The bacterial cell envelope is a remarkable and complex structure that guards bacteria from their surrounding environment. A defining feature of gram-negative bacteria is the presence of an outer membrane (OM) that encapsulates the peptidoglycan layer of these organisms. While the inner membrane (IM) is composed of glycerophospholipids (GPLs), the OM is a bilayer with extreme lipid asymmetry with GPL confined to the inner leaflet and lipopolysaccharide (LPS) localized to the outer leaflet. This unique membrane organization affords gram-negative bacteria protection from large polar molecules, as well as lipophilic compounds, serving as an essential innate barrier to a variety of antibiotics. However, once assembled, environmental factors can disrupt the LPS monolayer resulting in shedding of LPS, and as a consequence, migration of GPLs from the inner leaflet to the outer leaflet of the OM. Extensive formation of GPL rafts at the bacterial surface results in the loss of barrier function which leads to cell death. To prevent this, the cell must maintain the OM asymmetry even under extreme environmental stress. The overall objective of this application is to investigate the molecular mechanisms required for maintenance of OM asymmetry, including the role of the recently identified Mla retrograde GPL transport system. We will also investigate two additional systems, Pqi and Yeb systems, that may also serve in GPL transport. All three systems (Mla, Pqi, and Yeb) are highly conserved across Gram-negative bacteria and disruption of OM maintenance machinery has been shown to result in decreased virulence for many pathogens.
The Specific Aims of the current proposal are: (1) structure and functional analysis of Mla lipid binding proteins; (2) overall architecture and protein-protein interactions of Mla components; and (3) investigation of lipid binding by GPL transport systems in whole bacteria. Completion of the Aims will fill major gaps towards understanding maintenance of OM asymmetry and provide new avenues for the generation of novel antimicrobials.

Public Health Relevance

Gram-negative bacteria (e.g. Salmonella, Escherichia coli) are responsible for a number of human infectious diseases. Much like armor, these bacteria have a unique outer surface that prevents the use of a number of antibiotics. This proposal will help determine the molecular machinery used by gram-negative organisms to maintain their outer surface, possibly leading to novel therapies to fight multi-drug resistant bacterial pathogens.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI138576-02
Application #
9701121
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Ernst, Nancy L
Project Start
2018-06-01
Project End
2023-05-31
Budget Start
2019-06-01
Budget End
2020-05-31
Support Year
2
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Georgia
Department
Microbiology/Immun/Virology
Type
Schools of Veterinary Medicine
DUNS #
004315578
City
Athens
State
GA
Country
United States
Zip Code
30602