Lysophospholipid signaling coordinates outer membrane homeostasis in

The Gram-negative outer membrane (OM) is an asymmetric bilayer that protects cells from environmental stress and antibiotics. This asymmetry, with lipopolysaccharide (LPS) in the outer leaflet and glycerophospholipids (GPLs) in the inner leaflet, requires coordinated synthesis of both lipid classes. The committed step of LPS biosynthesis is catalyzed by LpxC, a prime antibiotic target. Here, we show that lysophospholipids (LPLs), considered byproducts of membrane turnover, act as signaling molecules restoring OM homeostasis when LPS synthesis is limited. In the presence of the LpxC inhibitor PF-5081090 (PF), loss of the LPL recycling system increased growth, suppressed envelope stress responses, improved OM asymmetry, and lowered GPL levels to maintain GPL-to-LPS balance. This recycling system includes the transporter LplT, which moves LPLs across the inner membrane, and the acyltransferase/acyl-ACP synthetase (Aas), which acylates them to regenerate GPLs. These protective effects required the OM phospholipase PldA that degrades mislocalized GPLs into LPLs and free fatty acids. Although previous work showed that PldA-generated fatty acids stabilize LpxC and promote LPS synthesis, our findings reveal a complementary role for LPLs in signaling reduced GPL synthesis when LPS is limiting. Genetic and chemical manipulation of fatty-acid flux altered PF resistance, confirming that decreased GPLs drives protection. The two PldA-derived signals, fatty acids that promote LPS synthesis and LPLs that suppress GPL synthesis, likely operate under different metabolic conditions to interpret membrane stress and restore OM balance. This lipid-feedback mechanism establishes the first signaling function for bacterial LPLs and reveals a new layer of regulation in envelope homeostasis.IMPORTANCEThe multilayered cell envelope of Gram-negative bacteria provides natural resistance to antibiotics. Understanding cell envelope synthesis and regulation is crucial for the identification of new antimicrobial targets and improved drug design. LpxC inhibitors, a new and promising class of antibiotics, impede function of the committed enzyme in lipopolysaccharide synthesis. Here, we characterize a new mechanism of resistance to the LpxC inhibitor PF-5081090, where the accumulation of lysophospholipids signals a reduction in cellular glycerophospholipid levels to repair outer membrane balance. This work proposes a new pathway to restore outer membrane asymmetry, which is a critical aspect of cell envelope integrity, and describes a role for lysophospholipids in bacterial cell signaling when lipopolysaccharide synthesis is disrupted.