Mechanisms of Action of Ribosome-Binding Antimicrobial Peptides

Today’s urgent need for better antibiotics calls for multifaceted approaches not only to search for new compounds but also to understand their mode of action to develop them as effective inhibitors of microbial growth. As contributors to this task, our lab focuses on deciphering the molecular mechanisms of antimicrobials that target the protein synthesis process in bacteria. Antimicrobial peptides (AMPs) have recently gained attention as promising candidates for new antibiotics. AMPs are produced by organisms of all kingdoms to protect themselves from bacterial infections. AMPs are structurally dissimilar and have a diverse array of bacterial targets. In these studies, we are demonstrating that two different types of AMPs, odilorhabdins (ODLs) and proline-rich antimicrobial peptides (PrAMPs), inhibit protein synthesis by targeting the ribosome. However, their binding sites within the ribosome and their strategies to stop protein synthesis are completely different and novel. The bacterium Xenorhabdus nematophila produces ODLs to kill competing bacteria co-residing in the nematode host. By binding to a unique location of the ribosome, ODLs can affect protein synthesis in two ways: at low concentrations, they cause ribosomes to make mistakes, while high concentrations block the assembly of new proteins. Importantly, the inhibitory action of ODLs effectively eradicates infections caused by drug-resistant bacteria in mice, a property that has advanced ODLs into the antibiotic development pipeline. As part of their immune systems, insects, crustaceans, and mammals produce PrAMPs. A common feature of PrAMPs is that they enter the exit tunnel through which newly made proteins leave the ribosome. Once inside the tunnel, while type I PrAMPs, i.e. Onc112 and Bac7, stop ribosomes from starting to make proteins, type II PrAMP Apidaecin allows assembly of the entire protein but impedes its release from the ribosome. Besides contributing to design of treatments for bacterial infections, the knowledge of the detailed mechanisms of action of AMPs extends beyond their application as pharmaceutical drugs. For example, Onc112 was used as a tool to discover new bacterial genes and we are taking advantage of the inhibitory properties of Apidaecin to develop molecular biology screens and to study translation regulation in a variety of organisms

Charting the sequence-activity landscape of peptide inhibitors of translation termination

Apidaecin (Api), an unmodified 18-amino-acid-long proline-rich antibacterial peptide produced by bees, has been recently described as a specific inhibitor of translation termination. It invades the nascent peptide exit tunnel of the postrelease ribosome and traps the release factors preventing their recycling. Api binds in the exit tunnel in an extended conformation that matches the placement of a nascent polypeptide and establishes multiple contacts with ribosomal RNA (rRNA) and ribosomal proteins. Which of these interactions are critical for Api's activity is unknown. We addressed this problem by analyzing the activity of all possible single-amino-acid substitutions of the Api variants synthesized in the bacterial cell. By conditionally expressing the engineered api gene, we generated Api directly in the bacterial cytosol, thereby bypassing the need for importing the peptide from the medium. The endogenously expressed Api, as well as its N-terminally truncated mutants, retained the antibacterial properties and the mechanism of action of the native peptide. Taking advantage of the Api expression system and next-generation sequencing, we mapped in one experiment all the single-amino-acid substitutions that preserve or alleviate the on-target activity of the Api mutants. Analysis of the inactivating mutations made it possible to define the pharmacophore of Api involved in critical interactions with the ribosome, transfer RNA (tRNA), and release factors. We also identified the Api segment that tolerates a variety of amino acid substitutions; alterations in this segment could be used to improve the pharmacological properties of the antibacterial peptide