A two-step screen identifies a small molecule that disrupts membrane voltage and is effective against growing and persister gram-negative bacteria

New antibiotics effective against gram-negative bacteria are difficult to discover because many small molecules are excluded by the cell envelope. There is a growing consensus that the proton motive force (PMF) represents a key target for the development of antimicrobial agents. Here, we report data that both support that consensus and demonstrate the utility of host-based screens in discovering novel antibiotics. Previous in-host screens of small-molecule libraries have identified compounds that inhibit <i>Salmonella enterica</i> serovar Typhimurium (<i>S</i>. Typhimurium) survival within macrophages but lack potency in standard broth alone. This suggests that infection conditions may facilitate penetration of small molecules due to host-mediated damage to the bacterial cell envelope, confirming the potential for streamlined host-based screens to identify new antibiotics. Here, we screened a library of drug-like compounds in broth with an outer membrane-permeabilizing agent. Eighty compounds were identified and selected for inhibiting bacterial proliferation in macrophages. CB1.11, a mildly lipophilic compound predicted to embed in the inner membrane, was particularly effective. We found that CB1.11 targets the inner membrane to weaken the PMF, disrupt voltage-dependent protein localization, and enhance the ability of the host cell to clear the pathogen. Additionally, CB1.11 reduces persister cell survival in broth and macrophages and demonstrates antibacterial activity in gram-negative and gram-positive bacteria. These data provide evidence that the PMF is a critical target for antimicrobial development and demonstrate the utility of using streamlined host-based screens to discover novel antibiotics.IMPORTANCEFinding new antimicrobials for gram-negative bacteria is challenging because their outer membrane is difficult to penetrate. Standard laboratory screens often miss promising candidates since they do not reflect real infection conditions, where the host environment weakens the gram-negative outer membrane and allows compounds better access to their targets. In this study, we developed a simple two-step screening method to enhance antimicrobial discovery in host-based screens. First, we tested compounds in broth that mimics host conditions to find those likely to work during infection. We then screened this smaller group in infected immune cells to identify molecules that limit pathogen survival. We identified a compound that disrupts the bacterial inner membrane and energy production, helping host cells clear the infection. The compound also effective against gram-positive bacteria and persister cells. These findings show the potential of developing antimicrobials that target bacterial energy production and the benefits of using host-based screening platforms.