Lipid A, the hydrophobic membrane anchor of lipopolysaccharide, is a glucosamine-based saccharolipid that constitutes the outer monolayer of the outer membrane of Gram-negative bacteria and protects bacteria from the external damage of detergents and antibiotics. It is also the active component of lipopolysaccharide that causes life-threatening Gram-negative septic shock. Lipid A is synthesized by nine enzymes of the Raetz pathway in E. coli. The first six enzymes of lipid A biosynthesis are required for the viability of virtually all Gram-negative bacteria and are novel antibiotic targets. After lipid A i generated, it is flipped from the cytosolic surface of the inner membrane to the periplasmic surface, where it can be further transformed by lipid A modification enzymes that are specific to individual bacterial species. These modifications help bacteria evade the host immune response, adapt to environmental changes, and generate altered lipid A molecules that display diverse bioactivities. Although detailed structural analyses of several lipid A biosynthetic and modification enzymes have been carried out, providing rich information for structure-aided inhibitor development, others require further characterization. This proposal focuses on biochemical and structural studies of the essential lipid A biosynthetic enzymes that convert UDP-diacylglucosamine (UDP-DAGn) to 2,3- diacyl-GlcN-1-P (lipid X) and the lipid A modification enzyme LpxE that is important for bacterial virulence and production of monophosphorylated lipid A (MPLA), a widely used immunotherapeutic agent. These studies are expected to generate fundamental insights into the structure and mechanism of lipid A enzymes as well as the biogenesis and function of the bacterial outer membrane, and ultimately contribute to the development of novel therapeutics to improve human health.

Public Health Relevance

The wide spread of multidrug-resistant Gram-negative pathogens, particularly those in hospitals with high numbers of vulnerable patients, is a major public health threat. Gram-negative bacteria are characterized by the enrichment of lipid A-anchored lipopolysaccharide in the outer monoleaflet of the outer membrane. Lipid A biosynthesis is a highly conserved pathway that is required for the survival of nearly all Gram-negative bacteria and represents an excellent antibiotic target. Lipid A modification enzymes, on the other hand, are specific to individual bacterial species and are not essential. However, they are major contributors to bacterial virulence and pathogenicity in human hosts. Modified lipid A molecules display diverse bioactivities and have been exploited as immunotherapeutic agents for prevention and treatment of cancer, allergy, and Alzheimer's disease. Bioengineered bacterial strains bearing altered lipid A show significantly attenuated infectivity and are being evaluated as live vaccines. Hence, the proposed biochemical and structural characterization of lipid A biosynthetic and modification enzymes will directly contribute to the development of novel therapeutics for the prevention and treatment of antimicrobial-resistant Gram-negative infections and other human diseases, such as cancer, immunological disorders, and neurodegenerative diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM115355-02
Application #
9230402
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Marino, Pamela
Project Start
2016-03-01
Project End
2020-02-29
Budget Start
2017-03-01
Budget End
2018-02-28
Support Year
2
Fiscal Year
2017
Total Cost
$271,178
Indirect Cost
$97,928
Name
Duke University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
Cho, Jae; Lee, Chul-Jin; Zhao, Jinshi et al. (2016) Structure of the essential Haemophilus influenzae UDP-diacylglucosamine pyrophosphohydrolase LpxH in lipid A biosynthesis. Nat Microbiol 1:16154
Titecat, Marie; Liang, Xiaofei; Lee, Chul-Jin et al. (2016) High susceptibility of MDR and XDR Gram-negative pathogens to biphenyl-diacetylene-based difluoromethyl-allo-threonyl-hydroxamate LpxC inhibitors. J Antimicrob Chemother 71:2874-82
Young, Hayley E; Zhao, Jinshi; Barker, Jeffrey R et al. (2016) Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in Chlamydia trachomatis. MBio 7:e00090
Wu, Qinglin; Coggins, Brian E; Zhou, Pei (2016) Unbiased measurements of reconstruction fidelity of sparsely sampled magnetic resonance spectra. Nat Commun 7:12281
Liang, Xiaofei; Gopalaswamy, Ramesh; Navas 3rd, Frank et al. (2016) A Scalable Synthesis of the Difluoromethyl-allo-threonyl Hydroxamate-Based LpxC Inhibitor LPC-058. J Org Chem 81:4393-8