bioRxiv2023
A promising but yet clinically unexploited antibiotic target in difficult-to-treat Gram-negative bacteria is LpxC, the key enzyme in the biosynthesis of lipopolysaccharides (LPS), which are the major constituents of the outer membrane. To gain insights into the mode of action of five different LpxC inhibitors, we conducted a comparative phenotypic and proteomic analysis. All five compounds bound to purified LpxC from Escherichia coli. Treatment of E. coli with these compounds changed the cell shape and stabilized LpxC suggesting that the FtsH-mediated turnover is impaired. LpxC inhibition sensitized E. coli to the cell wall antibiotic vancomycin, which typically does not cross the outer membrane. Four of the five compounds led to an accumulation of lyso-PE, a cleavage product of phosphatidylethanolamine (PE), generated by the phospholipase PldA. The combined results suggested an imbalance in phospholipid (PL) and LPS biosynthesis, which was corroborated by the global proteome response to treatment with the LpxC inhibitors. Apart from LpxC itself, FabA and FabB responsible for the biosynthesis of unsaturated fatty acids, were consistently upregulated. Our work also shows that antibiotics targeting the same enzyme do not necessarily elicit identical cellular responses. Compound-specific marker proteins belonged to different functional categories, like stress responses, nucleotide or amino acid metabolism and quorum sensing. These findings provide new insights into common and distinct cellular defense mechanisms against LpxC inhibition. Moreover, they support a delicate balance between LPS and PL biosynthesis with great potential as point of attack for antimicrobial intervention. ImportanceThe alarming spread of antimicrobial resistance among Gram-negative bacteria calls for novel intervention strategies. Inhibitors of LpxC, the first committed enzyme of lipopolysaccharide biosynthesis have been recognized as promising broad-spectrum antibiotics against Gram-negative pathogens. Despite the development of dozens of chemically diverse LpxC inhibitor molecules, it is essentially unknown how bacteria counteract LpxC inhibition. Our study provides comprehensive insights into the bacterial defense strategies against five different LpxC inhibitors. We show that the cellular response of Escherichia coli is compound-specific but shares a common pattern. Inhibition of LpxC is toxic, disrupts membrane integrity, and elicits a stress response, including upregulation of fatty acid biosynthesis proteins. Pre-treatment of E. coli with low doses of LpxC inhibitors increased the sensitivity to the cell wall antibiotic vancomycin suggesting new directions in combination therapies.