Secondary Acylation of Vibrio cholerae Lipopolysaccharide Requires Phosphorylation of Kdo

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.Lipopolysaccharide (LPS),² the major surface molecule in the outer membrane of Gram-negative bacteria, is composed of three domains: lipid A, core oligosaccharide, and O-antigen (1). The core oligosaccharide is further divided into two distinct regions: inner and outer core. The inner core consists of the Kdo sugars, which are responsible for linking the core region to the lipid A moiety of LPS. Lipid A is the hydrophobic anchor of LPS and is the only portion of LPS required for activating the host innate immune response by interacting with Toll-like receptor 4 and the accessory molecule, MD2.Kdo-lipid A biosynthesis is a well conserved and ordered process among Gram-negative bacteria; however, not all Gram-negative bacteria produce similar lipid A structures (2). In Escherichia coli, the biosynthesis of the Kdo-lipid A domain occurs via a nine-step process, resulting in the production of a hexa-acylated lipid A structure known as Kdo₂-lipid A. Kdo₂-lipid A has long been thought to be essential for the viability of E. coli; however, viable suppressor strains have been isolated that lack the Kdo sugar (3). The late steps of the biosynthetic pathway involve the addition of the Kdo sugars and the secondary or “late” acyl chains. The enzyme responsible for the addition of the Kdo sugars is the Kdo transferase (WaaA). In E. coli, this enzyme is bifunctional, transferring two Kdo sugars to the lipid A precursor, lipid IV_A (4); however, other Gram-negative bacteria have been shown to possess a monofunctional or trifunctional WaaA, as is the case in Haemophilus influenzae (5) or Chlamydia trachomatis (6), respectively.Previous reports have shown that in E. coli, the addition of the Kdo sugars is critical for the functionality of the secondary acyltransferases (LpxL, LpxM, and LpxP). The E. coli late acyltransferase LpxL catalyzes the transfer of laurate (C12:0) to the acyl chain linked at the 2′-position of Kdo₂-lipid IV_A (7). LpxM then catalyzes the addition of a myristate (C14:0) to the 3′-linked acyl chain of the penta-acylated lipid A precursor (8). When E. coli experience cold shock conditions (temperatures below 20 °C), the late acyltransferase LpxP transfers a palmitoleate (C16:1) to the 2′-position of Kdo₂-lipid IV_A, replacing the C12:0 acyl chain transferred by LpxL (9). Lipid A secondary acyltransferases have been shown to primarily utilize acyl-acyl carrier proteins (acyl-ACPs) as their acyl chain donor; however, a recent report by Six et al. (10) has shown that purified E. coli LpxL is capable of utilizing acyl-coenzyme A (acyl-CoA) as an alternative acyl donor at a lesser rate.The Gram-negative bacteria Vibrio cholerae is the causative agent of the severe diarrheal disease cholera. Cholera is transmitted via the fecal-oral route by ingestion of contaminated drinking water or food. The World Health Organization reported ～130,000 cases of cholera in 2005 with the majority occurring in Africa. There are two serogroups of V. cholerae capable of epidemic and pandemic disease: O1 and O139 (11). Previous structural analyses have revealed that these serogroups possess very different lipid A structures. The V. cholerae O1 lipid A structure was reported as hexa-acylated, bearing secondary acyl chains at the 2- and 2′-positions of phosphorylated Kdo-lipid A (11–13); however, V. cholerae O139 was reported as having an octa-acylated lipid A (see Fig. 1) (11, 14).Open in a separate windowFIGURE 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.Our report focuses on V. cholerae O1 El Tor, which is the predominant disease-causing strain worldwide. Because little attention has been given to the Kdo-lipid A domain of V. cholerae, we investigated the assembly of the inner core structure of V. cholerae O1 LPS and the late acylation steps. This report demonstrates the importance of a secondary negative charge on the primary Kdo sugar of lipid A for late acyltransferase activity in V. cholerae and in other Gram-negative bacteria. Also, we have identified the putative V. cholerae late acyltransferase, Vc0213 as the LpxL homolog, transferring a myristate (C14:0) to the 2′-position of V. cholerae lipid A. These initial findings provide us with the groundwork needed to investigate the modifications of the V. cholerae Kdo-lipid A structure, which may serve as attractive vaccine targets in future research.