• Biosynthesis and Modification of Helicobacter pylori Lipid A

      Stead, Christopher Michael; Department of Biochemistry and Molecular Biology (2010-05)
      The secondary acylation steps of Helicobacter pylori lipid A biosynthesis are poorly understood because H. pylori only has one homolog (Jhp0265) to the Escherichia coli secondary acyl transferases LpxL and LpxM. Jhp0265 was shown to be responsible for the transfer of a secondary C18 acyl chain to the 2′-linked acyl chain of lipid A, making Jhp0265 homologous to LpxL. An activity was also demonstrated for the addition of a secondary acyl chain to the 3′-linked acyl chain of H. pylori lipid A, although the enzyme responsible for the transfer remains unknown. After synthesis, H. pylori lipid A is modified by the action of five enzymes. Mutation of the candidate modification enzyme Jhp0634 demonstrated that the enzyme catalyzes the removal of the 3′-linked acyl chains of H. pylori lipid A, producing a tetra-acylated lipid A species. Continuing with the characterization of H. pylori lipid A modification enzymes, we were also able to demonstrate an activity for a Kdo trimming enzyme in vitro. Requirement for a Kdo hydrolase in vivo was confirmed after the Kdo transferase of H. pylori was shown to be bifunctional despite the presence of only one Kdo sugar in H. pylori lipopolysaccharide. Attempted identification of the Kdo hydrolase revealed that both Hp0579 and Hp0580 were required for the removal of the Kdo sugar, which occurred in the periplasm. A Kdo hydrolase mutant revealed two unexpected phenotypes related to interaction with the innate immune system. The first was an increased sensitivity to cationic antimicrobial peptides, which was explained by a downstream effect on modification to the 4′- phosphate group of lipid A. The second phenotype related to the expression of Oantigen on the bacterial cell surface. The Kdo hydrolase mutants produced a reduced amount of fully extended lipopolysaccharide and conversely, an increased amount of core-lipid A. The type of O-antigen epitope displayed was also affected by a Kdo hydrolase mutation, in a strain specific manner.
    • Biosynthesis of the Vibrio cholerae Kdo-lipid A Domain and its Role in Pathogenesis

      Hankins, Jessica V.; Department of Biochemistry and Molecular Biology (2011-05)
      Bacteria assemble remarkable surface structures that interface with their surrounding environment. One such structure is the glycolipid lipopolysaccharide (LPS) that covers the surface of Gram-negative bacteria. LPS is anchored to the bacterial cell by its lipid anchor known as lipid A. Since lipid A is the bioactive component of LPS, modulation of its structure can have a profound impact on disease by altering the host immune response. Additionally, LPS structure directly impacts the outer membrane permeability barrier and bacterial resistance to host antimicrobial peptides. Although the lipid A domain of Escherichia coli has been well characterized, the Vibrio cholerae lipid A biosynthetic pathway has received little attention. The late stages of lipid A biosynthesis include the transfer of the 3-deoxy-Dmanno- octulosonic acid (Kdo) sugars and the secondary acyl chains to the lipid A backbone. Here, the V. cholerae Kdo transferase (Vc0233) was shown to be monofunctional, transferring one Kdo residue to the lipid A precursor, lipid IVA. V. cholerae encode a Kdo kinase (Vc0227) responsible for the phosphorylation of the Kdo residue. The functionality of Vc0227 was shown to be required for the activity of the V. cholerae lipid A LpxL homologue, Vc0213. Interestingly, the addition of the phosphate group on the Kdo sugar was shown to be essential for lipid A secondary acylation in Haemophilus influenzae and Bordetella pertussis. Vc0213 was shown to catalyze the transfer of a myristate (C14:0) to the 2′-position of the V. cholerae phosphorylated Kdolipid A domain. A second protein, Vc0212, acts as an LpxM homologue and transfers 3- hydroxylaurate (3-OH C12:0) to the 3′-position creating hexa-acylated V. cholerae lipid A domain. Although lipid A secondary acyltransferases have been characterized among various Gram-negative bacteria, this is the first report of a lipid A secondary hydroxyacyltransferase. Further, the transfer of 3-hydroxylaurate (3-OH C12:0) was demonstrated to be essential for antimicrobial peptide resistance in V. cholerae and required for activation of the innate immune receptor TLR4.