Inter-membrane lipid signaling for preserving gram-negative outer membrane integrity and antibiotic resistance

Emory University's Office of Sponsored Programs received a $430,375 Project Grant from the National Institute of Allergy and Infectious Diseases (NIAID) under the Allergy and Infectious Diseases Research program (CFDA 93.855) effective August 1, 2025, with completion scheduled for July 31, 2027. The award funds investigator-initiated biomedical research examining inter-membrane lipid signaling mechanisms in gram-negative bacteria, specifically focusing on how Escherichia coli and Pseudomonas aeruginosa maintain outer membrane (OM) integrity and sustain antibiotic resistance. The research addresses the critical public health challenge of antibiotic-resistant bacterial infections by investigating how bacteria sense lipopolysaccharide (LPS) deficiency in the OM and activate compensatory LPS synthesis pathways through a novel phospholipase-mediated signaling mechanism.

The research will generate scientific knowledge regarding OM lipid asymmetry maintenance, bacterial homeostatic mechanisms, and potential vulnerabilities in resistance pathways that may be exploited by emerging antibiotics targeting LPS synthesis. Expected products include peer-reviewed publications, methodological advances in understanding inter-membrane lipid signaling, and preclinical findings that inform the development of next-generation antimicrobial therapeutics targeting intrinsic resistance mechanisms in gram-negative pathogens. The work will be conducted at Emory University's Atlanta, Georgia research facilities over the two-year project period.

Abstract

The alarming increase in antibiotic-resistant bacterial infections is a looming public health crisis. Gram-negative bacteria, including escherichia coli and pseudomonas aeruginosa, are leading causes of mortality attributed to antibiotic resistant infections. These bacteria build an outer membrane (OM) around their cell that acts as a shield, blocking the entry of many antibiotics and conferring potent intrinsic antibiotic resistance. Key to the OM's barrier function is the packing of lipopolysaccharide (LPS) molecules into the surface-exposed outer leaflet of the OM bilayer. Phospholipids (PLS) then make up the OM inner leaflet. An LPS deficit in the OM allows PLS to mislocalize to the surface (to fill the void) and this weakens the OM. OM lipid asymmetry is critical for gram- negative survival, virulence, and antibiotic resistance. Accordingly, bacteria employ active measures to maintain lipid asymmetry. We uncovered evidence for a novel inter-membrane lipid signaling mechanism by which e. Coli can sense LPS deficiency in the OM and trigger increased LPS synthesis inside the cell so that OM lipid asymmetry can be restored. Signaling originates from an OM phospholipase (plda) that degrades mislocalized PLS; lipid products from this reaction are internalized and notify the cell of the LPS deficiency in the OM. Our preliminary data suggest that plda plays a key homeostatic role. Deleting plda is lethal in mutant strains with slow transport of LPS to the OM. Moreover, plda activity can potently resist antibiotic inhibition of LPS synthesis. Recent novel antibiotic discovery efforts have focused on disrupting LPS synthesis and transport to the OM. Our data forewarn that plda may enable resistance to such drugs in the pre-clinical pipeline. However, our data also offer hope that interfering with plda lipid signaling may potentiate the action of these emerging novel antibiotics. We aim to test the role of plda in protecting e. Coli against LPS synthesis and transport defects induced genetically or with antibiotic inhibitors. Our goal is to characterize the plda lipid signaling pathway and unravel the steps leading to increased LPS production. We also aim to explore the conservation of inter-membrane lipid signaling by examining the newly discovered OM phospholipase of p. Aeruginosa and assessing if IT too can help counteract LPS deficits in the OM. Our study will establish a new inter-membrane lipid signaling paradigm in two important pathogens and will reveal new insights into how OM lipid asymmetry is continuously sensed and, if broken, rapidly repaired so that integrity of the OM antibiotic barrier can be continuously preserved.