Discovering Novel Peptide Inhibitors of LpxA and LpxD through Phage Display

Lipid A biosynthesis is essential for membrane integrity and survival of gram negative bacteria. Because of its essentiality, this pathway is a potential target for inhibition in the development of novel antimicrobial agents. LpxA is an enzyme that catalyzes the first step in lipid A biosynthesis, the transferring of an R-3-hydroxyacyl moiety of R-3-hydroxyacyl-ACP to the 3-OH position of UDP-GlcNAc. Likewise LpxD, which catalyzes the third reaction of lipid A Biosynthesis, transfers an R-3-hydroxyacyl moiety of R-3-hydroxyacyl-ACP to the amine of UDP-3-O-(R-3-OHC14)-GlcN. Using phage display, a 12 amino acid peptide library (Ph.D.-12; New England Biolabs) was screened against immobilized LpxA and LpxD from several species of gram negative bacteria. This method was successful in finding peptides that bind to E. coli LpxA and LpxD, N. meningitidis LpxA and LpxD, and P. aeruginosa LpxD. Phage that bound to the targeted proteins were amplified and their DNA sequenced in order to identify the peptides. The binding strength between ecLpxA-binding phage and ecLpxA, nmLpxA, ecLpxD, nmLpxD, and paLpxD were compared by performing HRP-Conjugated Anti-M13 Phage Coat Antibody binding assays. We show that phage # 18 obtained from the ecLpxA phage display screen, binds tightly not only with ecLpxA, but also with ecLpxD and paLpxD. Identifying different peptides that bind to the targeted enzyme(s) and determining their binding strength, will allow focused efforts on a subset of the peptides identified in our phage display screens to further test for inhibition of cell growth in gram-negative bacteria.