The lipid A moiety of lipopolysaccharide (LPS) forms the outer monolayer of the outer membrane of most Gram-negative bacteria. Escherichia coli lipid A is synthesized on the cytoplasmic surface of the inner membrane by a conserved pathway of nine constitutive enzymes, which were discovered by the PI. Following attachment of the core oligosaccharide, the core-lipid A complex is flipped to the outer surface of the inner membrane by the ABC transporter MsbA, where the O-antigen polymer is attached. Subsequent trafficking of nascent LPS to the outer membrane involves a second ABC transporter and two outer membrane proteins, encoded by the lpt genes. Many additional covalent modifications of lipid A may occur during its transit from the outer surface of the inner membrane to the outer membrane. Lipid A modification enzymes are therefore excellent reporters for LPS trafficking within the bacterial envelope. However, modification systems are quite variable between different Gram-negative organisms and are often regulated by environmental factors. Although not required for growth, the lipid A modification enzymes can modulate the virulence of some pathogens. Mutation or heterologous expression of the genes encoding the lipid A modification enzymes facilitates the re-engineering of lipid A structure in diverse bacterial strains and can enable the development of new vaccines, as illustrated by the PI's recent genetic studies with Francisella novicida, a mouse-specific model organism for human tularemia. Given the importance of Francisella and the fact that it contains many unprecedented lipid A modification enzymes, the specific aims for the coming grant period will be: 1) the purification and characterization of the lipid A phosphatases and glycosyltransferases of F. novicida;2) the biochemical and genetic analysis of LPS core assembly enzymes of F. novicida;3) the elucidation of the regulation of the free lipid A versus LPS content of F. novicida;and 4) the characterization of modification enzymes that hydroxylate or oxidize Kdo2- lipid A, a defined LPS substructure that supports E. coli growth and potently activates toll-like receptor 4 (TLR4). The availability of diverse modification enzymes and their structural genes will enable the large-scale, combinatorial biosynthesis of novel, Kdo2-lipid A derivatives in E. coli. In addition to creating new opportunities for vaccine development, these studies will provide fundamental insights into lipid A biology, chemistry and outer membrane biogenesis.

Public Health Relevance

Half of all human bacterial pathogens are classified as "Gram-negative";they appear as pale, pink rods in the light microscope because of their inability to take up a purple dye, called Gram-stain. Bacteria of this kind, which include all strains of Escherichia coli, Salmonella, Pseudomonas, and Francisella, contain an outer membrane that makes them impermeable to certain dyes and antibiotics. The outer surfaces of the outer membranes of Gram-negative bacteria contain large amounts of a unique substance known as lipopolysaccharide (LPS), which is held in place by lipid A. The enzymes that assemble the lipid A anchor of LPS are conserved and are excellent targets for designing new antibiotics with activity against Gram-negative pathogens that have become resistant to commercial antibiotics. In addition, the modification of lipid A structure in live bacteria by manipulation of the genes encoding the enzymes that assemble the lipid A domain of LPS are useful for attenuating bacterial pathogens so that they can be used as vaccines.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Marino, Pamela
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Duke University
Schools of Medicine
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Sohlenkamp, Christian; Raetz, Christian R H; Ingram, Brian O (2013) The calcium-stimulated lipid A 3-O deacylase from Rhizobium etli is not essential for plant nodulation. Biochim Biophys Acta 1831:1250-9
Sohlenkamp, Christian; Raetz, Christian R H; Ingram, Brian O (2013) The calcium-stimulated lipid A 3-O deacylase from Rhizobium etli is not essential for plant nodulation. Biochim Biophys Acta 1831:1250-9
Chung, Hak Suk; Raetz, Christian R H (2011) Dioxygenases in Burkholderia ambifaria and Yersinia pestis that hydroxylate the outer Kdo unit of lipopolysaccharide. Proc Natl Acad Sci U S A 108:510-5
Kong, Qingke; Six, David A; Roland, Kenneth L et al. (2011) Salmonella synthesizing 1-dephosphorylated [corrected] lipopolysaccharide exhibits low endotoxic activity while retaining its immunogenicity. J Immunol 187:412-23
Kong, Qingke; Six, David A; Liu, Qing et al. (2011) Palmitoylation state impacts induction of innate and acquired immunity by the Salmonella enterica serovar typhimurium msbB mutant. Infect Immun 79:5027-38
Lu, Yi-Hsueh; Guan, Ziqiang; Zhao, Jinshi et al. (2011) Three phosphatidylglycerol-phosphate phosphatases in the inner membrane of Escherichia coli. J Biol Chem 286:5506-18
Ingram, Brian O; Sohlenkamp, Christian; Geiger, Otto et al. (2010) Altered lipid A structures and polymyxin hypersensitivity of Rhizobium etli mutants lacking the LpxE and LpxF phosphatases. Biochim Biophys Acta 1801:593-604
Zhao, Jinshi; Raetz, Christian R H (2010) A two-component Kdo hydrolase in the inner membrane of Francisella novicida. Mol Microbiol 78:820-36
Ingram, Brian O; Masoudi, Ali; Raetz, Christian R H (2010) Escherichia coli mutants that synthesize dephosphorylated lipid A molecules. Biochemistry 49:8325-37
Raetz, Christian R H; Guan, Ziqiang; Ingram, Brian O et al. (2009) Discovery of new biosynthetic pathways: the lipid A story. J Lipid Res 50 Suppl:S103-8

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