Lipopolysaccharide (LPS) is present in the outer membrane of most Gram-negative bacteria, and plays a key role in constructing a proper cellular envelope for bacteria to survive in harsh environments. The tight packing of LPS in the outer membrane generates a network of charges and sugars, which selectively allow the entry of nutrient molecules, while limit the penetration of toxic compounds including detergents and antibiotics. Due to its critical importance in the biogenesis of bacterial membrane barrier, LPS biosynthesis and transport pathway is a particularly interesting target for developing novel antibiotics. LPS is also crucial in the host-pathogen interactions, and functions as a potent activator of innate immune response in the animals. LPS is a complex and highly variable glycolipid, composed of a lipid A moiety, a core oligosaccharide and a long-chain O-antigenic polysaccharide. The structure of lipid A and core oligosaccharide are relatively conserved, presumably due to their roles in maintaining the integrity of permeability barrier. In contrast, the O-antigen of LPS shows hypervariable structures, which is consistent with their functions in interacting with the outside environment and host defense. Gram-negative bacteria devote a large amount of energy and a sophisticated protein machinery to the efficient and proper production, transport and assembly of LPS molecules. The synthesis of LPS starts at the interface between the cytoplasm and the inner membrane, leading to the generation of lipid A-core oligosaccharide, also called rough LPS, which resides in the inner leaflet of the inner membrane. Rough LPS is flipped across the inner membrane by an ATP binding cassette (ABC) transporter, MsbA. The rough LPS in the periplasmic leaflet is further added with various lengths and forms of O-antigen, becoming a ?smooth? LPS. For the LPS transport across the periplasm and to the outer membrane, seven proteins named as Lpt A-G are involved. Several lines of evidence converge to suggest a model, in which the Lpt proteins form a continuous bridge connecting the two membranes. The ABC transporter, formed as LptB2FG, is thought to extract the LPS molecules from the inner membrane, and transport them to the tightly associated bitopic LptC, and to the periplasmic LptA. Multiple LptA proteins may form a continuous bridge to reach the LptDE complex in the outer membrane, which mediates the LPS insertion into the outer leaflet of the outer membrane. Here we propose a series of structural and functional studies on the LPS transport protein machinery using a variety of biochemical and cryo-EM techniques. A molecular understanding on the function and regulation of the LPS transport pathway will contribute to the understanding of the biogenesis of the outer membrane of many Gram-negative bacteria, and also aid the development of novel antibiotics that directly target the bacterial membrane barrier.

Public Health Relevance

Lipopolysaccharide (LPS) is present in the outer membrane of most Gram-negative bacteria, and plays a key role in constructing a proper cellular envelope for bacteria to survive in harsh environments. The tight packing of LPS in the outer membrane selectively allow the entry of nutrient molecules, while limit the penetration of toxic compounds. Thus, understanding the molecular mechanisms of LPS transport pathway will contribute to the development of novel antibiotics that interfere with the biogenesis of bacterial membrane barrier.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM122797-04
Application #
9923673
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Flicker, Paula F
Project Start
2017-08-01
Project End
2022-04-30
Budget Start
2020-05-01
Budget End
2021-04-30
Support Year
4
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Harvard Medical School
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Mi, Wei; Li, Yanyan; Yoon, Sung Hwan et al. (2017) Structural basis of MsbA-mediated lipopolysaccharide transport. Nature 549:233-237