Synthetic lipid biology for combinatorial engineering of endotoxin

#In 1892, Richard Pfeiffer introduced the revolutionary concept of bacterial endotoxin in his description of a non-proteinaceous, non-secreted toxin bound to the surface of Vibrio cholerae (Pfeiffer et al., 1892). This toxin, now known as lipopolysaccharide (LPS), is the major surface molecule of Gram-negative bacteria that triggers the host immune response during infection (Poltorak et al., 2000; Raetz et al. 2002). LPS is composed of lipid A, core oligosaccharide, and O-antigen (Raetz et al., 2007). The bioactive domain of LPS is lipid A, or endotoxin (Raetz et al. 2002). Lipid A is recognized by the innate immune system through the conserved pattern recognition receptor, Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD-2) complex, which initiates a robust signal cascade that leads to production of inflammatory cytokines This signaling is crucial for detection and clearance of infection, but can be potent enough to result in lethal endotoxic shock (Raetz et al. 2002). Such tremendous immunogenicity makes LPS an attractive therapeutic tool, but its toxicity is a major concern.

#Efforts have been made to dampen the toxicity of whole bacteria by altering the degree of LPS acylation. One approach has been to inactivate lpxM, a gene encoding the acyltransferase responsible for converting lipid A from a penta-acylated to a hexa-acylated species. LpxM mutants are under investigation in the development of meningococcal vaccines, oncolytic Salmonella strains that specifically target tumors, and bacterial strains designed for gene therapy. Other efforts to detoxify cells or outer membrane vesicles have included acyl chain modification by the enzymes PagL or PagP. However, no strains have been previously generated using a complex combinatorial approach to yield a diverse library in one species of bacterium.

#A collection of LPS molecules exhibiting a wide range of toxicity would be beneficial for many biotechnological applications.

#Some specific example embodiments of the disclosure may be understood by referring, in part, to the following description and the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

#FIG. 1 shows combinatorial engineering of lipid A anchors to generate diverse immune responses. The outer surface of E. coli strains varies in LPS structure when plasmids are expressed that contain combinations of lipid A modifying enzymes. The altered LPS molecules bind and activate the TLR4/MD-2 complex differentially, altering the nature of downstream cytokine production.

#FIG. 2 shows modification machinery used for generation of engineered lipid A molecules in whole bacteria. Lipid A structures of wild type E. coli K12, BN1, and BN2 are shown with the names of the 6 lipid A modifying enzymes represented next to the group that each enzyme modifies (a). LpxR, PagL, LpxE, and LpxF (as indicated) all remove the corresponding acyl chain or phosphate group. LpxO and PagP transfer the hydroxyl or acyl chain onto the molecule, respectively. The attachment site for remaining polysaccharide is indicated at the 6′-position of each molecule. The organism source, enzyme activity, and active site topology of each of the 6 enzymes is presented (b), and the chart shows the 61 combinatorial strains, organized according to acylation and phosphorylation patterns (c). Combinatorial strains were generated by transformation of BN1 and BN2 with a pQLinkN plasmid expressing combinations of the 6 lipid A modifying enzymes. Each enzyme is abbreviated by its final letter and ordered alphabetically in the plasmid name, i.e., LpxE is abbreviated E, LpxF is F, LpxR is R, PagP is P, PagL is L, and LpxO is O.

#While the present disclosure is susceptible to various modifications and alternative forms, specific example embodiments have been shown in the figures and are herein described in more detail. It should be understood, however, that the description of specific example embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, this disclosure is to cover all modifications and equivalents as illustrated, in part, by the appended claims.

#The features and advantages of the present invention will be apparent to those skilled in the art. While numerous changes may be made by those skilled in the art, such changes are within the spirit of the invention.

#The present disclosure generally relates to genetic engineering of bacteria. More particularly, the present disclosure relates to genetic engineering of Gram-negative bacteria expressing different species of lipid A on their surface.