Characterization of LpxC inhibitors and resistant mutants
LpxC, the deacetylase that catalyzes the second and committed step of lipid A biosynthesis in <italic>E. coli</italic>, is an essential enzyme for virtually all Gram-negative bacteria and one of the most promising novel antibiotic targets for the treatment of multidrug-resistant Gram-negative infections. Here, we report the characterization of two novel LpxC inhibitors that have apparent binding affinities for <italic>E. coli</italic> LpxC in the picomolar range. Furthermore, these compounds display broad spectrum activity against a plethora of Gram-negative pathogens.
In anticipation for the advancement of LpxC inhibitors in clinical trials, we undertook studies to probe potential bacterial resistance mechanisms to these compounds. In this study, we report a two-step isolation of spontaneously resistant <italic>E. coli</italic> mutants that have > 200-fold resistance to LpxC inhibitors. These mutants have two chromosomal point mutations that account for resistance additively and independently: one in <italic>fabZ</italic>, a dehydrase in fatty acid biosynthesis, and the other in <italic>thrS</italic>, the Thr-tRNA ligase.
For both enzymes, the isolated mutations result in reduced enzymatic activities <italic>in vitro</italic>. Most unexpectedly, we observed a decreased level of LpxC in bacterial cells harboring <italic>fabZ</italic> mutations, suggesting that the biosyntheses of fatty acids and lipid A are tightly regulated to maintain balance between phospholipid and lipid A. Additionally, we show that the mutation in <italic>thrS</italic> slows protein production and cellular growth, providing the first example that reduced protein biosynthesis confers a suppressive effect on inhibition of membrane biosynthesis. Altogether, our studies reveal an impressive compensatory ability of bacteria to overcome inhibition of lipid A biosynthesis by rebalancing cellular homeostasis, a unique mechanism of antibiotic resistance.
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 United States License.
Rights for Collection: Duke Dissertations