, phosphorylated Kdo-[4′-³²P]lipid IV_A (P-Kdo-[4′-³²P]lipid IV_A), or Kdo₂-[4′-³²P]lipid IV_A as the lipid acceptor. B, E. coli LpxL membranes (0.001 mg/ml) were assayed at 30 °C for 1 h using C12:0-ACP and either Kdo-[4′-³²P]lipid IV_A, P-Kdo-[4′-³²P]lipid IV_A, or Kdo₂-[4′-³²P]lipid IV_A. The origin and solvent front are not shown.

FIGURE 5.

Vc0213 is a myristoyl (C14:0) acyltransferase and utilizes C14:0-CoA as its preferred acyl donor. A, MKV15 (DE3) membranes (0.01 mg/ml) expressing either Bp3073, Hi1527, or Vc0213 were assayed with Kdo₂-[4′-³²P]lipid IV_A and individual acyl-ACPs (C12:0, C14:0, C16:0, or C18:0) for 30 min. The percentage of conversion was calculated based on the amount of Kdo₂-[4′-³²P]lipid IV_A converted to the acylated form. B, membranes expressing Vc0213, pBp3073, or pHi1527 (0.01 mg/ml) were assayed for 15 min. with either C14:0-ACP or C14:0-CoA using phosphorylated Kdo-[4′-³²P]lipid IV_A as the lipid acceptor. For assays containing E. coli LpxL, the concentration of membranes was 0.001 mg/ml and either C12:0-ACP or C12:0-CoA and Kdo₂-[4′-³²P]lipid IV_A as the lipid acceptor.

FIGURE 6.

MALDI-TOF mass spectrometry confirms Vc0213 is the E. coli LpxL homolog. Lipid A of E. coli MKV13 (lpxL and lpxP) expressing pBluescript (A) or pBSVc0213 (B) were analyzed by MALDI-TOF mass spectrometry in the negative ion mode. The major ion peak in A is m/z 1404.5 amu, which corresponds to the expected mass of lipid IV_A. The major peak in B is m/z 1824.6 amu, indicating that Vc0213 is adding a myristate (C14:0) to the 2′-position of the glucosamine disaccharide. Endogenous MKV13 LpxM adds a C14:0 to the 3′-position, thus producing the hexa-acylated lipid A species with a predicted mass of 1825.3 (inset structure). Minor peaks are explained below: at 1614.5 amu, the addition of a single C14:0; at 1324.6 amu, the loss of phosphate group at 1-position of the glucosamine disaccharide from parent ion; and at 1426.6 amu, the addition of a sodium adduct.

FIGURE 7.

Proposed pathway of V. cholerae inner core biosynthesis and secondary acylation of lipid A. The V. cholerae WaaA transfers a single Kdo moiety to the 6′-position of V. cholerae lipid IV_A (Fig. 2A). Vc0227, the V. cholerae KdkA, catalyzes the addition of a phosphate group to the Kdo sugar (Fig. 2B). Vc0213 then adds an acyl chain to the 2′-position, using either C14:0-ACP or C14:0-CoA as an acyl donor (Figs. 5 and 6). Additional lipid A modifying enzymes and late acyltransferases are necessary to generate the hexa-acylated lipid A structure of V. cholerae O1 previously reviewed by Chatterjee and Chaudhuri (11).