Viable gram negative bacteria with reduced proteolytic activity lacking outer membrane agonists of TLR4/MD-2

摘要

Viable Gram-negative bacteria or components thereof comprising outer membranes that substantially lack a ligand, such as Lipid A or 6-acyl lipidpolysaccharide, that acts as an agonist of TLR4/MD-2. The bacteria may comprise reduced activity of arabinose-5-phosphate isomerases and one or more suppressor mutations, for example in a transporter thereby increasing the transporter's capacity to transport lipid IVA or in membrane protein YhjD. One or more genes (e.g., lpxL, lpxM, pagP, lpxP, and/or eptA) may be substantially deleted and/or one or more enzymes (e.g., LpxL, LpxM, PagP, LpxP, and/or EptA) may be substantially inactive. The bacteria may be competent to take up extracellular DNA, may be donor bacteria, or may be members of a library. The present invention also features methods of creating and utilizing such bacteria.

主权项

1. An isolated viable Gram-negative bacterium with substantially reduced bacterial proteolytic activity, wherein said bacterium is Escherichia coli and comprises (a) a mutation in at least one of the genes selected from the group consisting of kdsA, kdsC, kdsB, and waaA, or a mutation in each of the kdsD and gutQ genes, wherein said mutation results in a disruption in the biosynthesis of (Kdo)2-lipid IVA, (b) a mutation in each of the genes pagP and eptA wherein said mutation substantially inactivates the enzymatic activity of each of the proteins encoded by the pagP and eptA genes, and (c) a suppressor mutation in the msbA gene or the yjhD gene, such that the outer membrane of the bacterium substantially lacks a ligand that acts as an agonist of TLR4/MD-2 signaling, and TLR4/MD-2 signaling is substantially abrogated.