The Development of a Cell-Based Screen for Discovering Lipopolysaccharide Biogenesis Inhibitors

Abstract

Gram-negative bacteria are more difficult to kill than Gram-positive bacteria due to the presence of an outer membrane that prevents many antibiotics from reaching their cellular targets. The outer membrane is an asymmetric bilayer with the inner leaflet consisting of phospholipids and the outer leaflet consisting of lipopolysaccharide (LPS). LPS on the cell surface is responsible for creating this permeability barrier, which allows Gram-negative bacteria to become intrinsically insensitive to many antibiotics. Interfering with LPS biogenesis (biosynthesis and transport) has been shown to affect bacterial viability. However, targeting LPS biogenesis remains challenging since many of the components are membrane proteins with hard-to-assay activities. This dissertation utilizes the non-essentiality of this pathway in a Gram-negative pathogen, Acinetobacter baumannii, as the basis to develop a cell-based screen specific to LPS biogenesis. We used this screen to find a small-molecule inhibitor of MsbA, an essential ATP-dependent LPS flippase in the inner membrane, and validated it as an antibacterial target using a combination of genetics, biochemical and cellular assays. The discovery of a MsbA inhibitor further enabled us to study the cellular responses when LPS transport is hampered. The distinct LPS levels caused by MsbA inhibition and inactivation of a late-stage LPS transport step provide insights into an important question: why are certain steps of LPS biogenesis essential in Acinetobacter when the entire pathway can be removed?