The balance between phospholipids and lipopolysaccharides (LPS) in the outer membrane (OM) of Gram-negative bacteria is crucial for viability. Since an imbalance in OM homeostasis can lead to cell death, the LPS biosynthesis pathway is a promising antibiotic target for difficult-to-treat Gram-negative bacteria. The first enzyme in the LPS pathway is LpxA, which utilizes acyl-ACP and UDP-N-acetylglucosamine. Since this reaction is bidirectional, LpxC, the second enzyme in the pathway constitutes the first committed step. LPS levels are adjusted by tightly controlled FtsH-mediated proteolysis of LpxC. While LpxC leads to a deacetylation of UDP-N-acetylglucosamine, LpxD is responsible for adding the second fatty acid chain. The next synthesis step involves LpxH, which is the first membrane-bound protein. After the assembly of Kdo2-Lipid A by five other membrane-bound proteins and the attachment of the core oligosaccharides, the LPS precursor molecule is transported into the periplasmic space by the ABC transporter MsbA. Finally, the O-antigen is attached to the molecule and LPS is translocated into the outer layer of the outer membrane by the Lpt-complex.

To gain insights into the mode of action of inhibitors targeting five different enzymes in the LPS pathway, we conduct a comparative phenotypic analysis. Treatment of E. coli with these compounds changed the cell shape and the viability in different ways. Whereas inhibitors of early steps of LPS synthesis and the Lpt-complex led to cell lysis, the LpxH inhibitor and the MsbA inhibitor did not. Interestingly, LpxC, LpxA and MsbA inhibition sensitized E. coli to cell wall antibiotics, which typically do not cross the outer membrane, indicating an imbalance of the LPS to phospholipid ratio.

Our study shows that in addition to well-studied LpxC inhibitors, compounds targeting earlier or later steps of LPS synthesis deserve further attention for the development of antibiotics.