title: Rapid Inhibition Profiling in Bacillus subtilis to Identify the Mechanism of Action of New Antimicrobials.
abstract: Increasing antimicrobial resistance has become a major public health crisis. New antimicrobials with novel mechanisms of action (MOA) are desperately needed. We previously developed a method, bacterial cytological profiling (BCP), which utilizes fluorescence microscopy to rapidly identify the MOA of antimicrobial compounds. BCP is based upon our discovery that cells treated with antibiotics affecting different metabolic pathways generate different cytological signatures, providing quantitative information that can be used to determine a compound's MOA. Here, we describe a system, rapid inhibition profiling (RIP), for creating cytological profiles of new antibiotic targets for which there are currently no chemical inhibitors. RIP consists of the fast, inducible degradation of a target protein followed by BCP. We demonstrate that degrading essential proteins in the major metabolic pathways for DNA replication, transcription, fatty acid biosynthesis, and peptidoglycan biogenesis in Bacillus subtilis rapidly produces cytological profiles closely matching that of antimicrobials targeting the same pathways. Additionally, RIP and antibiotics targeting different steps in fatty acid biosynthesis can be differentiated from each other. We utilize RIP and BCP to show that the antibacterial MOA of four nonsteroidal anti-inflammatory antibiotics differs from that proposed based on in vitro data. RIP is a versatile method that will extend our knowledge of phenotypes associated with inactivating essential bacterial enzymes and thereby allow for screening for molecules that inhibit novel essential targets.
authors.affiliation: Division of Biological Sciences, University of California, San Diego , La Jolla, California, United States.
authors.forename: Anne
authors.initials: A
authors.lastname: Lamsa
authors.affiliation: Division of Biological Sciences, University of California, San Diego , La Jolla, California, United States.
authors.forename: Javier
authors.initials: J
authors.lastname: Lopez-Garrido
authors.affiliation: Department of Bioengineering, University of California, San Diego , La Jolla, California, United States.
authors.forename: Diana
authors.initials: D
authors.lastname: Quach
authors.affiliation: Division of Biological Sciences, University of California, San Diego , La Jolla, California, United States.
authors.forename: Eammon P
authors.initials: EP
authors.lastname: Riley
authors.affiliation: Division of Biological Sciences, University of California, San Diego , La Jolla, California, United States.
authors.forename: Joe
authors.initials: J
authors.lastname: Pogliano
authors.affiliation: Division of Biological Sciences, University of California, San Diego , La Jolla, California, United States.
authors.forename: Kit
authors.initials: K
authors.lastname: Pogliano
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D001412:Bacillus subtilis / Q000187:drug effects*; D001426:Bacterial Proteins / Q000378:metabolism; D004261:DNA Replication; D016002:Discriminant Analysis; D005227:Fatty Acids / Q000096:biosynthesis; D008826:Microbial Sensitivity Tests; D008856:Microscopy, Fluorescence; D014158:Transcription, Genetic
chemical_list: D000900:Anti-Bacterial Agents; D000894:Anti-Inflammatory Agents, Non-Steroidal; D001426:Bacterial Proteins; D005227:Fatty Acids