Regulation and antimicrobial inhibition of lipopolysaccharide biosynthesis in Escherichia coli

• Applicant: Professor Dr. Franz Narberhaus
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 543833581

The cell envelope of Gram-negative bacteria comprises three distinct layers, the cytoplasmic membrane containing phospholipids (PL), the cell wall composed of peptidoglycan (PGN), and the outer membrane characterized by lipopolysaccharides (LPS) in its outer leaflet. The biosynthesis of these layers is intricately connected through shared precursors, necessitating precise coordination. This project aims at understanding the regulatory mechanisms governing cell envelope biosynthesis, with a specific emphasis on LPS biosynthesis. In previous work, we have identified the membrane-anchored protein LapB as a crucial scaffold that recruits the initial cytoplasmic enzymes of LPS, PL and PGN biosynthesis to the membrane. We hypothesize that this protein interaction network is dynamic, responding to changes in the cell envelope status. The primary objective of this project is to identify all components in this cellular switchboard and elucidate whether and how these interactions respond to varying growth conditions. Understanding this process is relevant because both an excess and a deficiency of LPS molecules have detrimental effects. To gain insights into the molecular details of the dynamic LapB interactome, we will employ a range of genetic and biochemical assays, including the bacterial two-hybrid system, proximity labeling, pulldown experiments and microscale thermophoresis. Given that LPS biosynthesis is an essential process in most Gram-negative bacteria, enzymes involved in this pathway have emerged as appealing targets for antimicrobial intervention. While chemical compounds and antimicrobial peptides inhibiting LPS biosynthesis enzymes have been developed, the defense mechanisms of E. coli and other bacteria to such treatments are largely unknown. We have previously examined the response of E. coli and Pseudomonas aeruginosa to inhibitors of LpxC, the first committed enzyme in LPS biosynthesis. Our findings revealed distinct proteome response patterns, indicating that these bacteria employ different strategies to regulate LPS biosynthesis. In E. coli, for instance, LpxC inhibitors not only inactivate the enzyme but also stabilize it, creating a feedback loop that upregulates fatty acid biosynthesis. We now plan to extend this knowledge by comparing the responses to inhibiting early, intermediate, and late LPS biosynthesis steps using antimicrobial compounds that have become available only recently. To gain a comprehensive understanding of bacterial defense strategies against LPS biosynthesis inhibitors, we will analyze both the proteomic and transcriptomic responses. By unraveling how bacteria react to different inhibitors in the LPS biosynthesis pathway, we will learn about the crosstalk within this pipeline. Our study will also uncover the most vulnerable steps in LPS biosynthesis, offering valuable insights for the development of targeted antimicrobial strategies.

• DFG Programme: Research Grants