Intracellular Expression of Peptide Fusions for Demonstration of Protein Essentiality in Bacteria

Abstract

We describe a "protein knockout" technique that can be used to identify essential proteins in bacteria. This technique uses phage display to select peptides that bind specifically to purified target proteins. The peptides are expressed intracellularly and cause inhibition of growth when the protein is essential. In this study, peptides that each specifically bind to one of seven essential proteins were identified by phage display and then expressed as fusions to glutathione S-transferase in Escherichia coli. Expression of peptide fusions directed against E. coli DnaN, LpxA, RpoD, ProRS, SecA, GyrA, and Era each dramatically inhibited cell growth. Under the same conditions, a fusion with a randomized peptide sequence did not inhibit cell growth. In growthinhibited cells, inhibition could be relieved by concurrent overexpression of the relevant target protein but not by coexpression of an irrelevant protein, indicating that growth inhibition was due to a specific interaction of the expressed peptide with its target. The protein knockout technique can be used to assess the essentiality of genes of unknown function emerging from the sequencing of microbial genomes. This technique can also be used to validate proteins as drug targets, and their corresponding peptides as screening tools, for discovery of new antimicrobial agents.

Structure of UDP-N-acetylglucosamine acyltransferase with a bound antibacterial pentadecapeptide

“…Based on genetic evidence (43) and the toxicity associated with peptide 920 expression in living cells (25), E. coli LpxA is a validated antibiotic target. Peptide inhibitors are often poor drug candidates because they do not cross membranes and are subject to proteolysis.…”

“…Purity was assessed by using SDS͞PAGE, LpxA activity assays (8,23), and electrospray ionization mass spectrometry (45). Peptide 920 (NH 2 -SSGWMLDPIAGKWSR-COOH), a pentadecapeptide (25), was prepared at the University of North Carolina Peptide Synthesis Facility (Chapel Hill). Before crystallization, peptide 920 was added to a concentrated LpxA solution (20 mg͞ml) at a 25-fold molar excess (12.5 mM) and incubated overnight at 4°C.…”

“…2 A and B and 4B). Peptide 920 was derived from a library in which the P8 residue was fixed (25). In the structure of the complex, P8 (Fig.…”

“…Recently, a pentadecapeptide (termed peptide 920) with high affinity for LpxA was discovered by using phage display (25). When expressed in E. coli as a fusion protein with GST, bacterial growth was inhibited.…”

“…When expressed in E. coli as a fusion protein with GST, bacterial growth was inhibited. Specificity for LpxA was inferred from the resistance of cells overexpressing LpxA to killing by peptide 920 (25). Inhibition of LpxA in vitro was not investigated.…”