Antibacterial and plasmid elimination agents
Inhibitors of the tmRNA pathway have antibacterial activity with broad species specificity, including B. anthracis and other pathogens of military and civilian interest. Identified cyclic or linear peptides are further selected by in vivo selection methods, kill bacterial pathogens when added exogenously, and/or eliminate plasmids carrying antibiotic resistance or virulence genes. The molecular target of each cyclic peptide is in the tmRNA pathway and the tmRNA pathway is inhibited in vitro and in vivo by the addition of the peptides.
1. A method of identifying an agent that inhibits at least one step in a cell's tmRNA pathway, the method comprising the steps of: (a) providing at least one cell; (b) introducing into the cell a nucleic acid comprising a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter is operably linked to a nucleotide sequence encoding a luciferase, wherein the nucleotide sequence encoding the luciferase is operably linked to at least one nucleotide sequence encoding a tmRNA targeting sequence; (c) introducing into the cell at least one candidate agent for inhibiting at least one step in the cell's tmRNA pathway, wherein introduction of the candidate agent results in a luminescent signal if the candidate agent is an inhibitor of at least one step in the tmRNA pathway; and (d) analyzing the at least one cell for a luminescent signal.
2. The method of claim 1 , wherein the nucleotide sequence encoding the luciferase is operably linked to at least two nucleotide sequences, each encoding a tmRNA targeting sequence.
3. The method of claim 1 further comprising introducing into the cell a second nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding a luciferase, wherein the nucleotide sequence encoding the luciferase is operably linked at its 3′ end to a nucleotide sequence encoding a tmRNA peptide tag, prior to introduction of the at least one candidate agent.
4. The method of claim 1 , wherein the at least one candidate agent is a cyclic peptide.
5. The method of claim 1 , wherein step (c) of introducing into the cell at least one candidate agent for inhibiting at least one step in the cell's tmRNA pathway comprises introducing into the at least one cell a cyclic peptide-expressing library.