An Investigation of Lipopolysaccharide Degradation and the Role of Lipid A Modification on Innate Immune Response and Antibiotic Resistance
Open Access
- Author:
- Maybin, Michael
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology (PHD)
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 27, 2023
- Committee Members:
- Ken Keiler, Major Field Member
Timothy Miyashiro, Major Field Member
Timothy Meredith, Chair & Dissertation Advisor
Darrell Cockburn, Outside Unit & Field Member
Santhosh Girirajan, Program Head/Chair
Emily Weinert, Major Field Member - Keywords:
- lipopolysaccharide
innate immunity
polymyxin
antibiotic resistance
inserstion sequences
alkaline phosphatase
TLR4 - Abstract:
- Lipopolysaccharide (LPS) is a major component of the outer membrane (OM) in Gram-negative bacteria. Due to its location in the outer leaflet of the OM LPS acts as the first line of defense for the bacterial cell. Overall, LPS provides structural stability while also facilitating several interactions between the cell and its environment. These roles include maintaining membrane permeability, providing antibiotic resistance, and modulating immune responses all of which contribute to overall bacterial survival and pathogenesis. LPS is composed of three main structural regions consisting of lipid A, the core oligosaccharide, and the O-antigen. The lipid A region of LPS is the most conserved portion of LPS that bacteria regularly modify as a response to environmental factors. Modifications altering to the acylation state or overall charge of LPS, through additions of charged groups like phosphoethanolamine (PEtN) and 4-amino-4-deoxy-L-arabinose (Ara4N), alter membrane permeability or increase resistance to cationic antimicrobial peptides (AMPs) respectively. In addition, LPS recognition by the innate immune response is facilitated by the lipid A region whereby bacterial modifications modulate LPS immunogenicity. LPS is recognized by the Toll-like receptor 4/myeloid differentiation factor-2 (TLR4/MD2) complex where the acylation state and presence of the 1 and 4’-phosphate groups on lipid A are key for immunogenicity. This dissertation investigates the structure-activity relationship between lipid A and immune recognition, detoxification, and bacterial antibiotic resistance. Specifically, the substrate specificity of intestinal alkaline phosphatase against LPS is determined using chemically defined LPS chemotypes and demonstrates no appreciable activity against the key 1- and 4’- lipid A phosphate groups unless acyl chains adjacent to the phosphate groups are first removed. PEtN modifications on lipid A are demonstrated to enhance TLR4/MD2 agonist activity of underacylated LPS chemotypes. A mechanism of polymyxin resistance in a sub-population of Escherichia coli B is elucidated whereby increased lipid A modification with Ara4N due to insertion sequence mediated genome amplifications drives phenotypic resistance. Finally, the development and evaluation of three orthogonal assays for bacterial derived LPS degradation (LPS-ase) activity is described, and their applications are discussed.